document,summary,source
"over the past decade , protein separation techniques and peptide analysis by mass spectrometry have greatly improved , and qualitative and quantitative proteome comparisons have become powerful tools for defining the composition of complex proteomes and the functions of protein complexes.(1 ) current ion trap mass spectrometers , in particular , are very sensitive and have fast analysis times that enable the identification of hundreds of proteins in a single lc - ms / ms analysis . 
 however , obtaining comprehensive protein profiles from very complex samples , such as biological fluids or mammalian cell or tissue lysates , remains challenging due to the large number of proteins present in the sample over wide concentration ranges . to improve proteome coverage , 
 samples typically are fractionated at either the protein level or peptide level , or both . 
 regardless of the initial fractionation methods used , the final step in most strategies involves analysis of tryptic peptides by lc - ms / ms . 
 however ,  undersampling  will occur , that is , only a subset of the peptides will be identified , if the complexity of these tryptic digests exceeds the analytical capacity of the mass spectrometer ( e.g. , when more peptides elute from the hplc column per unit time than can be analyzed , or low - abundance peptides are below the instrument detection limit ) . 
 a common observation when complex proteomes are analyzed is that the lists of proteins identified are often quite variable , which has raised concerns about the general value of proteomics for most applications , if it actually does have poor reproducibility . 
 typically , the identifications of abundant proteins are reproducible , while most of the variability is observed in lower - abundance proteins . 
 one strategy of achieving more comprehensive proteome coverage actually exploits this variability by simply performing repetitive lc - ms / ms analyses on each sample . 
 other strategies for increasing the number of proteins identified include prefractionating the proteome based on protein physical properties , improving separation of the peptide mixture at the hplc separation step , modifying the esi interface to enhance ionization , or adding an ion mobility spectrometry ( faims ) interface before the ion trap.(19 ) proteome coverage also can be improved at the data analysis stage by using high - mass - accuracy data(20 ) or improved data - process algorithms . 
 other strategies include a two - step method that reanalyzes the same sample with an inclusion list ( eliminating redundant precursor selection in the second analysis ) and a  replay run  that analyzes a sample twice from a single injection with targeted analysis of undersampled features in the replay run.(26 ) although each of these methods can achieve increased sensitivity , it is difficult to assess the relative importance of strategies that have not been directly compared in a single study due to variations in samples , study design , mass spectrometry platforms , data - analysis strategies used , and other laboratory - to - laboratory variations . 
 consequently , a number of direct comparisons of alternative analysis strategies have been conducted to identify the best analysis approaches for specific types of samples such as serum and cell or tissue lysates . 
 factors that should be considered when comparing alternative analysis strategies are the amount of total protein required for the analysis and the total amount of mass spectrometry instrument time required by each method . 
 it is now well - established that repetitive lc - ms / ms analyses , longer hplc gradients , or additional fractionation  via either more fractions or more modes of separation of complex proteomes  will usually confer some benefit in terms of depth of analysis . 
 the key factor is to determine the most time- and resource - effective strategies for achieving a high depth of analysis , as these factors directly affect sample throughput and proteome analysis cost . in this study 
 , we compared the effects of two commonly used strategies : gelc - ms / ms with repetitive lc - ms / ms analysis and additional protein prefractionation and assessed their effects on depth of analysis and reproducibility using cancer cell lysates . 
 neither strategy requires expensive special hardware or reagents , and both have been integrated into diverse analyses of complex proteomes by multiple laboratories . 
 because of its simplicity , repetitive analysis often has been used to increase protein identifications after different fractionation schemes , most of which involve two dimensions of separation at either the peptide or protein levels , such as mud - pit or gelc . 
 utilizing a cell extract of the human metastatic melanoma cell line 1205lu,(31 ) the gelc - ms / ms analysis with and without repetitive injections was compared and these data sets were compared to a 3-d method that included the prefractionation of the cell lysate using microscale solution isoelectrofocusing ( microsol ief ) prior to the sds - page separation . to balance the total number of lc - ms / ms runs and total instrument time between the repetitive 2-d method and the 3-d method , the 20 fractions from the sds - page fractionation each were analyzed four times and the cumulative result was compared with the 80-fraction proteome analysis using the 3-d approach . 
 other factors such as hplc gradient , mass spectrometry method , and data processing were kept uniform to demonstrate clearly the relative effects of repetitive analyses versus an additional prefractionation step at the protein level . 
 these comparisons showed excellent reproducibility between the methods , and substantially improved proteome coverage using the 3-d method compared with the repetitive analysis of fractions from the 2-d method . 
 rpmi-1640 cell culture medium , sypro ruby stain , and nupage precast gels were from invitrogen corporation , carlsbad , ca . 
 ultra pure urea , thiourea , dtt , and chaps were from ge healthcare , ltd . , giles , u.k . 
 the bradford protein assay kit was from thermo fisher scientific , waltham , ma , and sequencing - grade porcine trypsin was from promega corporation , madison , wi . 
 the human melanoma 1205lu cell line was maintained in rpmi-1640 medium supplemented with 10% fetal calf serum . 
 cells were harvested on ice by scraping in cold phosphate - buffered saline ( pbs ) with inhibitors ( 0.3 mm pmsf , 2 mm sodium orthovanadate , 50 mm sodium fluoride , 1 g / ml leupeptin , 1 g / ml pepstatin ) when 7090% confluence was reached . 
 the tissue culture plates then were washed three times in cold pbs with inhibitors ; the washes were collected , combined with the initial scraped cells , collected by centrifugation , and the cell pellet was quick frozen in liquid nitrogen followed by storage at 80 c . 
 cell lysates were prepared using 800 l of lysis buffer ( 25 mm tris , ph 8.0 , 8 m urea , 2 m thiourea , 4% chaps , 1 mm dtt ) per cell pellet collected from a 15-cm tissue culture dish . 
 the lysate was sonicated with a probe sonicator on ice with a duty cycle setting of 50% and an output level < 5% . 
 insoluble material was removed by centrifugation at 100  000 g for 60 min and was estimated to be < 1% of the total protein based on staining intensity of colloidal coomassie sds gels . 
 the supernatant was reduced using dtt and alkylated with n , n - dimethylacrylamide ( dma ) , as previously described.(11 ) total protein in the supernatant was measured using the bradford method . 
 cell lysates ( 1.5 mg ) were separated into four ph ranges by microsol ief using a zoom ief fractionator ( invitrogen corporation , carlsbad , ca ) . for these separations , 
 the fractionator contained five immobiline gel disks , each sustaining a ph of 3.0 , 5.4 , 6.2 , 7.0 , and 12 , respectively . 
 these disks delineated four separation chambers and the cell lysate in a sample buffer consisting of 8 m urea , 2 m thiourea , 4% chaps , 1% dtt , 1% ph 37 zoom focusing buffer , and 1% ph 712 zoom focusing buffer was loaded into the middle two separation chambers . 
 the outer chambers were loaded with sample buffer only and standard electrode solutions were used . 
 samples were focused at a constant 750 v and a maximum of 1 ma for 3 h. the solution in each sample chamber was removed and measured . each chamber was rinsed with a small volume of sample buffer so that , when combined with the original solution removed from that chamber , the final volume was 700 l . proteins trapped in the immobiline gel disks were recovered using two sequential 175 l extractions for 30 min each using 10 mm tris , ph 8 , 1% sds for 30 min . 
 the cell lysate and microsol ief fractions were initially analytically separated on 10% nupage minigels ( with mes running buffer ) using standard separation conditions . 
 preparative sds - page was run using unfractionated cell lysate and the four microsol ief fractions in an analogous manner to the analytical run , except the separation distance was limited to 40 mm to minimize required numbers of fractions and gel volume per fraction . 
 typically , six lanes  two lanes from the gelc ( 2-d ) method containing 60 g each of cell lysate and one lane each from the 3-d method  were sliced into 4 mm  1 mm  1 mm thick gel pieces . 
 two adjacent pieces in each lane were combined in a digestion well and were digested overnight with trypsin . 
 corresponding digestions from the duplicate 2-d method lanes were combined , generating a total of 20 digestions for the 2-d method . 
 trypsin digestions were injected into a 75 m i.d .  15 cm picofrit ( new objective , inc . , 
 woburn , ma ) column packed with 5 m magic c18 resin and peptides were separated by nano - hplc ( eksigent technologies , dublin , ca ) interfaced with a ltq linear ion trap mass spectrometer ( thermo fisher scientific , waltham , ma ) . for each analysis , 
 8 l of trypsin digest was loaded onto the column using solvent a ( 0.1% formic acid in milli - q [ millipore corporation , billerica , ma ] water ) . 
 peptides were subsequently eluted using the following gradient conditions with 0.1% formic acid in acetonitrile as solvent b : 128% b over 42 min , 2850% b over 25.5 min , 5080% b over 5 min , and 80% b for 5 min . to minimize carryover 
 twenty 2-d samples were analyzed first , followed by another replicated injection of the same 2-d samples . 
 then , 80 3-d samples were analyzed , followed by the third and fourth replicated injections of the 2-d samples . 
 the ltq mass spectrometer was operated with dynamic exclusion enabled for 30 s , full scans from 4002000 m / z , and data - dependent ms / ms analysis on the six most intense ions . 
 28 , rev . 13 , university of washington , seattle , wa ) in bioworks ( ver . 
 3.1 , thermo fisher scientific , waltham , ma ) , and the human uniref 100 ( ver . 
 may , 2007 ) protein database , which was downloaded from the protein information resource at georgetown university . to generate the decoy database , the protein amino acid sequence for each database entry 
 was reversed and the entire reversed database was appended in front of the original forward sequences . 
 the data were searched against the combined forward / reverse database using partial trypsin specificity with a 2.5 da precursor mass tolerance and 1 da fragment ion mass tolerance . 
 consensus protein lists were generated by dtaselect ( ver 1.9 , licensed from scripps research institute , la jolla , ca ) after applying the following filters : full tryptic boundaries , xcorr  1.8 ( z = 1 ) , 2.1 ( z = 2 ) , 3.25 ( z = 3 ) , cn  0.05 . 
 custom software was then used to ensure that each unique peptide sequence was used only once in assembling the protein list . to identify common and unique proteins found by 2-d and 
 3-d methods , protein and peptide data were put into a relational database ( mysql ) and matched using custom software . 
 the strategy used to compare ( 1 ) the conventional gelc - ms / ms method ( protein separation by sds gel + reverse - phase peptide separation ) , ( 2 ) gelc - ms / ms with repetitive injections , and ( 3 ) a 3-d method that used microsol ief fractionation prior to the sds - page step ( protein ief + sds gel + reverse - phase peptide separation ) is summarized in figure 1 . to directly compare the relative impact on proteome coverage of repetitive injections versus an additional protein separation step 
 , equal amounts of the same cell lysate were fractionated and an equal number of lc - ms / ms analyses were used . 
 a cell lysate of 1205lu cell line was processed in such a manner that each common step was comparable . 
 the 3-d method consisted of microsol ief , sds - page , and lc - ms / ms . 
 subsequently , the final minimum consensus protein lists from the three data sets were compared to evaluate the ability of enhancing sensitivity of repetitive analysis and additional fractionation strategies . 
 microsol ief initially was developed to prefractionate complex proteomes prior to narrow ph range 2-d gels(33 ) and was subsequently used prior to 1-d sds - page as part of a 4-d strategy for fractionating serum or plasma.(11 ) in this study , we evaluated its utility for prefractionating cell lysates prior to gelc - ms / ms , since this separation mode is orthogonal to sds - page and does not suffer from many of the limitations of 2-d gels . 
 because the dynamic range of protein concentrations in cell lysates is substantially less than in plasma or serum , the microsol ief separation was designed to yield four final fractions rather than the larger number of fractions typically used for plasma . 
 the immobiline membrane disks between chambers were extracted because , as shown previously , proteins with pi values close to the ph of the disk remain in the disk but can be recovered in high yield . 
 extracts from disks with extreme ph values ( 3.0 and 12 ) showed only trace amount of proteins , as few proteins have pi s near these values . 
 extractions from disks located between separation chambers contained a mixture of proteins unique to that disk and proteins present in adjacent chambers as previously observed . 
 when similar samples were separated by microsol ief and analyzed on 2-d gels , comparisons of solution fractions and membrane fractions showed that most proteins recovered from membrane disks were either unique to the membrane or had pi s slightly lower than the ph of the membrane ( data not shown ) . since the goal in this experiment was to fractionate the entire proteome into a small number of fractions at the microsol ief step , disk extracts were combined with the fraction to its right to maximize recovery ( figure 2 ) . 
 the extraction from the ph 12 disk was discarded as it contained a negligible amount of protein . 
 thus , after microsol ief fractionation , the cancer cell proteome was divided into four fractions of roughly similar complexity and with a simpler protein content . 
 separation of the melanoma 1205lu cell extract by microsol ief and pooling to produce four fractions . 
 equal portions of each fraction and separation membrane disk extract were analyzed by sds - page to evaluate separation and relative amounts of total protein in each fraction . 
 the solution recovered from individual chambers was pooled with the adjacent membrane disk extract on the low ph side of the pool , as shown at the bottom of the gel , to produce four fractions . 
 the extract from the ph 12 membrane disk had negligible protein and was not used further . for the conventional gelc - ms / ms method , 
 60 g of the original cell lysate , which was close to a maximum load while avoiding band distortion , was loaded into each of two lanes of a nupage gel ( figure 3a ) . 
 proteins were separated until the tracking dye migrated 40 mm . after staining the gel with colloidal coomassie , the 40-mm lane was divided into 20 equal fractions and digested with trypsin 
 . digests from corresponding positions in the two replicate lanes were combined to yield sufficient volume for four 8 l injections . 
 sds - page separation of melanoma 1205lu cell lysate and microsol fractions for proteome analysis . 
 samples were electrophoresed until the tracking dye migrated 4 cm ; gels were stained with colloidal coomassie and individual lanes were cut into 20 equal - sized slices , as shown . 
 ( a ) for the 2-d method , the unfractionated lysate of the 1205lu cells was separated in two lanes ( 60 g / lane ) . 
 ( b ) microsol ief fractions derived from 120 g of cell lysate were separated for the 3-d method . 
 for the 3-d method , the amount of each fraction loaded onto the preparative gel was the protein recovered from 120 g of total cell lysate ( figure 3b ) . 
 hence , the total amount of protein in the four gel lanes in figure 3b should be close to the amount of protein in the two lanes in figure 3a , provided sample losses during the microsol ief procedure were low , as has been previously demonstrated . 
 as noted above , the gel lanes from the microsol fractions were cut and digested in the same manner as the gel slices from figure 3a , except in this case there were no duplicate gel lanes to be combined . 
 hplc and mass spectrometer performance were carefully monitored to ensure consistent performance through these experiments . 
 to further minimize effects of minor instrument performance variations , samples were injected in an order described in materials and methods . 
 consistent performance of the autoinjector was monitored by weighing each sample vial before and after injection . 
 there was no significant difference in the injection amount between the two methods ( t - test p > 0.05 ) . 
 all data were consistently analyzed and filtered as described in materials and methods , which resulted in estimated false - positive rates ( fpr ) calculated by dividing reverse - hit peptide counts by forward - hit peptide counts of 1.6% and 1.4% for 2-d and 3-d data , respectively . 
 figure 4 shows the nonredundant peptide and protein counts for the 3-d data set as well as differing numbers of replicates for the 2-d sample set . 
 similar numbers of peptide and protein identifications were obtained for all individual 2-d data sets ( data not shown ) . as expected , 
 the total number of nonredundant peptides increased moderately as additional replicate data sets were combined , and the incremental increase diminished as each new replicate was added . 
 it was evident from the curve shown in figure 4a that the data set resulting from combining four replicate runs was approaching a plateau . 
 parallel trends were observed for the peptide data sets defined by proteins identified by 3 , 2 , or 1 peptide(s ) . 
 a total of 25  641 nonredundant peptides were identified after combining all four replicates of the 2-d analysis , which was 26% less than the 32  216 peptides identified by the 3-d method . 
 ( a ) nonredundant peptide counts from a single 2-d analysis , combined data from increasing numbers of replicate analyses , and the 3-d method . 
 ( b ) corresponding nonredundant protein counts for the same data sets as shown in panel a. the total number of lc - ms / ms runs that each data set contains is shown at the bottom of the figure . 
 protein counts showed a similar trend as the peptide counts ( figure 4b ) , that is , adding a second replicate increased the number of nonredundant proteins more markedly than adding a third and fourth replicate . 
 for example , the number of proteins identified by two or more peptides increased by 594 , 305 , and 162 when two , three , and four replicates were combined . the most appropriate comparison of the 3-d and 2-d methods is between the 3-d data set and four replicates of the 2-d samples because they both involve a total of 80 lc - ms / ms runs using equal amounts of initial cell lysate . 
 when protein identifications based on two or more peptides were counted , the 3-d method identified 3486 proteins compared with 2850 proteins for the 2-d method . 
 these 636 additional proteins ( 22.3% increase ) indicate a clear advantage of the 3-d method compared with the 2-d / replicate run method , even when equal amounts of mass spectrometer time are utilized . 
 the proteomes produced by the 2-d / four repetitive - run and the 3-d methods were compared using the lists of proteins identified by two or more peptides . 
 this comparison was facilitated by the fact that both data sets were searched using the same database and that dtaselect lists all protein names identified in the search . 
 if a protein identification carried one protein name that could be found in the other data set , we deduced that both data sets had identified the same protein . 
 this comparison at the protein name level showed extensive overlap between the two data sets even at this first level comparison ( figure 5 ) . 
 specifically , the two data sets shared 2555 proteins , which corresponded to 90% of the smaller 2-d / repetitive - run proteome . 
 the 295 proteins that appeared to be unique to the less in - depth 2-d / replicate run data set were further investigated to determine the basis for this apparent lack of reproducibility between in - depth analyses of the same biological sample . 
 further analysis showed that 184 of these proteins were present in the 3-d data set as single - hit proteins . 
 a majority ( 135 ) of these 184 proteins were two - peptide proteins in the 2-d data , indicating that the 3-d method identified these proteins by one less peptide . 
 when the unfiltered data for the 3-d proteome was examined using randomly selected proteins in this group , we found that a substantial number of these proteins had been identified by the same second peptide as in the 2-d data set , but one or more of the filtering parameters was slightly lower than the cutoff values selected for global data filtering . as a result 
 , these moderate changes in scoring parameters reduced the two- or three - hit protein identification in the 2-d data set to a 1-hit protein in the 3-d data set . 
 we also further evaluated the 111 proteins found exclusively in the 2-d method . more than three - quarters ( 88 ) of them were two - peptide proteins . among these identifications , 
 the 11 proteins identified by more than three unique peptide sequences were a particular concern , as the number of peptide identifications suggested that these proteins were not near the detection threshold and should have been identified in the more in - depth 3-d proteome . 
 we found that the majority of the peptide sequences associated with these 11 proteins in the 2-d data set also existed in the 3-d data set , but they were assigned to highly homologous proteins ( supplemental table 1 ) . although a large number of common sequences for these proteins were observed in both data sets , a small number of unique sequences led the dtaselect program to assign these peptides to different proteins for each data set . 
 of the 385 peptides assigned to the 111 proteins unique to the 2-d / replicate run data set , 226 peptides were present in the filtered 3-d data 
 the two methods identified 2555 common proteins with two or more peptides per protein ( 90% of the total proteins identified in the smaller 2-d / replicate - run data set ) . 
 of the 295 apparently unique proteins in the 2-d method , 184 proteins were identified in the 3-d data set by one peptide . 
 the pie charts in the lower panels show the number of peptide hits for the 2-d method for the proteins that were not directly identified in the 3-d data set ( 111 proteins or 3.9% of the proteins in the 2-d data set ) and those identified by a single peptide in the 3-d data set ( 183 proteins or 6.5% of the proteins in the 2-d data set ) . 
 these proteins , which were identified by two or three peptides in the 2-d / replicate data set , were identified by a single peptide in the 3-d data set . in each case , a second peptide was detected in the 3-d data set but failed to pass the data filter cutoff due to slightly lower values for xcorr or cn ( bolded values in table ) . 
 the filtering cutoff values used were xcorr  1.8 ( + 1 ) ; 2.1 ( + 2 ) ; 3.25 ( + 3 ) , cn  0.05 . the bold values indicate they are below cutoff value .  -  indicates  not found . 
 to a first approximation , those proteins identified in the 2-d / repetitive - runs data set by only two or three peptides can be regarded as likely low - abundance proteins in the sample analyzed , since sequence coverage is usually a rough indicator of protein abundance level . 
 the relative depth of coverage for these putative low - abundance proteins was compared in the 2-d / repetitive - run and the 3-d methods . as shown in figure 6 , 
 for those proteins identified in both data sets , the majority showed greater sequence coverage in the 3-d data set . 
 that is , of the 491 common proteins identified by two peptides in the 2-d data , the 3-d method found more peptides for 317 proteins ( 64.6% ) and the remaining proteins in this group were identified with an equal number of peptides . furthermore , in 114 cases , the 3-d method found at least three additional unique peptides , indicating a substantially greater depth of analysis . 
 of the 394 common proteins identified by the 2-d method with three peptides , 325 proteins ( 82.5% ) were identified by an equal or larger number of peptides in the 3-d method . 
 these data clearly indicate that , in most cases , the 3-d method had the ability to detect more peptides for low - abundance proteins than did the 2-d method . 
 comparison of the number of peptides identified in the 2-d / repetitive and 3-d methods . 
 ( a ) among proteins common to both data sets , 491 proteins were identified by two peptides in the 2-d / repetitive data set . 
 the 3-d method found an equal number of peptides for 174 proteins and more peptides for 317 proteins . 
 ( b ) among 394 proteins identified with three peptides by the 2-d method , the 3-d method found one less peptide for 69 proteins ( i.e. , + 1 for 2-d ) , an equal number of peptides for 99 proteins , and more peptides for 226 proteins . 
 in this study , we systematically evaluated the relative merits of repetitive lc - ms / ms runs compared with introduction of an additional protein level separation step for increasing proteome coverage of cancer cell lysates . 
 factors that affect apparent reproducibility between proteome analyses performed on the same sample also were evaluated . the basic analysis platform used for repetitive analyses 
 was the commonly utilized gelc - ms / ms method , which can be considered to be a 2-d proteomics method as it involves two dimensions of separation , that is , protein separation using sds - page and reverse - phase hplc separation of tryptic peptides . 
 this method was compared to a 3-d method consisting of solution ief at the protein level followed by the gelc - ms / ms method . 
 it is important when comparing alternative analysis platforms to consider the total number of lc - ms / ms runs per proteome , because improved proteome coverage typically can be achieved by lengthening the hplc gradient or by repeating lc - ms / ms analysis of complex samples . 
 similarly , many separation modes prior to lc - ms / ms can be at least incrementally improved by simply increasing the number of fractions collected , provided that the resolution of the separation method exceeds the initial fraction size used . 
 but in some cases , increases in protein coverage may be too small to be considered advantageous when total analysis time per proteome is considered . 
 hence , evaluation of the merits of greater depth of analysis , particularly small improvements , must be constantly weighted relative to overall throughput . 
 furthermore , total mass spectrometer instrument time frequently is the limiting resource , and when fractionation prior to the lc - ms / ms step is used , it is the rate - limiting step in proteome analysis throughput . hence , the most meaningful comparisons are those where the total mass spectrometer analysis time per proteome is held constant . in this study , we used a consistent gradient time and 80 lc - ms / ms runs for both the 3-d method and the 2-d / repetitive - run method ( four repeat injections ) . 
 similarly , all other experimental variables were held as constant as possible , including use of replicate aliquots of a single cell lysate preparation , gel separation lengths , gel volumes per trypsin digestion reaction , instrument tuning , and data - analysis methods . 
 our goal was to determine , quantitatively , which method represents the more efficient utilization of mass spectrometer time when analyzing complex proteomes . 
 robust proteome analysis methods should be reproducible in addition to identifying the majority of proteins present in a biological sample 
 . one major cause of variations in proteins identified in replicate analyses of the same proteome is undersampling in the mass spectrometer , as discussed above . 
 therefore , high proteome coverage should be linked to good reproducibility of proteome analysis results because extensive proteome coverage will occur only if undersampling is minimized . a second factor that will contribute to poor reproducibility between proteome protein lists is use of data filtering conditions that result in high false - peptide and protein - identification rates , since false positives usually are random . 
 hence , data filtering stringency is another tradeoff that must be considered when selecting a proteome analysis strategy . 
 while low stringency filters contribute to noise and low reproducibility , excessively stringent filters will greatly diminish the number of protein identifications and hence the value of the experiment . in the current study 
 , data filters were used that yielded peptide false - positive rates between 1 and 2% as estimated using a decoy reverse database , thereby minimizing apparent poor reproducibility between data sets . 
 this level of stringency results in very few false positives for proteins identified by two or more peptides and , while there are some false positives within the one - hit protein list , a majority of these identifications are correct . 
 the repetitive analyses of 2-d data showed increased peptide and protein counts ( figure 4 ) indicative of undersampling in the basic gelc - ms / ms method used here . 
 the overall gain from four repetitive analyses of proteins identified by two or more peptides was 1061 ( 59% ) compared to the initial single analysis . a similar increase ( 61% ) 
 was observed in repetitive mudpit using nine analyses.(10 ) as expected , the greatest positive impact on proteome coverage was use of a second replicate run , which increased the number of proteins identified by two or more peptides by 33% while doubling instrument time . 
 in contrast , adding a third and fourth replicate only increased protein coverage by 13% and 6% , respectively . 
 these data indicate that performing a second analysis of each fraction when using gelc - ms / ms would be a positive tradeoff between instrument time and protein coverage . 
 however , further doubling of instrument time by performing four repetitive runs is unlikely to represent optimal use of instrument time for most types of experiments . of course 
 , an alternative to performing a repetitive analysis of gel fractions would be to obtain more slices per gel lane . 
 in analogous experiments where gel lanes were divided into 40 or 60 fractions , we observed increases in the number of proteins identified that were similar to those obtained in this study for duplicate and triplicate analyses of the 20 fractions per gel lane ( data not shown ) . 
 although producing more fractions per gel lane increases the number of in - gel digestions , the overall increase in total analysis time for a proteome is minor . 
 hence , we generally prefer to use more fractions per lane rather than to replicate analyses when greater depth of analysis is desired using gelc - ms / ms experiments . 
 longer gels and larger numbers of fractions per lane were not used in the current study because we wanted to keep total mass spectrometer time per proteome ( approximately 160 h per proteome ) within practical limits while simultaneously matching gel lengths , gel volumes , and other parameters . that is , extrapolating from other 2-d and 3-d experiments that we have performed , we expect that the total number of proteins for each data set ( 2-d , 2-d / repetitive runs , and 3-d ) 
 would have increased moderately if we would have used 40 or 60 slices per gel lane for all samples . but 
 use of 40 or 60 fractions per gel lane would have increased total instrument time to about 320 and 480 h per proteome , which represents an impractically low throughput for most studies . 
 interestingly , as we increase the number of fractions per gel lane to 40 or 60 fractions , the incremental increases in new proteins identified diminish , analogous to the diminishing benefits of adding each additional replicate in the repetitive - run approach ( figure 4a ) . 
 although similar trends are observed for these two approaches , the mechanisms for increasing protein coverage are quite different . that is , using a larger number of gel fractions increases protein separation and simplifies the mixture of proteins present in each fraction , while repetitive runs exploit subtle variations in peptide separations in replicate hplc runs and subtle variations in data - dependent selection of low - level ions for ms / ms fragmentation and analysis . 
 the 3-d method clearly provided superior protein and peptide coverage compared with the 2-d / repetitive method , which indicates that adding an additional protein separation step represents a more efficient use of mass spectrometer instrument time . 
 this method identified 3486 proteins with two or more peptides , which is 22% more than the 2-d / repetitive method that used equal instrument time . at the peptide level 
 , the 3-d method identified 30  385 high - confidence , nonredundant peptides , which is nearly 2.5 times more than that found in a single survey using the 2-d method ( 12  160 ) and 28% more than the cumulative count in four repetitive analyses ( 23  648 ) . 
 furthermore , more unique peptides were found for most low - abundance proteins in the 3-d method data compared with the cumulative 2-d method data ( figure 6 ) . 
 it is not surprising that adding solution ief as an additional orthogonal protein separation step to a gelc - ms / ms method is an efficient strategy for increasing proteome coverage and sequence coverage of lower - abundance proteins . 
 microsol ief separates the proteins that would normally be in a single gel slice into four gel slices ( see figure 3 ) . 
 , the simpler samples should decrease ion suppression effects and reduce dynamic range within each digest . finally , in some cases , improved scores for ms2 spectra in the 3-d method probably resulted from a lower probability of interfering ions being isolated with the target ion for fragmentation . 
 while the repetitive analysis strategy also improved proteome coverage , it had neither a built - in mechanism to reduce repeated sampling of abundant ions between replicates , nor could it explore the ions below the ms2 triggering threshold . 
 an alterative technique that has sometimes been used to improve replicate runs is to scan different mass ranges in each replicate . 
 however , pilot experiments suggested that this approach was less productive than the simple repetitive analysis method used here 
 . one frequent criticism of proteomics methods is that the proteins identified on repeat analyses often are not very reproducible . 
 a recent study suggested good reproducibility was achievable across 27 laboratories on a simple 20-protein mixture after uniform data processing was used.(35 ) but this simple sample of abundant proteins at the same concentration does not reflect real biological complexity . 
 hence , in the current study , we compared the reproducibility between different analysis methods using a very complex sample of biological interest , that is , a human cancer cell lysate . among four replicate analyses of the 2-d samples , 
 this indicated at least 76% of the proteins observed in one analysis were reproducibly detected despite significant undersampling . 
 more importantly , at least 90% of the proteins observed in the 2-d / four - replicate data set based on two or more peptides directly matched to a corresponding protein 3-d data set , and most of the apparent mismatches were caused by trivial data analysis issues . 
 a more rigorous comparison of the two comprehensive data sets showed that greater than 96% of the proteins identified in the 2-d / repetitive - run proteome were actually observed within the complete 3-d data set . 
 one reason for the initially apparent , lower reproducibility when protein names were compared was slight variations in peptides scores together with use of rigid data filter cutoff values ( see above and figure 5 ) . 
 that is , the 10% of proteins that were apparently unique to the 2-d / repetitive - run data set included 184 proteins ( 6.4% of the 2-d / repetitive protein list ) that were identified by a single peptide in the 3-d data set . reasons why these proteins were only identified by a single peptide in the 3-d data set include run - to - run variations in automated selection of low - abundance signals for ms / ms and run - to - run variations in sequest scores coupled with use of rigid data filters . 
 a second contributing factor to the initially apparent , lower reproducibility at the protein list level is database redundancy and limitations of current software for consistently producing consensus protein lists from identified peptides . of the 111 proteins apparently unique to the 2-d 
 / repetitive - run data set and not identified by a single - hit protein in the 3-d data set , most were highly homologous to proteins identified in the 3-d data set ( see results and supplemental table 1 ) . among the 385 peptides belonging to the 111 unique proteins in the 2-d data , 
 although these 111 unique proteins comprised 4% of all proteins identified , the 159 unique peptides were only 0.7% of all 2-d filtered peptides . 
 this illustrates that very small variations in identified peptides can have a proportionally higher  apparent 
 since we used each unique peptide a single time during assembly of consensus protein lists , the common sequences were assigned to the protein with the most unique sequences . 
 consequently , for a group of proteins that have high sequence identity , one or two unique peptides could determine which protein in the protein family emerged in the final consensus protein list . 
 this illustrates that better software tools are needed for identifying and displaying putative unique proteins within protein families . 
 similarly , improved databases with uniform names or other labels that clearly indicate membership within a protein family would be beneficial . in conclusion , 
 additional prefractionation with microsol ief substantially increased proteome coverage and sequence coverage compared with a gelc - ms / ms - repetitive run method that utilized an equal amount of mass spectrometer time . 
 furthermore , the reproducibility of protein lists between the two methods was quite high because undersampling during data acquisition had been minimized . 
 most of the apparent differences in protein identifications were due to limitations of current sequence databases and protein naming conventions , as well as software limitations for filtering database search results and building consensus protein lists .","in - depth , reproducible coverage of complex proteomes is challenging because the complexity of tryptic digests subjected to lc - ms / ms analysis frequently exceeds mass spectrometer analytical capacity , which results in undersampling of data . in this study 
 , we used cancer cell lysates to systematically compare the commonly used gelc - ms / ms ( 1-d protein + 1-d peptide separation ) method using four repetitive injections ( 2-d / repetitive ) with a 3-d method that included solution isoelectric focusing and involved an equal number of lc - ms / ms runs . 
 the 3-d method detected substantially more unique peptides and proteins , including higher numbers of unique peptides from low - abundance proteins , demonstrating that additional fractionation at the protein level is more effective than repetitive analyses at overcoming lc - ms / ms undersampling . 
 importantly , more than 90% of the 2-d / repetitive protein identifications were found in the 3-d method data in a direct protein level comparison , and the reproducibility between data sets increased to greater than 96% when factors such as database redundancy and use of rigid scoring thresholds were considered . 
 hence , high reproducibility of complex proteomes , such as human cancer cell lysates , readily can be achieved when using multidimensional separation methods with good depth of analysis .",pubmed
"although perioperative mortality from tonsillectomy and submucosal uvulopalatopharyngoplasty ( smuppp ) with tonsillectomy has decreased significantly , postoperative bleeding is still an important complication . 
 the incidence of bleeding after tonsillectomy is approximately 0.5%-10% , with death occurring in 1 in 20,000 patients . 
 primary bleeding refers to that occurring within the first 24 h postoperatively , and bleeding occurring 5 - 10 days after surgery has been classified as secondary or delayed bleeding . 
 factors such as age , gender , and surgical technique have been evaluated as risk factors for posttonsillectomy bleeding . to date , no study has specifically compared the rate of bleeding after tonsillectomy and smuppp . 
 we reviewed the medical records of all patients at our institution who underwent unipolar cautery tonsillectomy alone or in conjunction with smuppp during the last 10 years to compare the incidence of secondary , delayed postoperative hemorrhage between the two procedures . 
 in this retrospective study , following approval from the institutional review board , we reviewed the medical records of 404 patients who underwent tonsillectomy and smuppp procedures at our institution between january 2001 and december 2010 . 
 the patients were divided into two groups according to the surgical intervention they had gone through . 
 group 1 , who underwent tonsillectomy , consisted of 198 patients ; group 2 , who underwent smuppp , consisted of 206 patients . 
 patients younger than 18 years of age or those who underwent a tonsillectomy as part of the treatment of carcinoma of the head or neck were excluded from the study . all postoperative secondary , delayed 
 ( 5 - 10 days after surgery ) hemorrhage which required intervention , such as electrocautery under local anesthesia or a second surgery under general anesthesia for both groups was recorded . 
 it is based on standard surgical principles of epithelial preservation and tension - free closure of the epithelium . 
 most of the mucosa of the soft palate and the anterior and posterior pillars are preserved . 
 elimination of redundant palatal and pharyngeal tissue is accomplished by subepithelial and muscular stretching and closure . 
 the smuppp technique comprises two steps : step 1 , tonsillectomy and excision of the soft palate and redundant tissue ; and step 2 , reconstruction . 
 a detailed description of the technique is as follows . the patient is orally intubated , and the head is hyperextended with a crowe - davis tonsillectomy mouth gag in place . 
 this helps to assess the lateral extent of the tonsil and the proper plane of dissection . 
 the needle tip is then used in 35 w cutting mode to expose the tonsil capsule via a curvilinear incision . to further identify the capsule , yankauer suction 
 the dissection proceeds using a bovie in 35 w coagulation mode to separate the tonsil capsule from the superior constrictor muscle and to cauterize the blood vessels entering the tonsil . 
 constant traction of the tonsil is maintained , allowing the tissue to be separated as it is cauterized by the bovie . 
 the removal of the tonsil proceeds within its anatomical boundaries , with the musculature of the tonsil fossa left intact . 
 dissection proceeds inferiorly toward the base of the tongue until the tonsil is free of all attachments . at the end of the tonsillectomy , 
 after the removal of both tonsils , the palate is addressed . the uvula is grasped with allis forceps and retracted anteriorly for an optimal approach to the posterior surface of the soft palate . 
 a curvilinear horizontal incision is made on the mucosa at the base of the uvula posteriorly , preserving almost the entire posterior soft palate mucosa . using the needle tip in cautery coagulation mode , 
 the mucosa is separated from the muscle , releasing the posterior soft palate mucosa . using the needle tip in cutting mode , 
 a trapezoid incision is outlined at the anterior mucosa of the soft palate at a level previously identified below the  dimpled  area , as visualized preoperatively on the awake patient . 
 the incision is extended bilaterally and horizontally across the soft palate to the downward slope of the anterior pillar . 
 the uvula and the submucosal tissue of the lower edge of the soft palate are excised . 
 the redundant pharyngeal folds are eliminated by approximation of the muscular tissue of the tonsillar fossa and the soft palate using interrupted 3 - 0 vicryl sutures through these muscles . 
 the mucosal flap edges are loosely approximated , without undermining , using 3 - 0 vicryl sutures . 
 particular attention is paid to the inferior parts of the tonsillar fossa , where the majority of secondary bleeding occurs . 
 bipolar cautery is used to cauterize these parts , after which the inferior parts of the tonsillar fossas are sutured using inferolateral pharyngoplasty . 
 inferolateral pharyngoplasty involves at least four interrupted sutures with 3 - 0 vicryl to tack the inferior pharyngeal redundancy laterally . 
 this is accomplished by suturing the muscle layer of the inferior tonsillar fossa laterally to the base of the tongue as the first layer . 
 the second layer of mucosal epithelial interrupted sutures then overlies this layer . during soft palate excision , 
 particular attention is paid to the base of the uvula on both sides where the uvular artery enters the soft palate . 
 bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have decreased the incidence of postoperative bleeding compared wth routine tonsillectomy . 
 the tonsillectomy technique used in group 1 patients is also the same electrocautery technique and instrumentation described by drezner , and the tonsillar fossae are left intact for secondary healing [ videos 1 - 2 ] . 
 the  test was used for statistical analysis . except in cases with expected sample sizes of fewer than five , 
 postoperative secondary bleeding from the tonsillectomy regions were recorded and compared . bleeding from the palate was excluded . the smuppp technique used in this study 
 it is based on standard surgical principles of epithelial preservation and tension - free closure of the epithelium . 
 most of the mucosa of the soft palate and the anterior and posterior pillars are preserved . 
 elimination of redundant palatal and pharyngeal tissue is accomplished by subepithelial and muscular stretching and closure . 
 the smuppp technique comprises two steps : step 1 , tonsillectomy and excision of the soft palate and redundant tissue ; and step 2 , reconstruction . 
 a detailed description of the technique is as follows . the patient is orally intubated , and the head is hyperextended with a crowe - davis tonsillectomy mouth gag in place . 
 this helps to assess the lateral extent of the tonsil and the proper plane of dissection . 
 the needle tip is then used in 35 w cutting mode to expose the tonsil capsule via a curvilinear incision . to further identify the capsule , yankauer suction 
 the dissection proceeds using a bovie in 35 w coagulation mode to separate the tonsil capsule from the superior constrictor muscle and to cauterize the blood vessels entering the tonsil . 
 constant traction of the tonsil is maintained , allowing the tissue to be separated as it is cauterized by the bovie . 
 the removal of the tonsil proceeds within its anatomical boundaries , with the musculature of the tonsil fossa left intact . 
 dissection proceeds inferiorly toward the base of the tongue until the tonsil is free of all attachments . at the end of the tonsillectomy , 
 and the muscle fibers are still covered by fascia . after the removal of both tonsils , 
 the uvula is grasped with allis forceps and retracted anteriorly for an optimal approach to the posterior surface of the soft palate . 
 a curvilinear horizontal incision is made on the mucosa at the base of the uvula posteriorly , preserving almost the entire posterior soft palate mucosa . using the needle tip in cautery coagulation mode , 
 the mucosa is separated from the muscle , releasing the posterior soft palate mucosa . using the needle tip in cutting mode , 
 a trapezoid incision is outlined at the anterior mucosa of the soft palate at a level previously identified below the  dimpled  area , as visualized preoperatively on the awake patient . 
 the incision is extended bilaterally and horizontally across the soft palate to the downward slope of the anterior pillar . 
 the uvula and the submucosal tissue of the lower edge of the soft palate are excised . 
 the redundant pharyngeal folds are eliminated by approximation of the muscular tissue of the tonsillar fossa and the soft palate using interrupted 3 - 0 vicryl sutures through these muscles . 
 the mucosal flap edges are loosely approximated , without undermining , using 3 - 0 vicryl sutures . 
 particular attention is paid to the inferior parts of the tonsillar fossa , where the majority of secondary bleeding occurs . 
 bipolar cautery is used to cauterize these parts , after which the inferior parts of the tonsillar fossas are sutured using inferolateral pharyngoplasty . 
 inferolateral pharyngoplasty involves at least four interrupted sutures with 3 - 0 vicryl to tack the inferior pharyngeal redundancy laterally . 
 this is accomplished by suturing the muscle layer of the inferior tonsillar fossa laterally to the base of the tongue as the first layer . 
 the second layer of mucosal epithelial interrupted sutures then overlies this layer . during soft palate excision , particular attention 
 is paid to the base of the uvula on both sides where the uvular artery enters the soft palate . 
 bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have decreased the incidence of postoperative bleeding compared wth routine tonsillectomy . 
 the tonsillectomy technique used in group 1 patients is also the same electrocautery technique and instrumentation described by drezner , and the tonsillar fossae are left intact for secondary healing [ videos 1 - 2 ] . 
 the  test was used for statistical analysis . except in cases with expected sample sizes of fewer than five , 
 the mean age of group 1 patients was 38.1 ( 2.58 ) years , and that of group 2 was 37.7 ( 2.2 ) years . 
 no statistically significant difference in age or gender distribution was found between groups 1 and 2 ( p > 0.05 ) . there were 198 patients in the tonsillectomy with unipolar electrocautery group ( group 1 ) and 206 in the smuppp group ( group 2 ) . 
 the incidence of secondary , delayed hemorrhage was 5.05% ( 10 patients ) in group 1 and 1.45% ( 3 patients ) in group 2 ( p = 0.05 ) showing statistically significant difference . 
 the incidence of delayed hemorrhage requiring surgical treatment was 4.54% ( nine patients ) in group 1 and 0.97% ( two patients ) in group 2 [ graph 1 ] . 
 the rate of post - tonsillectomy hemorrhage reported in the literature is variable due to the lack of consensus on what is considered significant postoperative hemorrhage . in many studies , only if surgical intervention under general anesthesia is required to control the bleeding will a case be considered as having significant hemorrhage , and the studies that take this view find lower hemorrhage rates . 
 but in our study , all patients who required medical attention for control of bleeding were considered as having postoperative hemorrhage , regardless of whether they required hemorrhage control under local anesthesia or full surgery under general anesthesia . 
 posttonsillectomy hemorrhage incidence in adults is reported in the literature to be 1.5%-18.0% , but the more commonly given range is between 3.0% and 5.0% . in our study , 
 the postoperative bleeding rate of the tonsillectomy group was similar to the rates mentioned in the literature . 
 rates of hemorrhage after smuppp have not been studied as extensively as those after tonsillectomy . 
 the range encountered in the literature varies from 0.6% to 14.0% , and in our study , the bleeding rate after smuppp was 1.45% which falls within that range . in the present study , 
 when electrocautery tonsillectomy and smuppp groups were compared , a significant difference in the rate of hemorrhage was apparent . 
 although the tonsillectomy portion of the procedures was performed in an identical manner for both of our study groups , the postoperative bleeding rates differed . 
 some studies have found an increased rate of posttonsillectomy hemorrhage in males compared with females , but the gender distribution did not differ between study groups , which agrees with several other studies . 
 the increased rate of hemorrhage in the tonsillectomy group could not be attributed to any factor other than the operative techniques used . 
 however , the bleeding rates for tonsillectomy and smuppp patients remained significantly higher among tonsillectomy patients whose muscular tissue of the tonsillar fossa was not approximated using interrupted 3 - 0 vicryl sutures through the tissue . 
 suturing both the muscular and mucosal layers of the tonsillar fossa in smuppp seemed to decrease the incidence of postoperative bleeding . furthermore , 
 bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have . decreased the incidence of postoperative bleeding . 
 suturing of the tonsillar pillars is the only influencing factor detected that may have lowered the bleeding incidence . 
 double - layer closure and approximating muscular and mucosal layers after tonsillectomy might help postoperative oropharyngeal healing , resulting in decreased bleeding incidence . 
 medical attention for control of bleeding were considered as having postoperative hemorrhage , regardless of whether they required hemorrhage control under local anesthesia or full surgery under general anesthesia . 
 posttonsillectomy hemorrhage incidence in adults is reported in the literature to be 1.5%-18.0% , but the more commonly given range is between 3.0% and 5.0% . in our study , 
 the postoperative bleeding rate of the tonsillectomy group was similar to the rates mentioned in the literature . 
 rates of hemorrhage after smuppp have not been studied as extensively as those after tonsillectomy . 
 the range encountered in the literature varies from 0.6% to 14.0% , and in our study , the bleeding rate after smuppp was 1.45% which falls within that range . in the present study , 
 when electrocautery tonsillectomy and smuppp groups were compared , a significant difference in the rate of hemorrhage was apparent . 
 although the tonsillectomy portion of the procedures was performed in an identical manner for both of our study groups , the postoperative bleeding rates differed . 
 some studies have found an increased rate of posttonsillectomy hemorrhage in males compared with females , but the gender distribution did not differ between study groups , which agrees with several other studies . 
 the increased rate of hemorrhage in the tonsillectomy group could not be attributed to any factor other than the operative techniques used . 
 however , the bleeding rates for tonsillectomy and smuppp patients remained significantly higher among tonsillectomy patients whose muscular tissue of the tonsillar fossa was not approximated using interrupted 3 - 0 vicryl sutures through the tissue . 
 suturing both the muscular and mucosal layers of the tonsillar fossa in smuppp seemed to decrease the incidence of postoperative bleeding . furthermore , bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have . 
 suturing of the tonsillar pillars is the only influencing factor detected that may have lowered the bleeding incidence . 
 double - layer closure and approximating muscular and mucosal layers after tonsillectomy might help postoperative oropharyngeal healing , resulting in decreased bleeding incidence . 
 submucosal tonsillectomy , which is a part of smuppp , has a lower incidence of postoperative delayed hemorrhage compared with tonsillectomy using unipolar cautery in adult patients . 
 we suggest routine use of the suturing technique for smuppp in all tonsillectomy patients to reduce the rate of postoperative hemorrhage .","background : the aim of this study was to compare the incidence of postoperative secondary hemorrhage for tonsillectomy and submucosal uvulopalatopharyngoplasty ( smuppp).materials and methods : in this retrospective case series , the medical records of 404 patients who underwent tonsillectomy with unipolar electrocautery and smuppp at our institution between january 2001 and december 2010 were reviewed . 
 the patients were divided into two groups : group 1 ( 198 patients ) underwent tonsillectomy ; group 2 ( 206 patients ) underwent smuppp . 
 main outcome measures were incidence of bleeding or complications after tonsillectomy and smuppp and the need for revision surgery.results:the mean age of group 1 patients was 38.1 ( 2.58 ) years and that of group 2 was 37.7 ( 2.25 ) years . 
 males were 51.3% of group 1 and 46.7% of group 2 . 
 no statistically significant difference in age or gender distribution was found between groups 1 and 2 . 
 the incidence of secondary , delayed hemorrhage was 5.05% ( 10 patients ) in group 1 and 1.45% ( three patients ) in group 2 ( p = 0.05 ) . 
 the incidence of delayed hemorrhage requiring surgical treatment was 4.54% ( nine patients ) in group 1 and 0.97% ( two patients ) in group 2.conclusion:in adults , smuppp , which includes tonsillectomy , has a lower incidence of postoperative delayed hemorrhage than does tonsillectomy with unipolar cautery .",pubmed
"the - and -lactam 
 scaffolds are found in various 
 biologically relevant compounds . additionally , these core structures 
 are crucial intermediates in the synthesis of various natural products 
 as well as pharmaceuticals , prompting the development of new methods 
 for the synthesis of these valuable structures . 
 our group has explored the synthesis 
 of -lactams by the reactions of aryl- , thioaryl- , and cyano - substituted anhydrides . 
 furthermore , we have demonstrated that one - pot four - component reactions 
 ( 4crs ) are possible in the case of thioaryl substitution . 
 recent computational studies of the cyanosuccinic anhydride reactions 
 demonstrate that they proceed by a mannich - like mechanism via the 
 enol tautomer of the anhydride . 
 related 
 reactions reported by castagnoli , cushman , and haimova likely proceed 
 by this mechanism as well . 
 consistent with the proposed mechanism , those 
 anhydrides are poorly enolizable and require forcing conditions . 
 two 
 decades later , cushman and haimova reported analogous reactions with 
 homophthalic anhydride , which is highly enolizable . 
 the high reactivity 
 of this anhydride at lower temperatures is consistent with the mannich - like 
 mechanism . 
 these results prompted us to 
 explore mannich - type reactions and 
 related 4crs of variously substituted anhydrides . 
 although aryl - substituted 
 succinic anhydrides showed some variation in reactivity based on the 
 substituents attached to the aromatic ring , direct attachment of an 
 electron - withdrawing group resulted in higher reactivity . 
 furthermore , in the case of cyanosuccinic anhydrides , 
 the diastereoselectivity was reversed relative to the aryl and thioaryl 
 cases and could be controlled by resident stereogenic centers . 
 we envisioned that the electron - withdrawing capability of a sulfone 
 would impart the anhydride with similar reactivity as the nitrile - substituted 
 anhydride , albeit with drastically different steric requirements ( scheme 1 ) . 
 herein we report the development of anhydride 
 mannich reactions ( amrs ) using sulfone - substituted anhydrides for 
 the synthesis of - and -lactams as well as a six - step 
 formal synthesis of ( )-isoretronecanol . 
 multigram - scale syntheses of 
 anhydrides 3a  d were achieved from 
 readily available starting materials . 
 anhydride 3a was synthesized by conjugate addition of 
 benzenesulfinic acid sodium salt ( 7 ) to maleic anhydride 
 to yield a diacid , which was cyclized 
 in one step ( scheme 2a ) . 
 the syntheses of anhydrides 3b  d were achieved by michael addition 
 of sulfone acetate 8 to various tert - butyl acrylates followed by ester cleavage and cyclization ( scheme 2b ) . 
 anhydride 3a readily 
 reacted with imines to form -lactams . 
 as with previous amrs 
 , the carboxylic acid 5 ( table 1 ) is the first - formed product . unlike all previous 
 related intermediates , these carboxylic acids were prone to facile 
 decarboxylation . 
 mild heating drove the decarboxylation to completion , 
 allowing isolation of the sulfone - substituted lactams in high yields 
 with only the anti diastereomer detectable by h nmr spectroscopy . in all cases , non - enolizable substrates 
 the lack of reactivity of ketones and enolizable 
 aldehydes in the amr probably stems from the rapid equilibration to 
 the enamine tautomer , which is unreactive in the mannich addition 
 step . 
 the stereochemical 
 configuration of the products was assigned by 
 x - ray crystallography . 
 lactam 10e has an anti configuration between the aromatic ring and the sulfonyl group . 
 the other lactams were assigned by comparison of the coupling constants 
 of the adjacent protons ( e.g. , 10f ; figure 1a ) . furthermore , acid intermediate 5f , which 
 leads to 10f by decarboxylation , was also crystalline 
 and was found to have an anti configuration between 
 the sulfone and aromatic substituents ( figure 1b ) . 
 our previous mechanistic work on the cyano - substituted anhydrides 
 suggests that the anti - configured carboxylic acids 
 are the kinetic products of the amrs . 
 subsequent 
 decarboxylation leads to the anti - sulfones , which 
 would also be expected to be thermodynamically preferred . 
 x - ray structures 
 of 5f , 10f , and 10 g and j values for 10e and 10j . 
 the stereochemical outcome of 
 the reactions of imines with 3a is adequately explained 
 by a transition state that is analogous 
 to what is operative for cyanosuccinic anhydrides 
 the iminium ion is attacked by the enolate of the anhydride , 
 and the cyclic transition state is stabilized by hydrogen bonding 
 to the sulfone . 
 after the mannich reaction , subsequent rapid transannular 
 acylation forms the lactam product.1 the carboxylic acid 
 intermediates could be trapped with trimethylsilyldiazomethane 
 ( tmschn2 ) . 
 after many esterification conditions 
 were tried , treatment of crude mixtures of 5 with a commercially 
 available solution of tmschn2 resulted in reasonable yields 
 of methyl esters 11a  c ( table 2 ) . 
 these intermediates 
 could also be smoothly desulfonylated with magnesium in methanol to 
 produce the corresponding esters 12 in good yield . 
 although 
 this reaction was successful for 11a and 11b , no ester product was observed in the attempted desulfonylation 
 of 11c . 
 unlike the analogous -lactams , these intermediates were impossible 
 to isolate , thus preventing assignment of the configuration of the 
 predominant diastereomer . 
 upon decarboxylation , sulfone lactams 13 were formed as single isomers in high yields ( table 3 ) . determined by h 
 this 
 change in diastereoselectivity upon decarboxylation suggests 
 the formation of a planar anion followed by selective protonation 
 from one face of the anion . 
 this result is in accordance with experimental 
 data and models of sulfone anions proposed by corey . similar to our results with the succinic anhydride , 
 the reaction 
 tolerated a variety of non - enolizable aldehydes and a variety of alkylamines 
 and anilines . 
 while some of our initial reactions gave low yields 
 ( 4050% ) , the use of commercially available imines or those 
 purified by distillation significantly increased the yields of the 
 lactam products . 
 methyl - substituted anhydrides 3c and 3d provided trisubstituted -lactams in high 
 yields with good 
 diastereomeric ratios ( scheme 3 ) . 
 these results 
 suggest that substitution on the anhydride provides excellent facial 
 selectivity , resulting in the formation of only two diastereomers 
 of the intermediate carboxylic acids . 
 studies are ongoing 
 in our laboratory to develop an enantioselective michael addition 
 with sulfone acetate 8 to provide the anhydrides as single 
 enantiomers , which would presumably provide enantiomerically pure 
 lactam products . 
 this new amr was applied to a formal synthesis of ( )-isoretronecanol 
 ( scheme 4 ) . 
 the reaction of imine 16 with anhydride 3a followed by ring - closing metathesis 
 provides the pyrrolizidine core of the natural product from cinnamaldehyde , 
 allylamine , and 3a in two steps . the elimination of 18 yields 21 , which has been converted to ( )-isoretronecanol 
 in two steps in low yield . 
 further 
 reduction yields lactam ester 22 , which has been converted 
 to ( )-isoretronecanol in one step . while this second route is redox - inefficient , it provides material in a much higher yield and avoids 
 the high - pressure conditions necessary to reduce pyrrole 21 . 
 this synthetic route showcases the versatility of the anhydride 
 michael reaction of the sulfone - substituted anhydride , exemplifying 
 its usefulness in the assembly of heterocyclic targets . 
 this synthesis 
 demonstrates that the amr of sulfone - substituted anhydrides will be 
 useful for the assembly of many heterocyclic targets , natural and 
 otherwise . 
 in 
 summary , we have successfully synthesized various sulfone - substituted 
 - and -lactams in good yields with high diastereoselectivities 
 ( > 95:5 ) by an anhydride mannich reaction . 
 we have previously studied 
 the reactivity of several different substituted succinic anhydrides , 
 but these are the first new results employing glutaric anhydyrides 
 since the original work of castagnoli . unlike previous 
 reactions of this type to date , the carboxylic acid products were 
 prone to decarboxylation . although the -lactam acids derived 
 from the substituted succinic anhydride could be trapped as esters , 
 the analogous -lactam products could not be intercepted . 
 another 
 important difference was observed in the diastereoselectivity of the 
 initial mannich - type reaction . 
 although the reactions of succinic 
 anhydyrides were very selective , the acid products derived from the 
 glutaric anhydrides were generally formed as diastereomeric mixtures . 
 in both cases , 
 we have shown that this methodology is highly robust and 
 have showcased it in the formal synthesis of a biologically active 
 natural product . 
 unless otherwise specified , all 
 commercially available reagents were used as received . all reactions 
 using dried solvents 
 were carried out under an atmosphere of argon 
 in flame - dried glassware with magnetic stirring . 
 dry solvent was dispensed 
 from a solvent purification system that passes solvent through two 
 columns of dry neutral alumina . 
 h nmr spectra and proton - decoupled c nmr spectra were obtained on a 400 or 600 mhz nmr spectrometer . 
 chemical shifts (  ) are reported in parts per million ( ppm ) 
 relative to residual solvent ( chcl3 , s ,  7.26 ) . 
 multiplicities are given as s ( singlet ) , d ( doublet ) , t ( triplet ) , 
 dd ( doublet of doublets ) , m ( multiplet ) , br m ( broad multiplet ) , or 
 br s ( broad singlet ) ; c nmr chemical shifts are reported 
 relative to cdcl3 ( t ,  77.23 ) unless otherwise noted . 
 high - resolution mass spectra were recorded in positive esi mode in 
 methanol or acetonitrile . 
 silica gel chromatographic purifications 
 were performed by flash chromatography with silica gel packed in glass 
 columns . 
 the eluting solvent for each purification was determined 
 by thin - layer chromatography ( tlc ) on glass plates coated with silica 
 gel and visualized by uv light or by staining with ceric ammonium 
 molybdate ( cam ) followed by gentle heating . 
 the following abbreviations 
 are used throughout : ethyl acetate ( etoac ) , hexanes ( hex ) , dichloromethane 
 ( dcm ) , triethylamine ( tea ) , diisopropylethylamine ( dipea ) . 
 benzenesulfinic acid sodium salt ( 10.0 
 g , 60.9 mmol ) was dissolved 
 in 100 ml of h2o at 0 c . 
 then 10% hcl was added , and the mixture was extracted two 
 times with etoac . 
 the combined organic layers were dried over na2so4 , and the solvent was removed in vacuo to yield 
 a white solid , which was dissolved in 50 ml of toluene . 
 ac2o ( 70 ml , 20 mmol ) was added , and the reaction mixture was heated 
 at reflux for 3 h. the solvent was evaporated in vacuo , and the resulting 
 brown solid was washed several times with cold dcm and collected in 
 a fritted funnel , yielding a white to light - brown solid ( 6.8 g , 46% 
 yield over the two steps ) . 
 mp 132.4133.1 c ( dec . ) ; h nmr ( 400 mhz , cdcl3 )  7.96 ( dd , j = 8.4 , 1.3 hz , 2h ) , 7.867.76 ( m , 1h ) , 7.727.63 
 ( m , 2h ) , 4.52 ( dd , j = 10.3 , 4.4 hz , 1h ) , 3.68 ( dd , j = 19.9 ( gem ) , 4.4 hz , 1h ) , 3.39 ( dd , j = 19.7 ( gem ) , 10.6 hz , 1h ) ; c nmr ( 100 mhz , cdcl3 )  166.0 , 162.9 , 135.8 , 135.4 , 129.9 , 129.7 , 64.4 , 
 30.1 ; ir ( neat ) 1782 , 1739 ( c = o ) , 1169 ( s = o ) cm ; hrms ( esi - tof ) 
 m / z [ m + h ] calcd for c10h9o5s 241.0171 , found 241.0165 . 
 the amine and 
 the aldehyde were 
 dissolved in thf ( 0.10 m ) , and 3 equiv of triethyl orthoformate was 
 added . 
 the anhydride was added , and then the reaction mixture was allowed 
 to stir for 3 h. water was added to the mixture ( final concentration 
 0.05 mmol ) , followed by the addition of 3 equiv of k2co3 and heating at reflux for 4 h , unless otherwise stated . 
 the 
 reaction mixture was allowed to cool to rt and then extracted two 
 times with etoac . 
 the solvent was evaporated 
 in vacuo , and the crude mixture was purified by flash chromatography 
 using an etoac/3050% gradient , unless otherwise stated . 
 general procedure a was used with sulfone 
 anhydride 3a ( 0.24 g , 0.99 mmol ) , benzylamine ( 109 l , 
 0.99 mmol ) , and benzaldehyde ( 102 l , 1.0 mmol ) to yield 10a ( 0.32 g , 83% ) as a foam ( 50:50 etoac / hex , rf = 0.59 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.687.64 ( m , 2h ) , 7.62 ( tt , j = 
 7.5 , 1.3 hz , 1h ) , 7.487.41 ( m , 2h ) , 7.327.23 ( m , 6h ) , 
 7.097.06 ( m , 2h ) , 6.836.79 ( m , 2h ) , 5.09 ( d , j = 14.9 hz , 1h ) , 4.70 ( d , j = 3.3 hz , 
 1h ) , 3.66 ( ddd , j = 9.9 , 4.4 , 3.3 hz , 1h ) , 3.44 ( d , j = 14.9 hz , 1h ) , 3.03 ( dd , j = 18.2 ( gem ) , 
 4.3 hz , 1h ) , 2.95 ( ddd , j = 18.2 ( gem ) , 9.9 , 1.2 
 hz , 1h ) ; c nmr ( 101 mhz , cdcl3 )  170.5 , 
 137.7 , 136.4 , 134.7 , 134.4 , 129.5 , 129.3 , 128.9 , 128.7 , 128.7 , 128.4 , 
 127.8 , 126.1 , 63.5 , 60.6 , 44.5 , 30.7 ; ir ( thin film ) 1692 ( c = o ) , 
 1322 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h21no3sna 414.1140 , found 414.1120 . 
 general procedure a was used with sulfone 
 anhydride 3a ( 0.30 g , 1.26 mmol ) and ( e)-n - methyl-1-phenylmethanimine ( 156 l , 1.26 
 mmol ) . because the commercially available imine was used in this experiment , 
 the anhydride was added immediately after the imine was dissolved 
 instead of waiting for 3 h or overnight . 
 the reaction yielded 10b ( 0.332 g , 83% ) as a foam ( 50:50 etoac / hex , rf = 0.35 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.89 ( d , j = 7.5 hz , 2h ) , 7.69 ( t , j = 7.5 hz , 1h ) , 7.58 ( t , j = 7.8 hz , 2h ) , 
 7.367.31 ( m , 3h ) , 7.067.02 ( m , 2h ) , 4.97 ( d , j = 3.5 hz , 1h ) , 3.66 ( dt , j = 9.8 , 4.1 
 hz , 1h ) , 2.90 ( dd , j = 18.1 ( gem ) , 4.7 hz , 1h ) , 2.82 
 ( dd , j = 18.1 ( gem ) , 9.8 hz , 1h ) , 2.64 ( s , 3h ) ; c nmr ( 150 mhz , cdcl3 )  170.6 , 138.2 , 136.8 , 
 134.6 , 129.7 , 129.5 , 129.0 , 128.9 , 126.1 , 64.3 , 63.6 , 31.0 , 28.4 ; 
 ir ( thin film ) 1690 ( c = o ) , 1308 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c17h17no3sna 338.0827 , 
 found 338.0818 . 
 general procedure a was used with sulfone 
 anhydride 3a ( 0.240 g , 1.00 mmol ) , prop-2-yn-1-amine 
 ( 64.0 l , 1.00 mmol ) , and benzaldehyde ( 102 l , 1.00 mmol ) 
 to yield 10c ( 0.182 g , 54% ) as a foam . the residue was 
 purified by flash column chromatography ( 50:50 etoac / hex , rf = 0.53 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.89 ( m , 2h ) , 7.68 ( t , j = 7.4 
 hz , 1h ) , 7.607.53 ( t , 2h ) , 7.357.29 ( m , 3h ) , 7.077.02 
 ( m , 2h ) , 5.23 ( d , j = 4.0 hz , 1h ) , 4.51 ( dd , j = 17.6 , 2.6 hz , 1h ) , 3.75 ( ddd , j = 9.4 , 
 5.1 , 4.0 hz , 1h ) , 3.253.20 ( m , 1h ) , 2.96 ( dd , j = 18.2 ( gem ) , 5.1 hz , 1h ) , 2.89 ( ddd , j = 18.2 
 ( gem ) , 9.8 , 1.1 hz , 1h ) , 2.25 ( t , j = 2.5 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 )  169.9 , 137.3 , 136.6 , 
 134.6 , 129.6 , 129.4 , 129.0 , 128.9 , 126.4 , 76.2 , 73.3 , 63.9 , 60.9 , 
 31.1 , 30.5 ; ir ( thin film ) 1699 ( c = o ) , 1414 , 1322 ( s = o ) 
 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h17no3sna 362.0827 , found 362.0831 . 
 general procedure a was used with sulfone 
 anhydride 3a ( 0.24 g , 0.99 mmol ) , propylamine ( 82 l , 
 0.99 mmol ) , and furan-2-carbaldehyde ( 83 l , 1.00 mmol ) to yield 10d ( 0.167 g , 50% ) as a foam . 
 the residue was purified by 
 flash column chromatography ( 50:50 etoac / hex , rf = 0.47 ) . 
 h nmr ( 600 mhz , cdcl3 )  
 7.907.86 ( m , 2h ) , 7.707.65 ( m , 1h ) , 7.57 ( dd , j = 8.3 , 7.2 hz , 2h ) , 7.32 ( d , j = 1.8 
 hz , 1h ) , 6.27 ( dd , j = 3.3 , 1.9 hz , 1h ) , 6.19 ( d , j = 3.3 hz , 1h ) , 5.07 ( d , j = 4.1 hz , 1h ) , 
 3.96 ( ddd , j = 8.9 , 6.1 , 4.1 hz , 1h ) , 3.40 ( ddd , j = 13.9 , 8.8 , 7.1 hz , 1h ) , 2.952.85 ( m , 2h ) , 2.65 
 ( ddd , j = 13.9 , 8.6 , 5.2 hz , 1h ) , 1.471.37 
 ( m , 1h ) , 1.331.24 ( m , 1h ) , 0.82 ( t , j = 7.4 
 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 )  169.9 , 
 149.4 , 143.5 , 136.7 , 134.5 , 129.6 , 128.6 , 110.6 , 109.8 , 60.8 , 55.2 , 
 42.7 , 31.2 , 20.1 , 11.1 ; ir ( thin film ) 1690 ( c = o ) , 1416 , 1308 
 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c17h19no4sna 356.0932 , found 356.0929 . 
 general procedure a was used with sulfone 
 anhydride 3a ( 0.3 g , 1.2 mmol ) , propylamine ( 100 l , 
 1.2 mmol ) , and benzaldehyde ( 126 l , 1.2 mmol ) to yield 10e ( 0.36 g , 87% ) as a solid ( 50:50 etoac / hex , rf = 0.36 ) . 
 mp 149.8150.4 c ; h nmr ( 400 mhz , cdcl3 )  7.89 ( dt , j = 8.5 , 1.4 hz , 2h ) , 7.767.54 ( m , 3h ) , 7.437.24 ( m , 
 3h ) , 7.136.98 ( m , 2h ) , 5.09 ( d , j = 3.5 hz , 
 1h ) , 3.703.56 ( m , 2h ) , 2.972.76 ( m , 2h ) , 2.54 ( dddd , j = 13.7 , 8.4 , 5.1 , 1.1 hz , 1h ) , 1.551.31 ( m , 2h ) , 
 0.85 ( td , j = 7.3 , 1.2 hz , 3h ) ; c nmr 
 ( 100 mhz , cdcl3 )  170.6 , 138.4 , 136.7 , 134.5 , 129.6 , 
 129.3 , 128.8 , 128.7 , 126.0 , 63.8 , 61.1 , 42.6 , 30.9 , 19.9 , 11.1 ; ir 
 ( thin film ) 1688 ( c = o ) cm ; hrms ( esi - tof ) m / z [ m  h ] calcd 
 for c19h20no3s 342.1164 , found 342.1155 . 
 general procedure a ( except that the decarboxylation 
 was performed at room temperature overnight ) was used with sulfone 
 anhydride 3a ( 0.3 g , 1.2 mmol ) , propylamine ( 102 l , 
 1.2 mmol ) , and 4-cyanobenzaldehyde ( 0.164 g , 1.3 mmol ) to yield 10f ( 0.36 g , 80% ) as a foam ( 50:50 etoac / hex , rf = 0.35 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.89 ( dt , j = 8.5 , 1.8 hz , 2h ) , 7.777.54 
 ( m , 5h ) , 7.26 ( m , 2h ) , 5.20 ( d , j = 3.6 hz , 1h ) , 
 3.67 ( dt , j = 13.7 , 8.0 hz , 1h ) , 3.57 ( dt , j = 10.0 , 4.3 hz , 1h ) , 2.88 ( dd , j = 18.2 
 ( gem ) , 4.5 hz , 1h ) , 2.76 ( dd , j = 18.1 ( gem ) , 10.0 
 hz , 1h ) , 2.52 ( ddd , j = 13.8 , 8.6 , 5.0 hz , 1h ) , 1.44 
 ( m , 2h ) , 0.87 ( t , j = 7.3 hz , 3h ) ; c 
 nmr ( 600 mhz , cdcl3 )  170.7 , 144.1 , 136.6 , 134.9 , 
 133.3 , 129.9 , 128.9 , 127.2 , 118.1 , 113.1 , 63.8 , 60.6 , 43.1 , 31.3 , 
 20.1 , 11.2 ; ir ( thin film ) 1692 ( c = o ) , 1445 , 1412 ( s = o ) 
 cm ; hrms 
 ( esi - tof ) m / z [ m + na ] calcd for c20h20n2o3sna 391.1092 , found 391.1098 . 
 general procedure a was used with sulfone 
 anhydride 3a ( 88 mg , 0.36 mmol ) , propylamine ( 30 l , 
 0.36 mmol ) , and aldehyde ( 50 mg , 0.37 mmol ) to yield 10 g ( 87 mg , 65% ) as a solid ( 50:50 etoac / hex , rf = 0.17 ) . 
 mp 125.3126.1 c ; h nmr 
 ( 600 mhz , cdcl3 )  7.927.86 ( m , 2h ) , 7.697.62 
 ( m , 1h ) , 7.55 ( t , j = 7.9 hz , 2h ) , 7.28 ( m , 1h ) , 
 6.936.82 ( m , 3h ) , 5.16 ( s , 1h ) , 3.80 ( d , j = 9.8 hz , 1h ) , 3.69 ( s , 3h ) , 3.49 ( dt , j = 13.5 , 
 8.1 hz , 1h ) , 2.96 ( dd , j = 17.9 ( gem ) , 4.0 hz , 1h ) , 
 2.85 ( dd , j = 17.9 ( gem ) , 9.9 hz , 1h ) , 2.48 ( ddd , j = 13.7 , 8.5 , 5.2 hz , 1h ) , 1.461.28 ( m , 2h ) , 0.82 
 ( t , j = 7.3 hz , 3h ) ; c nmr ( 151 mhz , 
 cdcl3 )  171.2 , 157.5 , 137.7 , 134.2 , 130.5 , 129.5 
 ( 2c ) , 129.0 ( 2c ) , 125.7 , 121.1 , 111.5 , 62.8 , 55.6 , 42.8 , 31.7 , 20.3 , 
 11.3 ; ir ( thin film ) 1681 ( c = o ) cm ; hrms 
 ( esi - tof ) m / z [ m + na ] calcd for c20h23no4sna 396.1245 , 
 found 396.1252 . 
 general 
 procedure a was used with sulfone anhydride 3a ( 0.18 
 g , 0.73 mmol ) , benzylamine ( 76 l , 0.69 mmol ) , and aldehyde 
 ( 0.1 ml , 0.79 mmol ) to yield 10h ( 0.25 g , 82% ) as a foam 
 ( 50:50 etoac / hex , rf = 0.36 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.817.74 ( m , 2h ) , 
 7.657.14 ( m , 13h ) , 6.12 ( d , j = 15.8 hz , 
 1h ) , 5.74 ( dd , j = 15.6 , 8.5 hz , 1h ) , 4.93 ( d , j = 15.0 hz , 1h ) , 4.38 ( dd , j = 8.8 , 4.3 
 hz , 1h ) , 3.90 ( d , j = 15.0 hz , 1h ) , 3.65 ( dt , j = 9.9 , 5.1 hz , 1h ) , 3.01 ( dd , j = 18.0 
 ( gem ) , 5.5 hz , 1h ) , 2.86 ( dd , j = 18.1 ( gem ) , 10.0 
 hz , 1h ) ; c nmr ( 151 mhz , cdcl3 )  170.1 , 
 136.8 , 135.3 , 135.2 , 135.0 , 134.4 , 129.5 , 128.8 , 128.7 , 128.7 , 128.4 , 
 127.7 , 126.8 , 126.7 , 125.3 , 61.7 , 59.9 , 44.7 , 30.8 ; ir ( thin film ) 
 1690 ( c = o ) , 1146 ( s = o ) cm ; hrms 
 ( esi - tof ) m / z [ m + na ] calcd for c25h23no3sna 440.1296 , 
 found 440.1282 . 
 general procedure a was used with sulfone 
 anhydride 3a ( 0.3 g , 1.26 mmol ) , isopropylamine ( 100 
 l , 1.22 mmol ) , and benzaldehyde ( 128 l , 1.26 mmol ) to 
 yield 10i ( 0.297 g , 70% ) as a foam ( 50:50 etoac / hex , rf = 0.42 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.987.86 ( m , 2h ) , 7.72 ( tt , j = 7.2 , 1.5 hz , 1h ) , 7.687.56 ( m , 2h ) , 7.407.26 ( m , 
 3h ) , 7.176.97 ( m , 2h ) , 5.10 ( d , j = 2.3 hz , 
 1h ) , 4.203.98 ( m , 1h ) , 3.57 ( ddd , j = 9.6 , 
 2.6 , 2.1 hz , 1h ) , 2.93 ( ddd , j = 18.2 ( gem ) , 9.6 , 
 1.9 hz , 1h ) , 2.882.75 ( m , 1h ) , 1.21 ( dd , j = 6.8 , 2.0 hz , 3h ) , 0.85 ( dd , j = 6.9 , 1.9 hz , 
 3h ) ; c nmr ( 151 mhz , cdcl3 )  170.7 , 
 140.7 , 136.6 , 134.5 , 129.6 , 129.2 , 128.8 , 128.7 , 125.9 , 64.5 , 60.1 , 
 45.5 , 31.2 , 20.8 , 19.5 ; ir ( thin film ) 1693 ( c = o ) , 1145 ( s = o ) 
 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h21no3sna 366.1140 , found 366.1147 . 
 general procedure a was used with sulfone 
 anhydride 3a ( 0.30 g , 1.26 mmol ) , allylamine ( 93 l , 
 1.24 mmol ) , and benzaldehyde ( 128 l , 1.26 mmol ) to yield 10j ( 0.33 g , 78% ) as a foam ( 50:50 etoac / hex , rf = 0.42 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.88 ( dt , j = 8.5 , 2.0 hz , 2h ) , 7.777.67 
 ( m , 1h ) , 7.59 ( m , 2h ) , 7.437.27 ( m , 3h ) , 6.99 ( m , 2h ) , 5.55 
 ( dddd , j = 17.6 , 10.0 , 7.8 , 4.3 hz , 1h ) , 5.235.15 
 ( m , 1h ) , 5.145.02 ( m , 2h ) , 4.40 ( ddt , j = 
 15.4 , 3.9 , 1.9 hz , 1h ) , 3.753.65 ( m , 1h ) , 2.95 ( m , 3h ) ; c nmr ( 151 mhz , cdcl3 )  170.42 , 138.2 , 
 136.8 , 134.5 , 131.0 , 129.7 , 129.5 , 129.4 , 128.9 , 126.2 , 119.0 , 63.9 , 
 60.7 , 43.4 , 31.0 ; ir ( thin film ) 1691 ( c = o ) , 1145 ( s = o ) 
 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h19no3sna 364.0983 , found 364.0974 . 
 general procedure a was used with sulfone 
 anhydride 3a ( 0.30 g , 1.2 mmol ) , propylamine ( 100 l , 
 1.2 mmol ) , and 4-methoxybenzaldehyde ( 151 l , 1.2 mmol ) to yield 10k ( 0.38 g , 84% ) as a foam ( 50:50 etoac / hex , rf = 0.27 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.927.86 ( d , j = 7.8 hz , 2h ) , 
 7.69 ( m , 1h ) , 7.58 ( t , j = 7.8 hz , 2h ) , 6.96 ( dd , j = 9.2 , 2.5 hz , 2h ) , 6.84 ( dd , j = 9.0 , 
 2.5 hz , 2h ) , 5.04 ( d , j = 3.5 hz , 1h ) , 3.80 ( sharp 
 s , 3h ) , 3.663.53 ( m , 2h ) , 2.90 ( dd , j = 18.2 
 ( gem ) , 4.7 hz , 1h ) , 2.852.76 ( dd , j = 18.2 
 ( gem ) , 10.2 hz , 1h ) , 2.52 ( m , 1h ) , 1.511.32 ( m , 2h ) , 0.880.79 
 ( t , j = 7.8 hz , 3h ) ; c nmr ( 150 mhz , 
 cdcl3 )  170.4 , 159.9 , 136.9 , 134.4 , 130.3 , 129.6 , 
 128.8 , 127.4 , 114.7 , 64.1 , 60.7 , 55.3 , 42.5 , 31.1 , 20.0 , 11.1 ; ir 
 ( thin film ) 1687 ( c = o ) , 1354 , 1144 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no4sna 396.1245 , 
 found 396.1252 . 
 n - benzylidenebenzylamine 
 ( 0.1 ml , 0.53 mmol ) was added to a round - bottom flask containing na2so4 ( 0.37 g , 2.6 mmol ) and thf ( 6 ml ) , and then 
 sulfone anhydride 3a ( 0.13 g , 0.54 mmol ) was added . 
 the 
 reaction mixture was allowed to stir for 1.5 h , after which the na2so4 was removed by filtration and the solvent was 
 removed in vacuo . 
 the residue was redissolved in etoac , transferred 
 to a separatory funnel , and washed with hcl ( 1.2 m or 10% ) and brine . 
 the combined organic layers were dried over na2so4 , and the solvent removed in vacuo . 
 the resulting acid was redissolved 
 in a mixture of toluene and methanol ( 12 ml:7 ml ) and cooled to 0 
 c . to the cold solution , tmschn2 ( 0.53 ml , 1.1 mmol , 
 2 m solution in hexanes ) 
 after 2 h the solvent was removed in vacuo , and 
 the resulting material was purified by column chromatography ( 3060% 
 etoac : hex ) to yield 11a ( 0.157 g , 66% over the two steps ) 
 as an amorphous solid ( 50:50 etoac / hex , rf = 0.47 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.647.18 
 ( m , 15h ) , 5.21 ( d , j = 14.5 hz , 1h ) , 4.89 ( s , 1h ) , 
 3.70 ( d , j = 18.7 ( gem ) hz , 1h ) , 3.383.30 
 ( m , 2h ) , 3.13 ( s , 3h ) ; c nmr ( 150 mhz , cdcl3 )  170.0 , 164.9 , 134.9 , 134.8 , 134.8 , 134.4 , 131.1 , 129.6 , 
 129.5 , 129.5 , 129.0 , 128.9 , 128.9 , 128.2 , 76.2 , 63.9 , 52.7 , 44.9 , 
 35.0 ; ir ( thin film ) 1783 , 1784 ( c = o ) , 1124 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c25h23no5sna 472.1195 , found 472.1176 . 
 propylamine ( 68 l , 0.83 mmol ) and 
 3,5-dimethoxybenzaldehyde ( 0.14 g , 0.84 mmol ) were dissolved in thf 
 ( 8 ml ) . 
 na2so4 ( 0.59 g , 4.2 mmol ) was added , 
 and the mixture was allowed to stir for 1.5 h. sulfone anhydride 3a ( 0.20 g , 0.83 mmol ) was added , and the reaction was allowed 
 to stir for an additional 1 h. the na2so4 was 
 filtered off , and the solvent was removed in vacuo . 
 the residue was 
 redissolved in etoac , transferred to a separatory funnel , and washed 
 with hcl ( 1.2 m or 10% ) and brine . 
 the combined organic layers were 
 dried over na2so4 , and the solvent was removed 
 in vacuo . 
 the resulting crude acid was dissolved in a mixture of toluene 
 and methanol ( 13 ml:7 ml ) and cooled to 0 c . to the cold solution , 
 tmschn2 ( 0.83 ml , 
 1.7 mmol , 2 m solution in hexanes ) was 
 added dropwise over a period of 5 min , and the reaction was monitored 
 by tlc for the disappearance of starting material . 
 after 2 
 h the solvent was removed in vacuo , and the resulting material was 
 purified by column chromatography ( 3060% etoac : hex ) to yield 11b ( 0.22 g , 57% over the two steps ) as an amorphous solid 
 ( 50:50 etoac / hex , rf = 0.31 ) . 
 h nmr ( 600 mhz , cdcl3 )  8.017.83 ( m , 2h ) , 
 7.817.68 ( m , 1h ) , 7.687.52 ( m , 2h ) , 6.39 ( d , j = 2.3 hz , 1h ) , 6.22 ( s , 2h ) , 5.38 ( s , 1h ) , 3.74 ( s , 6h ) , 
 3.663.59 ( m , 1h ) , 3.55 ( d , j = 18.4 ( gem ) 
 hz , 1h ) , 3.26 ( m , 3h ) , 3.14 ( d , j = 18.5 ( gem ) hz , 
 1h ) , 2.58 ( ddd , j = 13.8 , 8.7 , 5.2 hz , 1h ) , 1.651.32 
 ( m , 2h ) , 0.92 ( t , j = 7.3 hz , 3h ) ; c 
 nmr ( 150 mhz , cdcl3 )  169.9 , 164.7 , 160.9 , 137.3 , 
 135.3 , 134.9 , 130.4 , 129.2 , 100.8 , 76.7 , 64.3 , 55.4 , 52.8 , 43.2 , 35.8 , 
 20.2 ( 2c ) , 11.3 ; ir ( thin film ) 1748 , 1702 ( c = o ) , 1144 , 1457 
 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h27no7sna 484.1406 , found 484.1404 . 
 n - benzylidenemethylamine 
 ( 0.1 ml , 0.81 mmol ) was added to a round - bottom flask containing na2so4 ( 0.57 g , 4.0 mmol ) and thf ( 8 ml ) , and then 
 sulfone anhydride 3a ( 0.19 g , 0.80 mmol ) was added . 
 the 
 reaction mixture was allowed to stir for 1.5 h , after which the na2so4 was removed by filtration and the solvent removed 
 in vacuo . 
 the residue was redissolved in etoac , transferred to a separatory 
 funnel , and washed with hcl ( 1.2 m or 10% ) and brine . 
 the combined 
 organic layers were dried over na2so4 , and the 
 solvent was removed in vacuo . 
 the crude acid was redissolved in a 
 mixture of toluene and methanol ( 13 ml:7 ml ) and cooled to 0 c . 
 to the cold solution , tmschn2 ( 0.81 ml , 1.6 mmol , 2 m solution 
 in hexanes ) 
 was added dropwise over a period of 5 min , and the reaction 
 was monitored by tlc for the disappearance of starting material . 
 after 
 2 h the solvent was removed in vacuo , and the resulting material 
 was purified by column chromatography ( 3060% etoac : hex ) to 
 yield 11c ( 0.20 g , 67% over the two steps ) as an amorphous 
 solid ( 50:50 etoac / hex , rf = 0.28 ) . 
 h nmr ( 400 mhz , cdcl3 )  7.88 ( dt , j = 8.6 , 2.2 hz , 2h ) , 7.727.63 ( m , 1h ) , 7.647.50 
 ( m , 2h ) , 7.367.23 ( m , 4h ) , 7.05 ( d , j = 7.2 
 hz , 2h ) , 5.31 ( s , 1h ) , 3.49 ( d , j = 18.4 ( gem ) hz , 
 1h ) , 3.15 ( d , j = 2.3 hz , 3h ) , 3.01 ( d , j = 18.5 ( gem ) hz , 1h ) , 2.58 ( s , 2h ) ; c nmr ( 100 mhz , 
 cdcl3 )  169.75 , 164.51 , 135.32 , 135.1 , 134.3 , 130.2 , 
 129.4 , 129.3 , 128.7 , 127.6 , 76.7 , 66.5 , 52.9 , 35.5 , 28.3 ; ir ( thin 
 film ) 1736 , 1690 ( c = o ) , 1177 , 1360 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h19no5sna 396.0882 , 
 found 396.0887 . 
 ester 11a ( 0.104 g , 0.23 
 mmol ) was dissolved in anhydrous meoh ( 12 ml ) , and magnesium turnings 
 ( 169 mg , 6.9 mmol , flame - dried under argon ) were added to the reaction 
 mixture . 
 the mixture was heated to 50 c for 15 min and then 
 to 64 c for 1.5 h , cooled to rt , poured into an erlenmeyer flask 
 containing 10% hcl ( 20 ml ) , and extracted with diethyl ether . 
 the 
 organic layer was dried over na2so4 , and the 
 solvent was removed in vacuo . 
 purification by flash chromatography 
 ( 3050% etoac / hex ) yielded 12a as an oil ( 0.54 
 g , 76% ) . 
 h nmr data for the major diastereomer ( 600 mhz , 
 cdcl3 ) :  7.417.33 ( m , 4h ) , 7.307.23 
 ( m , 3h ) , 7.187.13 ( m , 1h ) , 7.057.01 ( m , 2h ) , 5.11 
 ( d , j = 14.7 hz , 1h ) , 4.634.56 ( d , j = 5.4 hz , 1h ) , 3.64 ( s , 3h ) , 3.48 ( d , j = 14.9 hz , 1h ) , 3.07 ( m , 1h ) , 2.942.75 ( m , 2h ) . the h nmr data are in accordance with the literature . 
 ester 11b ( 75.4 mg , 0.163 
 mmol ) was dissolved in anhydrous meoh ( 12 ml ) , and magnesium turnings 
 ( 162 mg , 6.67 mmol , flame - dried under argon ) were added to the reaction 
 mixture . 
 the mixture was heated to 50 c for 15 min and then 
 to 64 c for 1.5 h , cooled to rt , poured into an erlenmeyer flask 
 containing 10% hcl ( 30 ml ) , and extracted with diethyl ether . 
 the 
 organic layer was dried over na2so4 , and the 
 solvent was removed in vacuo . 
 purification by flash chromatography 
 ( 3050% etoac / hex ) yielded 12b as an oil ( 0.35 
 g , 67% ) . 
 h nmr ( 400 mhz , cdcl3 )  6.42 
 ( t , j = 2.2 hz , 1h ) , 6.35 ( d , j = 
 2.3 hz , 2h ) , 4.83 ( d , j = 5.3 hz , 1h ) , 3.79 ( s , 6h ) , 
 3.74 ( s , 3h ) , 3.66 ( dt , j = 13.6 , 8.1 hz , 1h ) , 3.04 
 ( ddd , j = 9.6 , 7.0 , 5.2 hz , 1h ) , 2.84 ( dd , j = 17.2 , 9.8 hz , 1h ) , 2.76 ( d , j = 7.0 
 hz , 1h ) , 2.61 ( ddd , j = 13.6 , 8.3 , 5.2 hz , 1h ) , 1.541.37 
 ( m , 2h ) , 0.84 ( t , j = 7.4 hz , 3h ) ; c 
 nmr ( 100 mhz , cdcl3 )  173.1 , 172.7 , 161.5 , 142.0 , 
 104.5 , 100.1 , 64.2 , 55.5 , 52.6 , 45.9 , 42.5 , 33.8 , 20.2 , 11.3 ; hrms 
 ( esi - tof ) m / z [ m + na ] calcd for c17h23no5na 344.1474 , 
 found 344.1465 . to a 500 ml round - bottom flask were added 
 benzenesulfinic 
 acid sodium salt ( 5.1 g , 30.8 mmol ) , tert - butyl bromoacetate 
 ( 3.8 ml , 25.6 mmol ) , and 170 ml of ethanol . 
 the crude mixture 
 was suspended in et2o ( 150 ml ) and washed with water ( 2 
  150 ml ) and brine ( 150 ml ) . 
 the organic layer was dried over 
 sodium sulfate and concentrated in vacuo to yield 8 ( 6.31 
 g , 96% ) as a clear oil . 
 h nmr ( 300 mhz , cdcl3 )  7.90 ( m , 2h ) , 7.68 ( m , 1h ) , 7.55 ( m , 2h ) , 4.03 ( s , 2h ) , 
 1.33 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 )  
 161.2 , 138.8 , 134.1 , 129.1 , 128.5 , 83.6 , 62.0 , 27.6 . 
 these data are 
 in accordance with the literature . to a dry 250 ml round - bottom flask were 
 added 8 ( 6.31 g , 24.6 mmol ) , cesium carbonate ( 400 mg , 
 1.23 mmol ) , tert - butyl acrylate ( 3.6 ml , 24.6 mmol ) , 
 and 80 ml of acetonitrile . 
 the reaction mixture was heated to 50 c 
 overnight and then diluted with water and extracted with ethyl acetate 
 ( 3  100 ml ) . 
 the crude product 
 was purified on silica gel ( 20:80 etoac / hex ) to yield 3ba ( 4.91 g , 52% ) as an amorphous solid . 
 h nmr ( 300 mhz , 
 cdcl3 )  7.89 ( d , j = 7.3 hz , 2h ) , 
 7.68 ( m , 1h ) , 7.57 ( dd , j = 8.4 , 6.9 hz , 2h ) , 3.99 
 ( m , 1h ) , 2.24 ( m , 4h ) , 1.42 ( s , 9h ) , 1.35 ( s , 9h ) ; c 
 nmr ( 75 mhz , cdcl3 )  170.9 , 164.4 , 137.4 , 134.1 , 
 129.4 , 129.0 , 83.5 , 81.0 , 70.1 , 32.1 , 28.0 , 27.7 , 22.3 ; ir ( thin film ) 
 2979 , 1730 , 1309 , 1138 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h28o6sna 407.1504 , found 407.1502 . to a dry 250 ml round - bottom flask were 
 added 8 ( 3.00 g , 11.7 mmol ) , cesium carbonate ( 382 mg , 
 1.17 mmol ) , tert - butyl crotonate ( 1.90 ml , 11.7 mmol ) , 
 and 40 ml of acetonitrile . 
 the reaction mixture was heated to reflux 
 overnight and then diluted with water and extracted with ethyl acetate 
 ( 3  100 ml ) . 
 the crude product 
 was purified on silica gel ( 20:80 etoac / hex ) to yield 3ca ( 3.75 g , 80% ) as an amorphous solid ( mixture of diastereomers ) . 
 nmr data for one diastereomer : h nmr ( 600 mhz , cdcl3 )  7.90 ( m , 3h ) , 7.64 ( m , 2h ) , 7.53 ( m , 3h ) , 4.10 ( d , j = 8.0 hz , 1h ) , 2.71 ( m , 2h ) , 2.43 ( m , 1h ) , 1.42 ( s , 16h , 
 overlap with minor diastereomer ) , 1.27 ( s , 14h , overlap with minor 
 diastereomer ) , 1.12 ( d , j = 6.7 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 )  170.7 , 164.4 , 138.4 , 134.0 , 
 129.2 , 128.9 , 83.3 , 80.8 , 73.7 , 39.9 , 29.6 , 28.1 , 27.6 , 17.1 ; ir ( thin 
 film ) 2980 , 1729 , 1325 , 1135 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h30o6sna 421.1661 , found 421.1653 . to a dry 250 ml round - bottom flask 
 were 
 added 8 ( 3.00 g , 11.7 mmol ) , cesium carbonate ( 382 mg , 
 1.17 mmol ) , tert - butyl methacrylate ( 1.90 ml , 11.7 
 mmol ) , and 40 ml of acetonitrile . 
 the reaction mixture was heated 
 to reflux for 48 h and then diluted with water and extracted with 
 ethyl acetate ( 3  100 ml ) . 
 the crude product was purified on silica gel ( 20:80 etoac / hex ) to 
 yield 3da ( 4.20 g , 90% ) as an amorphous solid ( mixture 
 of diastereomers ) . 
 nmr data for one diastereomer : h nmr 
 ( 400 mhz , cdcl3 )  7.89 ( m , 2h ) , 7.69 ( m , 1h ) , 7.58 
 ( m , 2h ) , 4.03 ( dd , j = 11.9 , 3.3 hz , 1h ) , 2.28 ( m , 
 2h ) , 1.99 ( m , 1h ) , 1.43 ( s , 9h ) , 1.37 ( s , 9h ) , 1.16 ( d , j = 6.6 hz , 3h ) ; c nmr ( 101 mhz , cdcl3 )  
 174.2 , 164.7 , 137.6 , 134.3 , 129.5 , 129.2 , 83.6 , 81.1 , 69.6 , 38.2 , 
 30.6 , 28.2 , 27.8 , 18.4 ; ir ( thin film ) 2978 , 2936 , 1727 , 1325 , 1138 
 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h30o6sna 421.1661 , found 421.1662 . to a dry 250 ml flask 
 were added 3ba ( 4.91 g , 12.7 mmol ) , ch2cl2 ( 50 ml ) , and trifluoroacetic acid ( 50 ml ) . 
 the reaction mixture 
 was stirred for 1 h and then concentrated in vacuo and azeotropically 
 distilled with ch2cl2 ( 3  10 ml ) . 
 the 
 crude mixture was taken up in 45 ml of trifluoroacetic anhydride and 
 stirred overnight . 
 the reaction mixture was concentrated in vacuo 
 and azeotroped with toluene ( 3  10 ml ) . 
 filtering the off - white 
 solid with diethyl ether yielded 3b ( 3.01 g , 93% ) as 
 a white solid . 
 mp 122.2122.8 c ; h nmr ( 600 
 mhz , cd3cn )  7.97 ( m , 2h ) , 7.83 ( m , 1h ) , 7.70 ( m , 
 2h ) , 4.55 ( m , 1h ) , 3.06 ( m , 1h ) , 2.87 ( m , 1h ) , 2.50 ( m , 2h ) ; c nmr ( 150 mhz , cd3cn )  165.3 , 160.7 , 137.3 , 
 135.0 , 129.5 , 129.0 , 63.4 , 27.1 , 17.1 ; ir ( thin film ) 3098 , 2929 , 
 1816 , 1746 1322 , 1152 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c11h10o5sna 277.0147 , found 277.0145 . 
 to a dry 250 ml flask 
 were added 3ca ( 3.73 g , 9.30 mmol ) , ch2cl2 ( 50 ml ) , and trifluoroacetic acid ( 50 ml ) . 
 the reaction mixture 
 was stirred for 1 h and then concentrated in vacuo and azeotropically 
 distilled with ch2cl2 ( 3  10 ml ) . to the 
 crude mixture 
 was added acetic anhydride ( 33 ml ) , and the mixture 
 was taken up in 100 ml of toluene and stirred overnight . 
 the mixture 
 was concentrated in vacuo and azeotroped with benzene ( 3  10 
 ml ) . 
 trituration with diethyl ether afforded 3c ( 2.31 
 g , 92% ) as a solid ( mixture of diastereomers ) . 
 nmr data for one diastereomer : h nmr ( 600 mhz , cd3cn )  7.93 ( m , 2h ) , 7.84 
 ( m , 1h ) , 7.70 ( m , 2h ) , 4.40 ( m , 1h ) , 3.29 ( m , 1h ) , 3.03 ( m , 1h ) , 2.75 
 ( m , 1h ) , 1.18 ( m , 3h ) ; c nmr ( 151 mhz , cd3cn )  164.8 , 160.4 , 135.2 , 129.6 , 129.3 , 128.9 , 69.6 , 33.9 , 
 24.4 , 19.4 ; ir ( thin film ) 2991 , 2957 , 1814 , 1758 , 1311 , 1146 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c12h12o5sna 291.0293 , found 291.0303 . to a dry 250 ml flask 
 were added 3da ( 4.00 g , 10.0 mmol ) , ch2cl2 ( 50 ml ) , and trifluoroacetic acid ( 50 ml ) . 
 the reaction mixture 
 was stirred for 1 h and then concentrated in vacuo and azeotropically 
 distilled with ch2cl2 ( 3  10 ml ) . to the 
 crude mixture 
 were added acetic anhydride ( 33 ml ) and 100 ml of toluene , 
 and the resulting mixture was stirred overnight , concentrated in vacuo , 
 and azeotroped with benzene ( 3  10 ml ) . 
 trituration with diethyl 
 ether afforded 3d ( 2.53 g , 94% ) as a solid ( mixture of 
 diastereomers ) . 
 nmr data for one diastereomer : h nmr ( 400 
 mhz , cdcl3 )  7.90 ( m , 2h ) , 7.74 ( m , 1h ) , 7.62 ( m , 
 3h , overlap with minor diasteromer ) , 4.22 ( dd , j = 
 3.0 , 9.0 hz , 1h ) , 2.95 ( ddd , j = 2.1 , 6.1 , 15.2 hz , 
 1h ) , 2.762.58 ( m , 1h ) , 2.13 ( ddd , j = 6.2 , 
 13.2 , 15.2 hz , 1h ) , 1.44 ( s , 3h ) ; c nmr ( 100 mhz , cdcl3 )  167.8 , 160.3 , 137.1 , 135.2 , 129.8 , 129.4 , 64.8 , 
 32.6 , 25.5 , 16.4 ; ir ( thin film ) 3066 , 2970 , 1803 , 1755 , 1310 , 1154 
 cm ; hrms ( esi - tof ) m / z [ m + h2o + na ] calcd for c12h14o6sna 309.0409 , found 309.0402 . to a dry 10 ml flask 
 were added 
 2 ml of acetonitrile , triethyl orthoformate ( 0.11 ml , 0.63 mmol ) , 
 amine ( 0.39 mmol ) , and aldehyde ( 0.39 mmol ) . 
 anhydride 3b ( 0.39 mmol ) in 2 
 ml of acetonitrile was added , and the reaction mixture was stirred 
 for 30 min at room temperature and then refluxed for 4 h. the mixture 
 was cooled to room temperature and concentrated in vacuo . 
 the product 
 was purified by flash column chromatography ( conditions given for 
 each compound ) . to a dry 10 
 ml flask 
 was added 
 4 ml of acetonitrile , triethyl orthoformate ( 0.11 ml , 0.63 mmol ) , 
 the prepared imine ( 0.39 mmol ) , and anhydride 3b  d ( 0.39 mmol ) . 
 the reaction mixture was stirred for 30 min 
 at room temperature and then refluxed for 4 h. the mixture was cooled 
 to room temperature and concentrated in vacuo . 
 the residue was purified 
 by flash column chromatography ( conditions given for each compound ) . 
 general procedure c. the residue was purified 
 by flash column chromatography ( 70:30 etoac / hex , rf = 0.40 ) to yield 13a as a solid ( 105 mg , 
 80% ) . 
 mp 152.6153.1 c ; h nmr ( 300 mhz , cdcl3 )  7.91 ( m , 2h ) , 7.69 ( m , 1h ) , 7.59 ( m , 2h ) , 7.32 ( m , 
 3h ) , 7.04 ( m , 2h ) , 5.10 ( s , 1h ) , 3.36 ( ddd , j = 5.0 , 
 4.9 , 2.3 hz , 1h ) , 2.84 ( s , 3h ) , 2.73 ( m , 1h ) , 2.45 ( ddd , j = 17.9 ( gem ) , 6.6 , 4.6 hz , 1h ) , 2.08 ( m , 2h ) ; c nmr 
 ( 75 mhz , cdcl3 )  169.3 , 139.0 , 137.3 , 134.4 , 129.6 , 
 129.3 , 128.6 , 128.4 , 126.0 , 65.0 , 60.7 , 34.4 , 27.9 , 17.8 ; ir ( thin 
 film ) 3057 , 2930 , 1635 , 1302 , 1142 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c18h19no3sna 352.0983 , found 352.0973 . 
 general procedure b. the residue was purified 
 by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13b as a foam ( 117 mg , 
 84% ) . 
 h nmr ( 300 mhz , cdcl3 )  7.92 ( m , 
 2h ) , 7.747.64 ( m , 1h ) , 7.60 ( m , 2h ) , 7.30 ( m , 3h ) , 7.15 ( m , 
 2h ) , 5.40 ( s , 1h ) , 4.68 ( m , 1h ) , 3.28 ( m , 1h ) , 2.78 ( m , 1h ) , 2.49 
 ( ddd , j = 12.3 , 11.9 , 9.8 hz , 1h ) , 1.92 ( m , 2h ) , 
 1.31 ( d , j = 6.8 hz , 3h ) , 0.89 ( d , j = 7.0 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 )  
 169.7 , 141.6 , 137.5 , 134.3 , 129.6 , 129.0 , 128.6 , 128.0 , 126.0 , 65.0 , 
 53.8 , 47.6 , 28.5 , 20.3 , 20.1 , 17.5 ; ir ( thin film ) 2967 , 2945 , 1641 , 
 1303 , 1143 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no3sna 380.1296 , found 380.1306 . 
 general procedure c. the residue was purified 
 by flash column chromatography ( 70:30 etoac / hex , rf = 0.48 ) to yield 13c as a foam ( 130 mg , 
 85% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.63 ( t , j = 7.4 hz , 1h ) , 7.39 ( m , 7h ) , 7.30 ( m , 3h ) , 7.25 ( t , j = 6.1 hz , 2h ) , 6.79 ( m , 2h ) , 5.76 ( d , j = 14.6 hz , 1h ) , 4.69 ( s , 1h ) , 3.22 ( d , j = 14.6 
 hz , 1h ) , 3.17 ( m , 1h ) , 3.07 ( m , 1h ) , 2.67 ( dd , j = 
 18.1 ( gem ) , 6.8 hz , 1h ) , 2.43 ( m , 1h ) , 2.18 ( m , 1h ) ; c nmr ( 150 mhz , cdcl3 )  169.0 , 138.8 , 137.0 , 136.1 , 
 134.0 , 129.5 , 129.4 , 129.3 , 128.6 , 128.6 , 128.5 , 127.7 , 125.9 , 63.9 , 
 57.2 , 47.6 , 28.3 , 16.1 ; ir ( thin film ) 3059 , 2936 , 1642 , 1307 , 1144 
 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c24h23no3sna 428.1296 , found 428.1303 . 
 general procedure c. the residue was purified 
 by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13d as a foam ( 103 mg , 
 75% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.99 ( m , 
 2h ) , 7.69 ( m , 1h ) , 7.59 ( m , 2h ) , 7.30 ( m , 3h ) , 6.98 ( dd , j = 7.3 , 2.0 hz , 2h ) , 5.40 ( d , j = 2.4 hz , 1h ) , 4.95 
 ( dd , j = 17.5 , 2.6 hz , 1h ) , 3.38 ( td , j = 5.0 , 2.5 hz , 1h ) , 3.30 ( dd , j = 17.5 , 2.5 hz , 
 1h ) , 2.81 ( ddd , j = 17.7 , 10.5 , 6.9 hz , 1h ) , 2.52 
 ( ddd , j = 18.0 ( gem ) , 6.7 , 4.3 hz , 1h ) , 2.28 ( m , 
 1h ) , 2.12 ( m , 2h ) ; c nmr ( 150 mhz , cdcl3 ) 
  168.7 , 138.4 , 137.3 , 134.4 , 129.5 , 129.3 , 128.9 , 128.6 , 126.2 , 
 77.7 , 73.1 , 64.6 , 57.9 , 34.3 , 28.3 , 17.3 ; ir ( thin film ) 3303 , 3055 , 
 1650 , 1267 , 1148 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h19no3sna 376.0983 , found 376.0977 . 
 general procedure c. the residue was purified 
 by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13e as a foam ( 124 mg , 
 89% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.92 ( m , 
 2h ) , 7.71 ( m , 1h ) , 7.60 ( m hz , 2h ) , 7.29 ( m , 3h ) , 6.96 ( dd , j = 7.5 , 1.9 hz , 2h ) , 5.83 ( dddd , j = 17.7 , 
 10.5 , 8.2 , 4.3 hz , 1h ) , 5.21 ( m , 2h ) , 5.11 ( s , 1h ) , 4.86 ( ddd , j = 15.0 , 4.3 , 2.0 hz , 1h ) , 3.31 ( td , j = 4.7 , 1.8 hz , 1h ) , 3.002.82 ( m , 2h ) , 2.54 ( ddd , j = 18.0 ( gem ) , 6.8 , 3.7 hz , 1h ) , 2.19 ( m , 2h ) ; c nmr ( 151 mhz , cdcl3 )  169.1 , 139.1 , 137.3 , 134.4 , 
 132.4 , 129.5 , 129.3 , 128.7 , 128.4 , 126.0 , 118.8 , 64.4 , 57.2 , 47.9 , 
 28.0 , 17.1 ; ir ( thin film ) 3054 , 2988 , 1641 , 1307 , 1148 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h21no3sna 378.1140 , 
 found 378.1136 . 
 general procedure c. the residue was purified 
 by flash column chromatography ( 70:30 etoac / hex , rf = 0.40 ) to yield 13f as a foam ( 110 mg , 
 72% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.92 ( m , 
 2h ) , 7.67 ( t , j = 7.5 hz , 1h ) , 7.56 ( m , 2h ) , 7.32 
 ( m , 2h ) , 7.27 ( m , 3h ) , 7.19 ( m , 5h ) , 5.54 ( s , 1h ) , 3.48 ( m , 1h ) , 2.95 
 ( m , 1h ) , 2.66 ( dt , j = 18.1 ( gem ) , 6.2 hz , 1h ) , 2.20 
 ( m , 2h ) ; c nmr ( 150 mhz , cdcl3 )  169.8 , 
 142.3 , 139.7 , 137.2 , 134.4 , 129.6 , 129.2 , 129.1 , 128.6 , 128.3 , 127.3 , 
 127.0 , 126.2 , 64.9 , 62.2 , 28.8 , 18.2 ; ir ( thin film ) 3066 , 3008 , 2946 , 
 1654 , 1304 , 1143 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h21no3sna 414.1140 , found 414.1131 . 
 general procedure c. the residue was purified 
 by flash column chromatography ( 70:30 etoac / hex , rf = 0.46 ) to yield 13 g as a foam ( 126 mg , 
 91% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.91 ( m , 
 2h ) , 7.69 ( m , 1h ) , 7.59 ( t , j = 7.8 hz , 2h ) , 7.31 
 ( dd , j = 8.4 , 6.6 hz , 2h ) , 7.25 ( m , 1h ) , 7.05 ( d , j = 7.4 hz , 2h ) , 5.22 ( s , 1h ) , 3.95 ( dtd , j = 13.6 , 6.8 , 3.3 hz , 1h ) , 3.32 ( td , j = 5.0 , 2.0 
 hz , 1h ) , 2.81 ( m , 1h ) , 2.43 ( m , 2h ) , 1.99 ( m , 2h ) , 1.61 ( m , 2h ) , 0.87 
 ( t , j = 7.4 hz , 3h ) ; c nmr ( 150 mhz , 
 cdcl3 )  169.2 , 139.7 , 137.4 , 134.3 , 129.6 , 129.2 , 
 128.5 , 128.3 , 126.1 , 64.7 , 57.9 , 48.0 , 28.0 , 20.3 , 17.6 , 11.3 ; ir 
 ( thin film ) 2967 , 2929 , 2871 , 1639 , 1308 , 1147 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no3sna 380.1296 , 
 found 380.1290 . 
 general 
 procedure b. the residue was purified by flash column chromatography 
 ( 60:40 etoac / hex , rf = 0.50 ) to yield 13h as an orange amorphous solid ( 101 mg , 68% ) . 
 h nmr ( 300 mhz , cdcl3 )  7.93 ( dd , j = 7.2 , 1.6 hz , 2h ) , 7.70 ( m , 1h ) , 7.60 ( dd , j = 
 8.2 , 6.6 hz , 2h ) , 7.32 ( m , 5h ) , 6.37 ( dd , j = 16.0 , 
 1.2 hz , 1h ) , 6.07 ( dd , j = 15.8 , 6.4 hz , 1h ) , 4.94 
 ( dt , j = 6.6 , 1.6 hz , 1h ) , 4.66 ( m , 1h ) , 3.35 ( td , j = 6.4 , 2.0 hz , 1h ) , 2.68 ( dt , j = 17.2 , 
 7.0 hz , 1h ) , 2.40 ( dt , j = 17.2 , 7.1 hz , 1h ) , 2.09 
 ( dt , j = 14.4 , 7.2 hz , 1h ) , 1.99 ( dt , j = 14.4 , 7.2 hz , 1h ) , 1.33 ( d , j = 6.8 hz , 3h ) , 
 1.18 ( d , j = 6.9 hz , 3h ) ; c nmr ( 75 
 mhz , cdcl3 )  169.7 , 137.6 , 135.4 , 134.2 , 132.0 , 
 129.8 , 129.6 , 128.8 , 128.6 , 128.4 , 126.5 , 63.5 , 51.8 , 47.3 , 29.1 , 
 20.7 , 20.2 , 18.78 ; ir ( thin film ) 3059 , 2975 , 1639 , 1446 , 1305 , 1145 
 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c22h25no3sna 406.1453 , found 406.1447 . 
 general procedure b. the residue was purified 
 by flash column chromatography ( 60:40 etoac / hex , rf = 0.42 ) to yield 13i as a foam ( 96 mg , 
 69% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.92 ( d , j = 8.4 hz , 2h ) , 7.69 ( t , j = 7.4 hz , 1h ) , 
 7.60 ( t , j = 7.8 hz , 2h ) , 6.97 ( d , j = 8.6 hz , 2h ) , 6.84 ( m , 2h ) , 5.18 ( m , 1h ) , 3.93 ( m , 1h ) , 3.77 ( s , 
 3h ) , 3.30 ( m , 1h ) , 2.81 ( m , 1h ) , 2.45 ( m , 2h ) , 2.01 ( m , 2h ) , 1.61 
 ( m , 2h ) , 0.88 ( t , j = 7.5 hz , 3h ) ; c 
 nmr ( 150 mhz , cdcl3 )  169.2 , 159.5 , 134.2 , 131.5 , 
 129.5 , 128.6 , 127.3 , 114.5 , 105.0 , 64.9 , 57.5 , 55.3 , 48.0 , 28.1 , 20.3 , 
 17.6 , 11.3 ; ir ( thin film ) 2964 , 2920 , 1633 , 1250 , 1144 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c21h25no4sna 410.1402 , 
 found 410.1400 . 
 general procedure c. the residue was purified 
 by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13j as a foam ( 133 mg , 
 87% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.89 ( m , 
 2h ) , 7.69 ( m , 1h ) , 7.59 ( m , 2h ) , 7.28 ( m , 2h ) , 7.01 ( m , 2h ) , 5.21 
 ( s , 1h ) , 3.92 ( ddd , j = 13.6 , 9.0 , 7.4 hz , 1h ) , 3.27 
 ( td , j = 5.1 , 2.2 hz , 1h ) , 2.78 ( ddd , j = 17.4 , 10.1 , 6.8 hz , 1h ) , 2.41 ( m , 2h ) , 1.96 ( m , 2h ) , 1.59 ( m , 
 2h ) , 0.86 ( t , j = 7.4 hz , 3h ) ; c nmr 
 ( 150 mhz , cdcl3 )  169.2 , 138.3 , 137.2 , 134.4 , 134.2 , 
 129.6 , 129.4 , 128.5 , 127.5 , 64.6 , 57.5 , 48.0 , 28.1 , 20.3 , 17.9 , 11.3 ; 
 ir ( thin film ) 2964 , 2937 , 1641 , 1306 , 1146 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h22clno3sna 
 414.0907 , found 414.0921 . 
 general procedure c. the residue was purified 
 by flash column chromatography ( 60:40 etoac / hex , rf = 0.42 ) to yield 13k as a foam ( 92 g , 61% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.95 ( d , j = 8.4 hz , 2h ) , 7.70 ( t , j = 7.4 hz , 1h ) , 
 7.61 ( t , j = 7.9 hz , 2h ) , 7.24 ( m , 1h ) , 7.17 ( m , 
 2h ) , 7.05 ( d , j = 7.8 hz , 1h ) , 5.51 ( s , 1h ) , 3.97 
 ( m , 1h ) , 3.14 ( m , 1h ) , 2.94 ( m , 1h ) , 2.53 ( dd , j = 
 18.3 ( gem ) , 7.4 hz , 1h ) , 2.42 ( m , 1h ) , 2.34 ( m , 2h ) , 2.07 ( m , 1h ) , 
 1.98 ( m , 1h ) , 1.69 ( m , 2h ) , 1.14 ( t , j = 7.5 hz , 
 3h ) , 0.92 ( t , j = 7.4 hz , 3h ) ; c nmr 
 ( 151 mhz , cdcl3 )  168.9 , 141.1 , 137.6 , 136.1 , 134.3 , 
 129.63 , 129.60 , 128.63 , 128.60 , 126.5 , 126.1 , 62.5 , 54.2 , 48.2 , 27.3 , 
 24.3 , 20.4 , 16.3 , 15.4 , 11.5 ; ir ( thin film ) 3006 , 2966 , 1639 , 1300 , 
 1144 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c22h27no3sna 408.1609 , found 408.1620 . 
 general procedure b. the residue was purified 
 by flash column chromatography ( 80:20 etoac / hex , rf = 0.50 ) to yield 13l as a foam ( 104 mg , 
 64% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.92 ( m , 
 2h ) , 7.69 ( t , j = 7.1 hz , 1h ) , 7.60 ( m , 2h ) , 6.32 
 ( s , 1h ) , 6.16 ( d , j = 2.1 hz , 2h ) , 5.14 ( d , j = 2.1 hz , 1h ) , 3.95 ( ddd , j = 13.4 , 9.6 , 
 6.5 hz , 1h ) , 3.72 ( s , 6h ) , 3.34 ( td , j = 4.8 , 1.9 
 hz , 1h ) , 2.79 ( m , 1h ) , 2.46 ( m , 2h ) , 2.03 ( m , 2h ) , 1.63 ( m , 2h ) , 0.89 
 ( t , j = 7.4 hz , 3h ) ; c nmr ( 150 mhz , 
 cdcl3 )  169.1 , 161.5 , 142.3 , 137.4 , 134.2 , 129.6 , 
 128.6 , 104.2 , 99.5 , 64.6 , 58.0 , 55.4 , 48.2 , 28.0 , 20.3 , 17.7 , 11.3 ; 
 ir ( thin film ) 2962 , 2937 , 1641 , 1594 , 1306 , 1145 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c22h27no5sna 440.1508 , 
 found 440.1517 . 
 general procedure b. the residue was purified 
 by flash column chromatography ( 70:30 etoac / hex , rf = 0.35 ) to yield 13 m as a foam ( 101 mg , 
 75% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.88 ( d , j = 7.2 hz , 2h ) , 7.65 ( m , 1h ) , 7.56 ( t , j = 7.7 hz , 2h ) , 7.27 ( dd , j = 1.9 , 1.0 hz , 1h ) , 
 6.24 ( m , 1h ) , 6.10 ( m , 1h ) , 5.16 ( d , j = 2.9 hz , 
 1h ) , 3.78 ( ddd , j = 13.6 , 9.6 , 6.4 hz , 1h ) , 3.59 
 ( td , j = 6.1 , 3.1 hz , 1h ) , 2.71 ( m , 2h ) , 2.43 ( dt , j = 17.5 , 6.6 hz , 1h ) , 2.09 ( m , 2h ) , 1.58 ( m , 1h ) , 1.49 
 ( m , 1h ) , 0.85 ( t , j = 7.4 hz , 3h ) ; c 
 nmr ( 150 mhz , cdcl3 )  169.4 , 151.3 , 143.0 , 137.3 , 
 134.2 , 129.5 , 128.5 , 110.6 , 108.4 , 61.8 , 52.9 , 47.8 , 28.5 , 20.4 , 19.0 , 
 11.3 ; ir ( thin film ) 2965 , 2933 , 2875 , 1647 , 1503 , 1306 , 1148 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c18h21no4sna 370.1089 , found 370.1076 . 
 the residue was purified by flash column chromatography 
 ( 70:30 etoac / hex , rf = 0.35 ) to yield 14 as a solid ( 111 mg , 87% ) . 
 mp 127.4128.1 c ; h nmr ( 600 mhz , cdcl3 )  7.99 ( m , 2h ) , 7.74 
 ( m , 1h ) , 7.65 ( m , 2h ) , 7.35 ( dd , j = 8.4 , 7.1 hz , 
 2h ) , 7.28 ( m , 1h ) , 7.05 ( d , j = 6.8 hz , 2h ) , 5.21 
 ( s , 1h ) , 3.45 ( d , j = 6.9 hz , 1h ) , 2.91 ( s , 3h ) , 
 2.53 ( m , 1h ) , 2.45 ( dd , j = 15.7 , 5.3 hz , 1h ) , 2.11 
 ( dd , j = 15.8 , 11.6 hz , 1h ) , 0.91 ( d , j = 6.7 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 )  
 171.1 , 139.3 , 137.0 , 134.5 , 129.6 , 129.4 , 129.1 , 127.9 , 125.3 , 73.7 , 
 60.3 , 38.1 , 34.4 , 29.1 , 22.4 ; ir ( thin film ) 2912 , 1658 , 1305 , 1130 
 cm ; hrms ( esi - tof ) m / z 
 the residue was purified by flash column chromatography 
 ( 80:20 etoac / hex , rf = 0.35 ) to yield 15 as a white solid ( 169 mg , 79% ) . 
 mp 143.7144.3 c ; h nmr ( 600 mhz , cdcl3 )  7.70 ( d , j = 7.5 hz , 2h ) , 7.50 ( t , j = 7.4 hz , 1h ) , 
 7.37 ( t , j = 7.7 hz , 2h ) , 5.89 ( s , 2h ) , 5.31 ( d , j = 9.5 hz , 1h ) , 4.22 ( ddd , j = 13.4 , 9.5 , 
 3.6 hz , 1h ) , 3.72 ( s , 3h ) , 3.71 ( s , 6h ) , 2.48 ( m , 4h , nch3 and 1 methylene h ) , 2.40 ( m , 1h ) , 1.84 ( m , 1h ) , 1.27 ( d , j = 6.9 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 )  173.1 , 161.4 , 158.6 , 137.4 , 133.4 , 128.50 , 128.48 , 106.8 , 
 90.6 , 61.9 , 55.6 , 55.2 , 51.9 , 35.0 , 31.8 , 29.3 , 17.1 ; ir ( thin film ) 
 3006 , 2939 , 1614 , 1286 , 1134 cm ; hrms ( esi - tof ) m / z [ m + h ] calcd for c22h28no6s 434.1637 , found 434.1649 . 
 allylamine ( 0.29 ml , 3.8 mmol ) and trans - cinammaldehyde 
 ( 0.53 ml , 4.2 mmol ) were added to a round - bottom flask containing 
 thf ( 38 ml ) and na2so4 ( 3.3 g , 23.0 mmol ) , and 
 the mixture was allowed to stir for 1 h. the anhydride ( 0.92 g , 3.8 
 mmol ) was added , and the resulting mixture was allowed to stir for 
 2 h. the na2so4 was filtered off , and the solvent 
 was evaporated in vacuo . 
 the residue was 
 triturated with hexanes , transferred to a fritted funnel , and washed 
 in portions with hexanes five times ( 5 ml ) and two times with a 9:1 
 mixture of cold hexanes and chcl3 ( 10 ml total ) , yielding 
 1.2 g of an off - white solid , which was 85% pure by nmr . 
 a 
 portion of the resulting acid ( 0.885 g , 2.2 mmol ) was redissolved 
 in anhydrous toluene and methanol ( 60 ml:30 ml ) , and tmschn2 ( 2.2 ml , 4.4 mmol , 2 m in hexanes ) was added at 0 c dropwise 
 over a period of 5 min . 
 the mixture was allowed to stir for 1.5 h 
 and monitored by tlc for disappearance of the starting material . 
 the 
 solvent was removed in vacuo , and the crude mixture was purified by 
 flash column chromatography ( etoac / hex 3060% ) , yielding 17 ( 0.85 g , 92% ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.44 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.987.89 ( m , 2h ) , 7.817.56 ( m , 3h ) , 
 7.417.24 ( m , 5h ) , 6.54 ( d , j = 15.8 hz , 1h ) , 
 5.845.72 ( m , 2h ) , 5.345.25 ( m , 2h ) , 5.02 ( d , j = 8.4 hz , 1h ) , 4.40 ( ddt , j = 15.3 , 4.7 , 
 1.7 hz , 1h ) , 3.713.42 ( m , 3h ) , 3.443.27 ( m , 2h ) , 3.13 
 ( d , j = 18.2 ( gem ) hz , 1h ) ; c nmr ( 150 
 mhz , cdcl3 )  169.4 , 165.3 , 136.7 , 135.8 , 135.2 , 
 135.0 , 131.7 , 130.6 , 129.3 , 128.9 , 128.9 , 126.8 , 121.9 , 119.4 , 76.1 , 
 62.0 , 53.4 , 43.8 , 35.6 . 
 ir ( thin film ) 1736 , 1715 , 1694 ( c = o ) , 
 1309 , 1148 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h23no5sna 448.1195 , found 448.1195 . 
 ester 17 ( 0.602 g , 1.41 mmol ) was dissolved in anhydrous benzene 
 ( 114 ml ) , which was degassed by bubbling with argon for approximately 
 5 min . 
 then grubbs second - generation catalyst ( 35 mg , 3 mol % ) was 
 added , and the mixture was heated to reflux for 9 h. the solvent was 
 evaporated in vacuo , and the residue was redissolved in etoac ( 30 
 ml ) . 
 saturated nh4cl ( 10 ml ) was added , and the mixture 
 was allowed to stir for 30 min . 
 the mixture was transferred to a separtory 
 funnel , and the organic layer was washed with brine , dried over na2so4 , and then purified ( etoac / hex 3060% ) , 
 yielding 18 ( 0.39 g , 86% ) as an amorphous solid ( 50:50 
 etoac / hex , rf = 0.16 ) . 
 h nmr 
 ( 600 mhz , cdcl3 )  8.027.93 ( m , 2h ) , 7.74 
 ( td , j = 7.4 , 1.3 hz , 1h ) , 7.61 ( t , j = 7.9 hz , 2h ) , 5.90 ( dq , j = 6.0 , 2.1 hz , 1h ) , 
 5.55 ( dq , j = 6.2 , 2.2 hz , 1h ) , 5.38 ( tt , j = 4.3 , 1.8 hz , 1h ) , 4.33 ( ddt , j = 15.7 , 
 4.2 , 2.1 hz , 1h ) , 3.783.67 ( m , 2h ) , 3.62 ( s , 3h ) , 3.00 ( d , j = 15.7 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 )  172.8 , 165.7 , 136.9 , 134.9 , 130.6 , 130.3 , 129.3 , 
 129.2 , 79.0 , 70.1 , 53.3 , 50.7 , 39.5 ; ir ( thin film ) 1735 , 1716 , 1687 
 ( c = o ) , 1311 , 1148 ( s = o ) cm ; hrms 
 ( esi - tof ) m / z [ m + na ] calcd for c15h15no5sna 344.0569 , 
 found 344.0568 . 
 alkene 18 ( 0.25 g , 0.77 mmol ) was dissolved in anhydrous meoh ( 30 
 ml ) , and the reaction mixture was degassed by bubbling with argon 
 for 5 min . 
 then pd / c ( 45 mg ) was added and h2 was bubbled 
 into the reaction flask , and the mixture was allowed to stir for 3 
 h ( monitored by tlc ) . 
 the pd / c was filtered off and washed with etoac 
 ( 3  10 ml ) . 
 the solvent was removed in vacuo , yielding 19 ( 0.246 g , 99% ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.1 ) . 
 h nmr ( 600 mhz , cdcl3 )  7.987.90 ( m , 2h ) , 7.797.70 ( m , 1h ) , 
 7.60 ( dd , j = 8.5 , 7.3 hz , 2h ) , 4.61 ( dd , j = 9.7 , 5.9 hz , 1h ) , 3.72 ( s , 3h ) , 3.623.49 ( m , 
 2h ) , 3.17 ( d , 
 j = 17.1 hz , 1h ) , 3.153.05 
 ( m , 1h ) , 2.071.93 ( m , 2h ) , 1.91 ( dtd , j = 
 12.2 , 6.0 , 3.9 hz , 1h ) , 1.25 ( dq , j = 12.6 , 9.3 hz , 
 1h ) ; c nmr ( 150 mhz , cdcl3 )  170.6 , 
 166.0 , 134.9 , 130.3 , 130.2 , 129.2 , 75.6 , 63.9 , 53.4 , 42.1 , 39.4 , 28.5 , 
 25.8 ; ir ( thin film ) 1734 , 1686 ( c = o ) , 1146 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c15h17no5sna 346.0725 , found 346.0730 . 
 to sulfone 19 ( 0.10 g , 0.31 
 mmol ) dissolved in 3 ml of chcl3 was added dbu ( 70 l , 
 0.46 mmol ) , and the reaction mixture was stirred overnight . the mixture 
 was concentrated in vacuo and purified by flash chromatography ( 70:30 
 etoac / hex , rf = 0.40 ) to yield 20 as a colorless oil ( 0.047 g , 84% ) . 
 h nmr ( 600 mhz , cdcl3 )  3.72 ( s , 3h ) , 3.58 ( t , j = 7.1 
 hz , 2h ) , 3.52 ( m , 2h ) , 2.93 ( m , 2h ) , 2.40 ( p , j = 
 7.4 hz , 2h ) ; c nmr ( 150 mhz , cdcl3 )  
 172.8 , 164.0 , 161.6 , 98.6 , 51.0 , 41.5 , 41.2 , 26.6 , 25.6 . these data 
 ester 18 ( 50 mg , 0.16 mmol ) 
 was dissolved in 2 ml of chcl3 , and dbu ( 36 l , 0.24 
 mmol ) was added . 
 after 90 min , 
 the solvent evaporated in vacuo , and the crude mixture was purified 
 by flash chromatography ( 20:80 etoac / hex , rf = 0.45 ) , yielding 21 as an oil ( 12 mg , 41% ) . 
 h nmr ( 600 mhz , cdcl3 )  7.04 ( d , j = 3.4 hz , 1h ) , 6.47 ( t , j = 3.5 hz , 1h ) , 6.17 ( d , j = 3.1 hz , 1h ) , 4.20 ( dd , j = 9 , 3 hz , 
 1h ) , 3.78 ( s , 3h ) , 3.48 ( dd , j = 19.8 ( gem ) , 3.6 
 hz , 1h ) , 3.19 ( dd , j = 18.6 ( gem ) , 9 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 )  170.6 , 169.6 , 136.1 , 
 119.2 , 112.2 , 106.6 , 52.9 , 38.1 , 38.0 . 
 alkene 20 ( 47 mg , 0.25 mmol ) 
 was dissolved in 2.2 ml of methanol 
 under an atmosphere of argon , and 10% pd / c ( 14 mg ) was added to the 
 vial . 
 the flask was purged with h2 gas via a balloon , and 
 the reaction mixture was stirred under an atmosphere of h2 overnight . 
 the reaction vessel was purged with argon , and then the 
 mixture was filtered through celite and concentrated to yield 22 as an oil ( 45 mg , 99% ) . 
 h nmr ( 600 mhz , cdcl3 )  4.09 ( ddd , j = 9.7 , 9.1 , 3.8 hz , 
 1h ) , 3.69 ( s , 3h ) , 3.57 ( q , j = 9.3 hz , 1h ) , 3.38 
 ( td , j = 8.5 , 4.8 hz , 1h ) , 3.03 ( t , j = 11.7 hz , 1h ) , 2.78 ( d , j = 8.9 hz , 2h ) , 2.05 
 ( q , j = 9.5 hz , 1h ) , 1.95 ( m , 1h ) , 1.82 ( m , 1h ) , 
 1.24 ( m , 1h ) ; c nmr ( 150 mhz , cdcl3 )  
 174.0 , 172.2 , 62.7 , 51.9 , 41.8 , 39.9 , 36.0 , 27.4 , 26.0 . these data","sulfone - substituted 
 - and -lactams have been prepared 
 in a single step with high diastereoselectivity . 
 sulfonylglutaric 
 anhydrides produce intermediates that readily decarboxylate to provide 
 -lactams with high diastereoselectivity . 
 substituents at the 
 3- or 4-position of the glutaric anhydride induce high levels of stereocontrol . 
 sulfonylsuccinic anhydrides produce intermediate carboxylic acids 
 that can be trapped as methyl esters or allowed to decarboxylate under 
 mild conditions . 
 this method has been applied to a short synthesis 
 of the pyrrolizidine alkaloid ( )-isoretronecanol .",pubmed
"the morbidity of breast cancer is gradually increasing , and a growing number of young people are now at risk for this disease . in china , 
 breast cancer continues to be the leading cause of death among women . with the improvement in diagnosis and development of drugs and radiotherapy technology 
 . therefore , early detection of the tumor and accurate analysis of molecular biology characteristics are still of great importance . 
 microrna ( mirna ) is a non - coding protein and single - stranded small rna . 
 it mainly inhibits translation or induces the degradation of some related mrnas through target mrna recognition , leading to changes in objective mrna at the protein expression level . 
 recent studies showed that some mirnas have functions similar to that of oncogenes or anti - oncogenes , which may play an important role in the invasion and development of breast cancer . 
 mir-125a , mir-206 , and mir-145 are similar to anti - oncogenes , and they are related to the occurrence of tumors . 
 thus , mir-206 plays a key role in adjustment during breast cancer proliferation . according to the research of scott and gk , mir-125a can affect the protein levels of her-2 and her-3 , thereby inhibiting the metastatic and invasive abilities of breast cancer cells . 
 moreover , lee   showed that mir-145 expression increases after mir-145 precursors transfect breast cancer cell lines , but can dose - dependently inhibit cell growth . mir-21 and mir-10b are similar to oncogenes , and related to the invasion and metastasis of tumors . in vitro and in vivo experiments showed that eliminating mir-21 gene fragments can inhibit the proliferation and invasion of mcf-7 and mda - mb-231 cells . in the 4t1 rat breast tumor metastasis model , mir-10b can effectively inhibit the metastasis of malignant breast cancer cells with high specificity . 
 the possible mechanism for this effect may be the inhibition of the entry of 4ti tumor cells into blood vessels . 
 however , the specific roles of mir-21 and mir-10b in the occurrence and development of breast cancer remain unclear . 
 the expression of mir-21 , mir-10b , mir-125a , mir-206 , and mir-145 in breast cancer tissues and para - carcinoma tissues was examined . 
 the expression of estrogen receptor ( er ) and progesterone receptor ( pr ) in breast cancer was analyzed by immunohistochemistry , whereas that of her-2 and topoisomerase 2a ( top 2a ) was analyzed by fluorescence in situ hybridization ( fish ) . 
 the relationship between the expression levels of mirnas and breast cancer clinical pathology was analyzed . 
 thus , this research aimed to investigate the function of mirnas in the invasion and development of breast cancer . 
 cancer tissues were obtained from these patients , who were pathologically confirmed to have invasive ductal carcinoma from january 1 2013 to december 1 2013 in the tumor hospital of the second affiliated hospital of xjtu . 
 all patients were female , with an age range of 4262 years ( mean age , 51 years ) . 
 these patients underwent modified radical mastectomy or reserved radical mastectomy , and had complete clinical pathology data . 
 the diameter of tumors ranged from 1.6 cm to 4.5 cm ( mean diameter , 2.2 cm ) . 
 one sample was cancer tissues , and the other sample was para - carcinoma normal gland tissues , which were more than 3 cm away from cancer tissues . 
 the samples were immediately flushed with 0.9% rna- and enzyme - free nacl , rapidly immersed in rna enzyme inhibitor , and incubated at 4c overnight . 
 the protective liquid was removed from the samples , which were stored in a refrigerator at 80c . 
 the patients were not treated before the operation , and neither neoadjuvant chemotherapy nor endocrine therapy was conducted . based on histological classification by the who ( 2003 ) standards , 
 m - mlv reverse transcriptase , rna enzyme inhibitor , oligo dt , dntps , kod - plus , and sybr mastermix were obtained from takara ( japan ) , and primers were from shanghai shenggong company ( china ) . 
 breast cancer her-2 , neu ( 17q12 ) , top 2a ( 17q21 ) , and csp 17 multicolor detection kits were purchased from guangzhou biping medical technology company . 
 total rna ( 500 ng ) was used for rt by mirna stem - loop rt primers . 
 the reaction was conducted in a 10-l system at the following reaction conditions : 16c for 30 min , 42c for 15 min , and 85c for 5 s. real - time qpcr was performed with a 20-l reaction system . 
 the mirna reaction detection system included 1 l of rt products , 1 sybr green i mastermix , 0.5 ixmol / l mirna - specific f - primer and 0.5 ixmol / l universal r - primer . 
 the conditions of immediate quantitative pcr were as follows : 95c for 10 min , followed by 40 cycles of 95c for 15 s and 60c for 1 min . 
 the circulation times in which real - time qrt - pcr reached the set - up threshold in every reaction tube ( ct data ) were recorded . according to some related studies , 
 the 2 method was used to express the gene expression of tumor tissues relative to that of the paired normal tissues . 
 moreover , ct=(ct mirna - ct u6 ) tumor  ( ct mirna - ct u6 ) was normal . 
 samples were fixed in formalin , embedded in paraffin , and sliced into 4-m - thick sections . 
 the results show positive expression of ep and er at 10% , and the cancer cell nucleus was brown . 
 samples were fixed in formalin , embedded in paraffin , and sliced into 4-m - thick sections . 
 sections were initially observed under the 40 objective with a suitable filter , and counted under the 100 objective . 
 the signals of gene - specific probe ( gsp ) her-2 ( red ) , gsp top 2a ( green ) , and centromeric probe ( csp ) 17 ( cyan ) were counted in 60 nuclei at the clear tumor areas . 
 the sum of signals of gsp her-2 , gsp top 2a , and csp 17 were recorded , and the data of gsp her-2/csp 17 and gsp top 2a / csp 17 were calculated . 
 the signals of her and top 2a were analyzed according to the following criteria : a ) the signal of gsp her-2 and top 2a ( red , green ) combined to bunches ; b ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) 2.2 and 2 ; c ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) < 1.8 and 0.8 ; and d ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) between 1.82.2 and 0.82.0 . if  a  or 
 if  d ,  new signal data should be counted in 20 other nuclei , or another researcher should recalculate the data . 
 researchers should indicate the consequence if the recalculated data were at the critical value . under situations with weak signal or high background , 
 signal judgment may be disturbed , so the experiment was repeated using another tissue slice . 
 differences in expression were analyzed by spss 13.0 . given the limited number of samples in this study , the data demonstrate an abnormal distribution . 
 all mirna expression data are shown as the median and interquartile range ( iqr ) . 
 the nonparametric wilcoxon test was used to rank matched - pair data , and the mann - whitney u test was used to compare data between the 2 groups . 
 cancer tissues were obtained from these patients , who were pathologically confirmed to have invasive ductal carcinoma from january 1 2013 to december 1 2013 in the tumor hospital of the second affiliated hospital of xjtu . 
 all patients were female , with an age range of 4262 years ( mean age , 51 years ) . 
 these patients underwent modified radical mastectomy or reserved radical mastectomy , and had complete clinical pathology data . 
 the diameter of tumors ranged from 1.6 cm to 4.5 cm ( mean diameter , 2.2 cm ) . 
 one sample was cancer tissues , and the other sample was para - carcinoma normal gland tissues , which were more than 3 cm away from cancer tissues . 
 the samples were immediately flushed with 0.9% rna- and enzyme - free nacl , rapidly immersed in rna enzyme inhibitor , and incubated at 4c overnight . 
 the protective liquid was removed from the samples , which were stored in a refrigerator at 80c . 
 the patients were not treated before the operation , and neither neoadjuvant chemotherapy nor endocrine therapy was conducted . based on histological classification by the who ( 2003 ) standards , 
 m - mlv reverse transcriptase , rna enzyme inhibitor , oligo dt , dntps , kod - plus , and sybr mastermix were obtained from takara ( japan ) , and primers were from shanghai shenggong company ( china ) . 
 breast cancer her-2 , neu ( 17q12 ) , top 2a ( 17q21 ) , and csp 17 multicolor detection kits were purchased from guangzhou biping medical technology company . 
 total rna ( 500 ng ) was used for rt by mirna stem - loop rt primers . 
 the reaction was conducted in a 10-l system at the following reaction conditions : 16c for 30 min , 42c for 15 min , and 85c for 5 s. real - time qpcr was performed with a 20-l reaction system . 
 the mirna reaction detection system included 1 l of rt products , 1 sybr green i mastermix , 0.5 ixmol / l mirna - specific f - primer and 0.5 ixmol / l universal r - primer . 
 the conditions of immediate quantitative pcr were as follows : 95c for 10 min , followed by 40 cycles of 95c for 15 s and 60c for 1 min . 
 the circulation times in which real - time qrt - pcr reached the set - up threshold in every reaction tube ( ct data ) were recorded . according to some related studies , 
 the 2 method was used to express the gene expression of tumor tissues relative to that of the paired normal tissues . 
 moreover , ct=(ct mirna - ct u6 ) tumor  ( ct mirna - ct u6 ) was normal . 
 samples were fixed in formalin , embedded in paraffin , and sliced into 4-m - thick sections . 
 the results show positive expression of ep and er at 10% , and the cancer cell nucleus was brown . 
 samples were fixed in formalin , embedded in paraffin , and sliced into 4-m - thick sections . 
 sections were initially observed under the 40 objective with a suitable filter , and counted under the 100 objective . 
 the signals of gene - specific probe ( gsp ) her-2 ( red ) , gsp top 2a ( green ) , and centromeric probe ( csp ) 17 ( cyan ) were counted in 60 nuclei at the clear tumor areas . 
 the sum of signals of gsp her-2 , gsp top 2a , and csp 17 were recorded , and the data of gsp her-2/csp 17 and gsp top 2a / csp 17 were calculated . 
 the signals of her and top 2a were analyzed according to the following criteria : a ) the signal of gsp her-2 and top 2a ( red , green ) combined to bunches ; b ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) 2.2 and 2 ; c ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) < 1.8 and 0.8 ; and d ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) between 1.82.2 and 0.82.0 . if  a  or 
 d ,  new signal data should be counted in 20 other nuclei , or another researcher should recalculate the data . 
 researchers should indicate the consequence if the recalculated data were at the critical value . under situations with weak signal or high background , 
 signal judgment may be disturbed , so the experiment was repeated using another tissue slice . 
 differences in expression were analyzed by spss 13.0 . given the limited number of samples in this study , the data demonstrate an abnormal distribution . all mirna expression data are shown as the median and interquartile range ( iqr ) . the nonparametric wilcoxon test was used to rank matched - pair data , and the mann - whitney u test was used to compare data between the 2 groups . 
 among the 35 samples , the relative quantity ( 2 ) of mir-125a was below 1 ( 80% ) in 28 cases and above 1 ( 20% ) in 7 cases . 
 the relative quantity ( 2 ) of mir-206 was below 1 ( 86% ) in 30 cases of breast cancer and above 1 ( 14% ) in 5 cases . 
 the relative quantity ( 2 ) of mir-145 was below 1 ( 60% ) in 21 cases of breast cancer and above 1 ( 40% ) in 14 cases . 
 the data ranged from 0.13 to 7.73 and the iqr was 0.511.72 . the median was 0.78 , which is 0.78 times that of normal tissue . 
 the relative quantity ( 2 ) of mir-21 was above 1 ( 94% ) in 33 cases of breast cancer and below 1 ( 6% ) in 2 cases . 
 the relative quantity ( 2 ) of mir-10b was above 1 ( 63% ) in 22 cases of breast cancer and below 1 ( 37% ) in 13 cases . 
 the difference was statistically significant ( p<0.05 ) . among the 35 patients , 16 cases ( 45.7% ) exhibited her-2 gene amplification , which was demonstrated as a red cluster or multiple red points , whereas 18 cases ( 51.4% ) exhibited top 2a gene amplification , which was demonstrated as a green cluster or multiple green points ( table 1 ) . 
 negative expression of her-2 and top 2a was observed in the 17 chromosome ( figure 1 ) . 
 the expression levels of mir-125a , mir-206 , and mir-145 in breast cancer tissues were all lower than those in para - carcinoma tissues 
 . low expression of mir-125a in the metastatic lymph node group and high negative expression of her-2 and top 2a in patients were unrelated to the tumor size , patient age , menstruation , negative er , and pr expression ( p<0.10 ) . 
 mir-206 was highly expressed in patients with small tumors , negative er , negative pr , and negative her-2 ( p<0.10 ) . 
 thus , mir-206 was irrelevant to patient age , menstruation , metastatic lymph node , and top 2a expression ( table 2 ) . 
 mir-145 was negatively correlated with the tumor size and lymph node metastasis ( p<0.05 ) , and had no correlation with patient age , menstruation , and pr expression . 
 however , mir-145 was correlated with negative er , negative her-2 , and negative top 2a ( p<0.05 ) ( table 3 ) . 
 the expression of mir-21 and mir-10b in breast cancer tissues was obviously higher than that in para - carcinoma tissues . 
 mir-21 exhibited high expression in older , postmenopausal patients with various symptoms , such as lymph node metastasis and positive her-2 ( p<0.10 ) , but it was not related to the tumor size and expression levels of top 2a , er , and pr . 
 the expression of mir-10b was related to the tumor size , lymph node metastasis , and positive top-2a ( p<0.05 ) , but it was not associated with age , menstruation , er , pr , and her-2 ( table 3 ) . 
 among the 35 samples , the relative quantity ( 2 ) of mir-125a was below 1 ( 80% ) in 28 cases and above 1 ( 20% ) in 7 cases . 
 the relative quantity ( 2 ) of mir-206 was below 1 ( 86% ) in 30 cases of breast cancer and above 1 ( 14% ) in 5 cases . 
 the relative quantity ( 2 ) of mir-145 was below 1 ( 60% ) in 21 cases of breast cancer and above 1 ( 40% ) in 14 cases . 
 the data ranged from 0.13 to 7.73 and the iqr was 0.511.72 . the median was 0.78 , which is 0.78 times that of normal tissue . 
 the relative quantity ( 2 ) of mir-21 was above 1 ( 94% ) in 33 cases of breast cancer and below 1 ( 6% ) in 2 cases . 
 the relative quantity ( 2 ) of mir-10b was above 1 ( 63% ) in 22 cases of breast cancer and below 1 ( 37% ) in 13 cases . 
 among the 35 patients , 16 cases ( 45.7% ) exhibited her-2 gene amplification , which was demonstrated as a red cluster or multiple red points , whereas 18 cases ( 51.4% ) exhibited top 2a gene amplification , which was demonstrated as a green cluster or multiple green points ( table 1 ) 
 . negative expression of her-2 and top 2a was observed in the 17 chromosome ( figure 1 ) . 
 the expression levels of mir-125a , mir-206 , and mir-145 in breast cancer tissues were all lower than those in para - carcinoma tissues . low expression of mir-125a in the metastatic lymph node group and high negative expression of her-2 and top 2a in patients were unrelated to the tumor size , patient age , menstruation , negative er , and pr expression ( p<0.10 ) . 
 mir-206 was highly expressed in patients with small tumors , negative er , negative pr , and negative her-2 ( p<0.10 ) . 
 thus , mir-206 was irrelevant to patient age , menstruation , metastatic lymph node , and top 2a expression ( table 2 ) . 
 mir-145 was negatively correlated with the tumor size and lymph node metastasis ( p<0.05 ) , and had no correlation with patient age , menstruation , and pr expression . 
 however , mir-145 was correlated with negative er , negative her-2 , and negative top 2a ( p<0.05 ) ( table 3 ) . 
 the expression of mir-21 and mir-10b in breast cancer tissues was obviously higher than that in para - carcinoma tissues . 
 mir-21 exhibited high expression in older , postmenopausal patients with various symptoms , such as lymph node metastasis and positive her-2 ( p<0.10 ) , but it was not related to the tumor size and expression levels of top 2a , er , and pr . 
 the expression of mir-10b was related to the tumor size , lymph node metastasis , and positive top-2a ( p<0.05 ) , but it was not associated with age , menstruation , er , pr , and her-2 ( table 3 ) . 
 recent studies have already proved that the expression of mirnas changes is closely related to various types of human cancer . 
 however , the functions of mirnas are complicated because they can effectively regulate and control hundreds of genes , including oncogenes and anti - oncogenes . 
 studies indicated that mir-125a ( has - mir-125a ) is located at chromosome 19q13.41 , including 2 mirnas that are in the same precursor ( mir-125a.5p and mir-125a.3p ) , and has the same origin as lin-4 in nematodes . 
 mir-125a can be detected as a phylogenetic mutation , which is associated with the occurrence of breast cancer . 
 mir-125a can also inhibit tumors by directly lowering the mrna and protein levels of some genes , such as erbb2 and rna binding protein hur . 
 mir-206 is located at human chromosome 6p12.2 , and is considered to be a type of mirna related to skeletal muscles and the myocardium . 
 it can boost muscle differentiation by lowering the p180 subunit of dna polymerase and muscle transcription factors ( e.g. , is13 and myor ) . 
 mir-206 can also inhibit the proliferation of breast cancer cells , mainly by lowering the mrna and protein expression levels of er and er co - regulatory proteins via the egf / mapk pathway [ lowering steroid acceptor nuclear co - activators ( e.g. , src-1 and src-3 ) and transcription factor gata-3 ] . 
 mir-206 can also act on the protein cx43 to inhibit the invasion and metastasis of cancer cells . 
 mir-206 can result in mutations at the rs6920648 locus , and is associated with the existence of breast cancer . 
 the expression of mir-145 decreases in many types of tumor tissues , including breast cancer , colon cancer , and lung cancer . 
 this phenomenon might be related to several factors , such as the location of mir-145 , ability of mir-145 to easily cause deficiency in tumor cells , and genetic changes in the receptor s surface . 
 similarly , mir-145 can inhibit hyperplasia in tumor cells by affecting target genes , such as c - myc , irs-1 , oct4 , sox2 , and klf4 . 
 mir-21 is an mirna located in the fra17b fragile area of chromosome 17q23.2 , and has an independent transcription unit . 
 moreover , mir-21 controls the expression of many anti - oncogenes , including tpm1 , programmed cell death 4 , maspin , and bcl-2 , to prompt the metastasis and hyperplasia of breast cancer cells . 
 mir-10b is located between the hoxd4 and hoxd8 genes of 2p31.1 , and its structure is 5-uacccuguagaaccgaauuugug-3. mir-10b is related to many types of tumors . 
 adjusting the twist - mir-10b - hoxd 10-rhoc pathway is the main mechanism by which mir-10b inhibits the metastasis of breast cancer cells . 
 mir-10b also facilitates the metastasis of breast cancer cells by regulating the expression of the target genes upar and syndecan-1 . 
 early diagnosis is essential to prevent and cure tumors . in this study , the relative transcript levels of mir-21 and mir-10b in patients tumor tissues were higher than those in para - carcinoma normal tissues . 
 by contrast , the relative transcript levels of mir-145 , mir-125a , and mir-206 in patients tumor tissues were apparently lower than those in para - carcinoma normal tissues . according to the literature [ 1214 ] , the relative transcript levels of mir-145 , mir-125a , and mir-206 in serum are also visibly abnormal compared with those in serum from healthy females . 
 therefore , these mirnas can be used as markers of breast cancer for early diagnosis . 
 our results show that mir-145 and breast cancer tumor size , as well as mir-145 and lymph node metastasis , was negatively correlated in 35 cases of breast cancer . 
 although mir-125a and mir-206 were expressed at low levels in the large mass group and lymph node metastasis group , statistical analysis showed no significant correlation between the expression of mir-125a and mir-206 and breast tumor size , as well as between the expression of mir-125a and mir-206 and lymph node metastasis . 
 we are currently further expanding the number of samples for analysis to determine the intrinsic correlations . 
 preliminary results show that mir-145 , mir-125a , and mir-206 were similar to tumor suppressor genes , and may be involved in the suppression of breast cancer invasion and metastasis ; these findings are consistent with those reported in the literature . 
 as indicated in the literature , mir-10b expression may indicate the invasion and development of breast cancer . 
 combined with the findings of lee , the results show that mir-21 may be correlated with old patients with lymph node metastasis . 
 hormone receptor - positive breast cancer , her-2-positive breast cancer , and triple - negative breast cancer ( tnbc ) ]  based on er , pr , and her-2 expression . 
 her-2-positive breast cancer is highly malignant , and can reappear and transfer easily . herceptin - targeted treatment has made significant progress for this type of cancer . 
 lee   showed that mir-21 is related to the metastasis of breast cancer lymph nodes and positive her-2 expression . 
 moreover , mir-21 is an independent prognostic factor of breast cancer . combined with cong s research 
 , the results showed that mir-21 is over - expressed in breast cancer cells resistant to trastuzumab , which causes the loss of pten expression . 
 a previous study   showed that mir-125a is negatively associated with her-2 , indicating that the her-2 gene may be under the regulation and control of mir-125a ; therefore , mir-125a can distinctly inhibit the expression of oncogenes her-2 and her-3 . 
 mir-125a expression is up - regulated when the hdac inhibitor entinostat is used to treat her-2-positive breast cancer cells , and has been shown to be an important mechanism of the functions of entinostat . 
 thus , mir-21 and mir-125a are significant to developing new anti - her-2 drugs and overcoming herceptin resistance in drugs . 
 tnbc is characterized by high malignancy , high possibility of reappearance and metastasis , and poor treatment methods . in this study , 
 mir-145 and mir-206 were found to be related to negative er and negative her-2 ( p<0.05 ) . 
 mir-145 was highly expressed in negative pr ( p>0.05 ) , whereas mir-206 was negatively correlated with negative pr ( p<0.10 ) . 
 showed that the metastasis of cancer cells could be inhibited by leading mir-145 and mir-206 into metastatic breast cancer cells . 
 mir-145 and mir-206 are expected to be the new target spots in tnbc treatment . besides inhibiting tnbc development , mir-206 can also inhibit positive er lines , and is considered as the switch of breast cancer when it is transferred to the base type from lumen - a . 
 top 2a codes dna topoisomerase ii , and is one of the components of the nuclear matrix . 
 top 2a can adjust the copy , transcription , recombination , and repair of dna , which is not only a hyperplasia index of tumor cells , but also a treatment target of anthracycline chemotherapy drugs . in breast cancer tissues , tumors with high expression of top 2a 
 this situation can be improved after using anthracycline alone or anthracycline combination chemotherapy . in this research , 
 mir-21 was found to be positively related to top 2a , whereas mir-125a and mir-145 were negatively associated with top 2a . 
 these findings show that mir-145 and mir-125a might inhibit the top 2a gene , and were connected with anthracycline drug resistance . therefore , mir-21 , mir-125a , and mir-145 may contribute to the chemotherapy effect of epirubicin , and are important in guiding the development of chemotherapy for breast cancer . 
 therefore , mir-125a , mir-206 , mir-145 , mir-21 , and mir-10b are highly expected to be novel tumor markers for the diagnosis and prognosis of breast cancer . mir-21 and mir-125a could contribute to the treatment of her-2-positive breast cancer cells , whereas mir-145 and mir-206 could accelerate the development of treatment methods for tnbc . 
 however , the detailed mechanism and whether these mirnas can be used as treatment targets in clinical applications should be verified in future investigations .","backgroundthis study aimed to investigate the expression and significance of 5 types of mirnas in breast cancer to provide a theoretical and practical foundation for using these mirnas in the diagnosis and treatment of breast cancer , thereby improving medical services.material/methodsstem-loop real - time rt - pcr was used to detect the expression levels of mir-145 , mir-21 , mir-10b , mir-125a , and mir-206 in 35 cases of breast cancer and adjacent normal breast tissues , and to analyze the relationship of mirnas expression with clinicopathological features of breast cancer . 
 the expression levels of estrogen receptor ( er ) and progesterone receptor ( pr ) were examined by immunohistochemistry . 
 fluorescence in situ hybridization was used for the detection of her-2 and top 2a.resultsthe expression levels of mir-145 , mir-125a , and mir-206 in breast cancer were lower than those in adjacent normal tissues . 
 mir-145 was negatively correlated with tumor size , lymph node metastasis , er , her-2 , and top 2a ( p<0.05 ) , regardless of age , menstruation , and pr . 
 mir-125a was correlated with negative node status , negative her-2 status ( p<0.05 ) , whereas tumor size , age , menstruation , er , and pr were independent factors . 
 mir-206 expression was correlated with negative er status , negative pr status , and negative her-2 status ( p<0.05 ) , regardless of age , menstruation , lymph node metastasis , and top 2a . 
 mir-21 and mir-10b expression in breast cancer tissues was significantly higher than that in adjacent tissues ( p<0.05 ) . 
 mir-21 in post - menstrual patients with lymph node metastasis was highly expressed ( p<0.05 ) , and had no correlations with tumor size , er , pr , and top 2a expression . 
 mir-10b expression was positively correlated with breast cancer tumor size , lymph node metastasis , and top 2a status ( p<0.05 ) , but had no correlations with age , menstruation , er , pr , and her-2.conclusionsmir-145 , mir-21 , mir-10b , mir-125a , and mir-206 may play important roles in breast cancer development and invasion .",pubmed
"however , transplantation is limited not only by organ shortage but also by its unfavorable long - term success due to chronic allograft injury ( cai ) [ 1 , 2 ] . chronic allograft vasculopathy ( cav ) , glomerulosclerosis , and interstitial fibrosis and tubular atrophy ( ifta ) are the dominating histopathological findings in cai [ 1 , 2 ] . 
 its pathogenesis is still poorly understood , but acute rejection episodes were defined as an important risk factor and experimental data suggest that they play an important pathogenetic role [ 15 ] . 
 transplantation of kidneys in fischer-344 ( f344 ) to lewis rat strain combination is a well - established experimental model for human cai . 
 we and others thoroughly characterized a variant of this model , where immunosuppression is omitted [ 710 ] . 
 this model allows investigation of a severe but reversible acute rejection episode , which peaks 9 days after allogenic transplantation and precedes the development of cai . 
 acute rejection of f344 to lewis renal allografts is characterized by a strong mononuclear infiltration of the allograft interstitium . at the same time , numerous leukocytes accumulate in the arterial , capillary , and venous blood vessels . 
 after resolution of acute rejection , the number of intravascular leukocytes decreases but remains chronically elevated compared to healthy kidneys . 
 a majority of blood leukocytes in day 9 allografts are monocytes displaying a unique state of partial activation regarding cell surface antigen and cytokine expression . 
 these cells interact with graft endothelial cells and might trigger cav , which is characterized by intimal hyperplasia . to identify factors contributing to the pathogenesis of cai during reversible acute rejection , we compared the transcriptome of mononuclear leukocytes isolated from renal allografts to isografts 9 days after transplantation . in this gene array experiment , 
 tissue transglutaminase ( transglutaminase 2 , tgm2 ) was found among the genes , which were upregulated by mononuclear leukocytes accumulating in the lumina of allograft blood vessels . 
 transglutaminases catalyze posttranscriptional modifications of proteins , resulting in cross - linking of proteins such as cytoskeleton or extracellular matrix ( ecm ) , as well as in modifications via amine incorporation into proteins and via deamidation [ 11 , 12 ] 
 . these modifications can have drastic consequences including increased mechanical stability , formation of autoantigens , and profound changes in protein function [ 11 , 13 , 14 ] . among the 9 transglutaminases present in humans , tgm2 is most intensively investigated and a bewildering functional diversity is described . 
 it acts as classical ca - dependent transglutaminase , as g - protein , as disulfide isomerase , and as protein kinase [ 16 , 17 ] . 
 in addition , it interacts with a plethora of other proteins and is involved in numerous cellular processes such as apoptosis and cell survival , phagocytosis , cell adhesion and migration , and cell signaling . 
 tgm2 is ubiquitously expressed and localizes to virtually all compartments of the cell including the cytoplasm , the nucleus , and mitochondria . 
 in addition , tgm2 can be attached to the surface and is secreted into the extracellular space , where it can cross - link itself to extracellular proteins [ 15 , 18 ] . 
 we are interested in tgm2 in the context of reversible acute rejection and subsequent chronic deterioration of renal allografts . 
 tgm2 might exert essential functions involved in rejection such as antigen presentation and t cell activation via dendritic cells , activation of the nfb pathway , and enabling the expression of proinflammatory factors , leukocyte adhesion to vascular endothelial cells , and leukocyte migration . during resolution of acute rejection , 
 its role in apoptosis , phagocytosis , and release of tgf- may be of importance [ 21 , 22 ] . 
 finally , during the development of cai , tgm2 might protect graft blood vessels as already evidenced in the context of atherosclerosis . 
 alternatively , tgm2 could contribute to vascular remodeling by increasing vascular stiffness and by promoting epithelial / endothelial to mesenchymal transition [ 24 , 25 ] , which was suggested to contribute to ifta . in this study 
 , we investigate tgm2 mrna and protein expression by mononuclear blood leukocytes isolated from lewis to lewis isografts and f344 to lewis allografts and by respective renal tissue tgm2 expressions compared to activated caspase-3 , a protease critically involved in apoptosis . 
 leukocytes and grafts are studied during acute rejection on day 9 after transplantation and after resolution of acute rejection on day 42 . 
 furthermore , allograft recipients were treated with cystamine during the first month after transplantation to assess the role of early extracellular transglutaminase activity in the pathogenesis of cai . 
 male lewis ( rt1 ) ( janvier , st . berthevin , france ) and f344 ( rt1 ) ( harlan winkelmann , borchen , germany ) rats weighing 270300  g were used for transplantation . 
 animals were kept under conventional conditions and received humane care according to the german law on the protection of animals and the  principles of laboratory animal care  formulated by the national society for medical research as well as the nih  guide for the care and use of laboratory animals . 
 experiments were approved by the local authorities ( permit number gi 20/10 number 23/2008 ; gi 20/27 number 51/2010 ) . 
 kidneys were transplanted orthotopically to totally nephrectomized lewis recipients as described previously , except that the ureter was anastomosed end to end . for allogenic transplantation , f344 rats and , for isogenic transplantation , 
 30  g ampicillin after surgery ( ratiopharm , ulm , germany ) ; no immunosuppressant was given . 
 total ischemic times remained below 30  min . on day 9 or 42 after transplantation 
 application of 60  mg / kg sodium pentobarbital ( narcoren , merial , hallbergmoos , germany ) and grafts were removed , cut in small pieces , and snap frozen in liquid nitrogen until use . 
 allograft recipients were treated with the transglutaminase inhibitor cystamine dihydrochloride ( sigma - aldrich , taufkirchen , germany ) for 28 days starting at the day of transplantation . 
 cystamine was diluted in sterile saline and delivered continuously using subcutaneously implanted osmotic minipumps ( 2ml4 alzet , cupertino , ca ) at a concentration of 12  mg per day . 
 animals were sacrificed 12 weeks after transplantation ; grafts were removed , fixed in 4% buffered paraformaldehyde , and embedded in paraffin . to study renal function , 
 isolation of mononuclear blood cells was described previously in detail [ 28 , 29 ] . 
 injection of 200  u of heparin ( liquemin n 5000 , roche , basel , switzerland ) , and kidneys were intensively perfused with cold ca- and mg - free phosphate buffered saline ( paa , pasching , austria ) , supplemented with 2.7  mm edta and 0.1% bovine serum albumin ( serva , heidelberg , germany ) . to deplete erythrocytes and granulocytes 
 total rna was isolated from 30  mg kidney tissue or from 5  10 mononuclear leukocytes harvested from the blood vessels of control kidneys , isografts , or allografts on day 9 or 42 after transplantation . 
 rna extraction was performed using the rneasy mini kit ( qiagen , hilden , germany ) according to the instructions of the supplier . 
 one g of total rna was reversely transcribed using the m - mlv h reverse transcriptase and 1  g of random hexamer primers ( promega , mannheim , germany ) . 
 the reaction was carried out at 40c for 1  h. real - time rt - pcr was used to assess the mrna gene expression of tgm2 . 
 reactions were performed in an abi 7900 sequence detection system ( applied biosystems , foster city , ca ) using platinum sybr green qpcr super mix - udg ( invitrogen , karlsruhe , germany ) . 
 the relative gene expression values were calculated by the equation 2 , where ct is the difference in ct values between the porphobilinogen deaminase ( pbgd ) housekeeping gene and tgm2 . 
 data for each experimental group are expressed in relation to the expression in the healthy control , which was set to 1 arbitrary unit . 
 all primers for real - time rt - pcr were synthesized by mwg biotech ( ebersberg , germany ) . 
 primer sequences are as follows : for tgm2 , 5-cca gcg tgg aca gac tta ca-3 ( sense ) , 5-ctg ctc cac atc gtc aga ca-3(antisense ) and for pbgd , 5-ggc gca gct aca gag aaa gt-3(sense ) , 5-agc cag gat aat ggc act ga-3(antisense ) . 
 pcr conditions included denaturation for 5  min at 95c , followed by 45 cycles of 20  s at 95c , 20  s at 60c , and 10  s at 72c . to confirm the production of a single amplicon with the expected molecular mass , 
 in addition , each product was sequenced ( seqlab , gttingen , germany ) to confirm the specificity of the pcr . 
 a negative control , where cdna was omitted , was included in every run . in negative controls , 
 protein concentrations were determined using micro bca protein assay kit ( pierce biotechnology , rockford , il ) . 
 equal amounts of protein ( 40  g of tissue extract or 8  g of cellular extract ) were resolved on 8% or 15% reducing sds - polyacrylamide gels and transferred onto polyvinylidene difluoride membranes ( millipore , billerica , ma ) . 
 membranes were blocked in 1x roti - block solution ( roth , karlsruhe , germany ) diluted in 50  mm tris - hcl , ph 7.6 , and 0.9% nacl for 60  min at rt and then incubated overnight at 4c with mouse monoclonal antibodies ( mabs ) to tgm2 ( clone tg100 ) ( thermo fisher scientific , fremont , ca ) diluted 1  :  4000 or 1  :  6000 for cellular extract or kidney tissue , respectively . 
 optionally , membranes were incubated overnight with rabbit anti - active caspase-3 abs ( abcam , cambridge , uk ) diluted 1  :  1000 in roti - block solution . to ensure equal protein loading , 
 membranes were blocked with 5% milk and incubated with mabs to gapdh ( novus biologicals , littleton , co ) , 1  :  20000 . 
 bound antibodies were visualized with horseradish peroxidase - conjugated secondary abs ( dako , glostrup , denmark ) , 1  :  5000 , using the chemiluminescent reagent lumi - light western blotting substrate ( roche , mannheim , germany ) . 
 densitometric analyses were performed using a digital gel documentation system ( biozym , hessisch oldendorf , germany ) . 
 all data of individual samples were divided by the values obtained for gapdh on the same blot . 
 the mean of the tgm2/gapdh or active caspase-3/gapdh ratio of the controls was set to 1 arbitrary unit and each individual value including control values was calculated accordingly . 
 histological sections were stained with acidic orcein 12 weeks after transplantation to visualize the internal and external elastic lamina of arteries and to evaluate a possible effect of cystamine on arterial remodelling . 
 at least 10 arteries of the muscular type were investigated per section . to estimate the relative thickness of arterial media and intima , the ratio of the total vessel diameter , including media and intima , and the diameter of the lumen of the artery were determined . additionally , the percentage of arteries exhibiting intimal hyperplasia was analyzed . 
 histological sections were stained with azocarmine / aniline blue ( azan ) 12 weeks after transplantation and tunica adventitia was measured . to estimate relative thickness of the tunica adventitia , the diameter of outer arterial diameter ( including tunica adventitia , media , intima , and lumen ) was determined and divided by diameter of inner arterial diameter ( including media , intima , and lumen ) . 
 sections of 6  m were dewaxed and rehydrated . to unmask tgm2 immunoreactivity in the fixed tissue , 
 antigen retrieval was performed in 0.01  m sodium citrate buffer ( ph 6.0 ) for 15  min at 120c in a steamer . sections were pretreated with protease xiv ( sigma - aldrich ) for 15  min at rt for detection of cd163 or active caspase-3 . 
 after washing in pbs , ph 7.2 , the paraffin sections were incubated for 30  min at rt with pbs supplemented with 1% bsa and 0.1% nan3 . 
 thereafter , mouse mabs to tgm2 diluted 1  :  1500 or ed2 ( anti - cd163 , serotec , oxford , uk ) abs diluted 1  :  200 or polyclonal rabbit abs to active caspase-3 ( abcam ) diluted 1  :  100 in pbs supplemented with 1% bsa ( serva ) were applied and incubated at 4c overnight . bound , primary tgm2 abs were detected using rabbit anti - mouse immunoglobulin and goat anti - rabbit hrp labeled abs ( dako ) and 3,3-diaminobenzidine ( dab ) ( sigma - aldrich ) . to amplify the signal for ed2 abs , additionally , anti - rabbit envision peroxidase system ( dako ) 
 was applied according to the manufacturer 's instructions . to detect abs bound to active caspase-3 , anti - rabbit envision peroxidase system ( dako ) was used . 
 sections were weakly stained with hemalum and coverslipped with pertex ( medite , burgdorf , germany ) . to detect tgm2-positive monocytes / macrophages , 
 sections were first stained with mabs to tgm2 as described , followed by protease xiv treatment for 15  min at rt and overnight incubation at 4c with mab ed1 directed to a cd68-like antigen ( serotec ) diluted 1  :  500 in pbs supplemented with 1% bsa and 0.1% nan3 . 
 to detect bound primary abs , rabbit anti - mouse immunoglobulin and alkaline phosphatase anti - alkaline phosphatase ( apaap ) ( both from dako ) were used in combination with fast blue ( sigma - aldrich ) as chromogen . 
 sections were evaluated with an olympus bx51 microscope and analysis software ( olympus , hamburg , germany ) . 
 data were analyzed , where applicable , by nonparametric kruskal - wallis test followed by the mann - whitney rank sum test using spss software ( spss software , munich , germany ) . 
 renal transplantation in the f344 to lewis rat strain combination results in accumulation of mononuclear leukocytes in allograft blood vessels [ 9 , 31 ] . to isolate these cells , 
 kidneys were intensively perfused on days 9 and 42 after transplantation . in agreement with the previously published data 
 , we observed a strong increase in the number of mononuclear leukocytes accumulating in the vasculature of allografts on day 9 compared to isografts . 
 furthermore , the number of intravascular leukocytes was reduced on day 42 ; however , it remained still increased compared to day 42 isografts . 
 the difference in cell number was also observed between perfusates from control kidneys and isografts on days 9 and 42 ( figure 1 ) . 
 the cellular composition of intravascular leukocytes obtained by perfusion on day 9 and day 42 was published before [ 9 , 31 ] . 
 the mrna expression of tgm2 was analyzed by real - time rt - pcr in mononuclear leukocytes isolated from renal blood vessels of control animals and isogenic and allogenic renal transplants . 
 real - time rt - pcr led to a single amplicon of the expected molecular mass in all samples analyzed . in negative controls , where cdna was omitted , 
 interestingly , an elevated level of tgm2 mrna was detected in cells collected from allografts on day 9 , as well as on day 42 after transplantation , when compared to the respective isografts ( figure 2(a ) ) . 
 tgm2 mrna expression was also increased in day 9 and day 42 allograft tissue compared to respective isografts . 
 in contrast to graft blood leukocytes , where the expression was stronger on day 9 compared to day 42 , tgm2 tissue expression did not differ between day 9 and day 42 ( figure 2(b ) ) . 
 protein expression of tgm2 was investigated by immunoblotting of lysates of mononuclear intravascular leukocytes isolated from control animals and isogenic or allogenic renal transplants ( figures 3(a ) and 4(a ) ) as well as of total tissue lysates ( figures 3(c ) and 4(c ) ) . 
 expression of tgm2 was detected in all groups investigated as a major band with expected molecular mass of ~70  kda . on day 42 
 densitometrical analyses revealed elevated levels of tgm2 expression in blood mononuclear leukocytes isolated from isografts on day 9 compared to cells from control kidneys . furthermore , an elevated expression level of tgm2 was detected in perfusates from allografts on day 9 compared to respective isograft samples ( figure 3(b ) ) . 
 in contrast to the results from real - time rt - pcr , no difference in the expression of tgm2 was detected in perfusates on day 42 after transplantation ( figure 4(b ) ) as well as in total kidney tissue on day 9 and day 42 after transplantation ( figures 3(d ) and 4(d ) ) . 
 so far , the expression of tgm2 was analyzed in the total intravascular population of mononuclear leukocytes . to examine tgm2 expression by graft monocytes , we performed immunohistochemical staining on paraffin sections from control kidneys , isografts , and allografts explanted on day 9 after transplantation ( figures 5(a)5(d ) ) . in line with previously published data describing accumulation of blood leukocytes , 
 tgm2-positive cells were most abundant in the vasculature of day 9 allografts ( figure 5(c ) ) . 
 in addition to intravascular leukocytes , blood plasma exhibited tgm2 immunoreactivity , predominantly in allografts . to identify monocytes expressing tgm2 , 
 double - staining experiments with mab ed1 detecting cd68-like antigen were performed ( figure 5(d ) ) . 
 in isograft tissue , tgm2 immunoreactivity was detected in the vascular endothelium of some arteries , veins , and capillaries . in the media of arteries , 
 smooth muscle cells were immunopositive in the adventitia extracellular material and cells with a fibroblast - like shape . in the renal parenchyma , renal tubules , most probably proximal tubules , 
 collecting ducts , and the outer layer of bowman 's capsule were stained with antibodies to tgm2 . 
 the staining was identical to the pattern seen in isografts but it was more intense all over . in the leukocytic infiltrate , no conspicuous additional staining was detected ( data not shown ) . 
 tgm2 was described as a marker for m2 macrophages ; we investigated if graft monocytes also express the classical m2 cell surface marker cd163 . to detect cd163-positive cells , immunohistochemical staining with mab ed2 
 as expected , we detected a very low number of positive cells in isografts ( figure 5(e ) ) . 
 in contrast , cd163-positive cells were more frequent in allografts ( figure 5(f ) ) . in both groups analyzed , no cd163-positive cells were detected in the lumina of blood vessels . 
 therefore , we analyzed if caspase-3 , a key protease directly involved in apoptosis , is also expressed by intravascular graft leukocytes . to examine activation of caspase-3 during reversible acute rejection , detection of active caspase-3 in renal mononuclear graft leukocytes as well as in graft tissue on days 9 and 42 after transplantation was performed by immunoblotting ( figures 6(a ) , 6(c ) , 7(a ) , and 7(c ) ) . 
 the appearance of the active p19 ( ~19  kda ) and p17 ( ~17  kda ) fragments of caspase-3 was detected . 
 densitometric analyses revealed increased levels of active caspase-3 in mononuclear blood leukocytes from allografts on day 9 ( figure 6(b ) ) . 
 active caspase-3 was also detected in perfusates from day 42 isografts and allografts , but no difference was observed among them ( figures 7(a ) and 7(b ) ) . also , in allograft tissue , elevated levels of active caspase-3 were measured on days 9 and 42 compared to respective isografts ( figures 6(c ) and 7(c ) ) . 
 furthermore , these results were corroborated by immunohistochemical staining of graft tissue on day 9 after transplantation . 
 active caspase-3-positive cells were detected in the graft blood vessels predominantly in allografts veins , whereas caspase-3 immunoreactivity was less prominent in leukocytes accumulating in arteries ( figure 6(d ) ) . to examine the role of tgm2 in the pathogenesis of cai , renal allograft recipients were treated with cystamine , a general inhibitor of tgm , or placebo . 
 creatinine clearance was monitored 4 , 8 , and 12 weeks after transplantation to determine renal function . 
 no differences were observed between placebo- ( 3.4  1.5 at 4 weeks ; 5.1  0.7 at 8 weeks ; 3.0  0.7 at 12 weeks ; n = 6 per group ; data are given as mean  standard deviation , ml / min ) and cystamine - treated animals ( 3.9  1.2 at 4 weeks ; 4.4  0.6 at 8 weeks ; 3.5  0.5 at 12 weeks ; n = 6 per group ; data are given as mean  standard deviation , ml / min ) independent of the time point investigated . at the end of the study , at 12 weeks after transplantation , animals were sacrificed and relative thickness of arteries was quantified on sections stained with acidic orcein . 
 no significant difference was observed between both experimental groups ( figures 8(a ) , 8(c ) , and 8(d ) ) . 
 furthermore , arteries with intimal hyperplasia were detected in the sham - treated group , whereas cystamine treatment had no effect on intimal remodeling ( figures 8(b ) , 8(c ) , and 8(d ) ) . 
 additionally , histological sections were stained with azocarmine / aniline blue ( azan ) 12 weeks after transplantation to detect interstitial fibrosis . 
 we demonstrate in this study that tgm2 mrna and protein are overexpressed by mononuclear leukocytes , predominantly by activated monocytes , which accumulate in the vascular bed of rat renal allografts ( f344 to lewis ) on day 9 after transplantation . 
 after resolution of acute rejection on day 42 , tgm2 mrna levels are lower but still moderately increased compared to respective isografts , which is not reflected in increased protein levels . 
 tgm2 mrna expression was also induced in graft tissue upon allogenic transplantation . on the protein level , however , no quantitative changes were detected . 
 we focused on the well - characterized population of mononuclear leukocytes , which accumulate in large numbers in the blood vessels of experimental allografts during acute rejection . 
 the number of leukocytes , which were isolated from control kidneys , isografts , and allografts 9 and 42 days after transplantation by intensively perfusing renal blood vessels , nicely corroborated data published previously [ 9 , 31 ] . 
 the acute rejection episode , which peaks at around day 9 after transplantation , is of major interest for the understanding of cai , since clinical and experimental data suggest that acute rejection is an important triggering factor [ 15 ] . as cav , a hallmark of cai , 
 is characterized by arterial intimal hyperplasia , leukocytes interacting with allogenic endothelial cells were suggested to play a pivotal pathogenic role . 
 the number of intravascular leukocytes decreases after reversion of acute rejection in the f344 to lewis rat strain combination but remains elevated for several weeks . 
 first hints regarding the overexpression of tgm2 by mononuclear leukocytes isolated from the blood vessels of allografts on day 9 after transplantation came from gene array data . 
 these data were confirmed by real - time rt - pcr and immunoblotting using antibodies , which detected a major band of the expected molecular mass . 
 immunohistochemistry with the same antibodies revealed that intravascular leukocytes in renal allografts were strongly tgm2-immunoreactive . on tissue sections 
 double - stained with mab ed1 directed to a cd-68-like antigen , an established marker for rat monocytes / macrophages , we identified graft monocytes as strongly tgm2-immunopositive . 
 it might be argued that the increase in tgm2 expression in mononuclear blood leukocytes from day 9 allografts is due to the increase in the proportion of monocytes , which is indeed seen . 
 however , isograft blood leukocytes contain about 50% monocytes and allografts about 70% , whereas tgm2 mrna expression levels increase by about 10-fold , suggesting that the increased proportion of monocytes is not the only explanation for the observed increase in tgm2 mrna levels . 
 interactions of monocytes with graft endothelial cells , which is a prerequisite for intravascular accumulations , may induce tgm2 expression as described previously for monocytes adhering to endothelial cells in vitro . 
 in addition , proinflammatory cytokines produced during acute rejection   are known inducers of tgm2 expression [ 14 , 15 , 33 , 34 ] . in graft tissue , which was investigated for comparison , mrna levels were elevated on both days 9 and 42 . 
 tgm2 protein was detected by immunoblotting but , in contrast to tgm2 mrna , no significant increase in protein signals was seen . however , some weak additional bands of lower molecular mass that were only detected in allograft perfusates and tissue would be compatible with an accelerated degradation of tgm2 in allografts . 
 another explanation for the discrepancy between mrna and protein is based on reports in which tgm2 acts as a substrate of itself and forms insoluble cross - linked complexes , which are not detected in conventional immunoblots . for the detection of tgm2 
 covalently bound to extracellular matrix , unfixed cryostat sections should be stained in future studies . in line with this idea and with our mrna data , tissue sections of renal day 
 the renal expression pattern of tgm2 was in line with other publications on rodent and human tgm2 expression . 
 recently , shrestha et al . reported upregulation of tgm2 mrna , protein , and transglutaminase activity in cai kidneys . 
 their model also is based on the f344 to lewis rat strain combination but , in contrast to our model , includes a 10-day course of suboptimal immunosuppression , which interferes with early acute rejection , followed by delayed contralateral nephrectomy . 
 our study and the study by these authors complement each other , as they predominantly investigate end - stage cai , whereas we focus on the early phase posttransplantation . 
 evidence increased mrna levels of tgm2 as well as increased tgm2 immunopositivity in allograft tissue sections compared to isografts . 
 of note , these authors also demonstrate that , in addition to blood leukocytes investigated only in our study , renal tubules and glomeruli also overexpress tgm2 in allografts . during the pathogenesis of cai , urinary secretion of the transglutaminase cross - link product (-glutamyl)-lysine is increased . in end - stage cai , this product is detected more abundantly in allografts compared to isografts . from both studies , however , it is difficult to conclude if tgm2 is a marker of cai reflecting changes in the cellular composition of the graft or actively contributes to the pathogenesis of cai as shown for diverse models of experimental renal fibrosis [ 3740 ] . 
 tgm2 was recently described to be a reliable marker of m2 macrophages , an anti - inflammatory subpopulation of differentiated macrophages . to the best of our knowledge , 
 tgm2 expression by blood monocytes in vivo has not been described before , although , similarly to macrophages , blood monocytes can be activated and express cytokine patterns and cell surface molecules resembling proinflammatory m1 macrophages and anti - inflammatory m2 macrophages . 
 we previously demonstrated that blood monocytes of f344 to lewis renal allografts are activated , acquire an intermediate state of differentiation , and express both cytokines , typical for m1 and for m2 macrophages , during acute rejection . 
 we do not know , however , if this population is a mixture of m1- and m2-like monocytes or if individual monocytes are characterized by an intermediate phenotype . to further characterize these monocytes 
 , we investigated expression of cd163 , a typical m2 cell surface marker , which was not detected on graft monocytes during the peak of acute rejection . 
 hence , we conclude that tgm2 can be expressed by activated monocytes but we can not decide if , similarly to macrophages , tgm2 expression is confined to m2-like monocytes . 
 the functional relevance of tgm2 expression by m2 macrophages , however , is still elusive and similarly its role in monocytes is unclear . 
 we described before that monocytes isolated from the blood vessels of renal allografts during fatal acute rejection undergo apoptosis when cultivated ex vivo . 
 as tgm2 is more strongly expressed in apoptotic cells [ 11 , 13 , 44 ] , we investigated if levels of activated caspase-3 correlate with expression of tgm2 in perfusate cells . 
 indeed , a stronger induction of activated caspase-3 was seen in leukocytes isolated from day 9 allografts compared to respective isografts . 
 this suggests that tgm2 expression by blood mononuclear cells might be linked to apoptosis . in line with the idea that tgm2 enhances apoptosis depending on a prolonged contact with endothelial cells in the inflamed allograft , activation of caspase-3 was more visible in veins compared to arteries . 
 tgm2 plays a dual role in apoptosis : expression of tgm2 can induce apoptosis but it also stabilizes the cytoskeleton of dying cells , which prevents cell rupture and release of proinflammatory cytoplasmic components [ 13 , 4446 ] . as a large number of monocytes accumulate in allograft vessels , their apoptosis might be a tremendous anti - inflammatory stimulus capable of reverting rejection . 
 this issue certainly deserves further investigation . in the tissue of day 9 and day 42 allografts , 
 immunohistochemistry , however , evidenced that tgm2 not only is present in the cytoplasm of graft blood monocytes but was also detected in the surrounding blood plasma . 
 due to rejection - associated vascular damage and the high number of leukocytes accumulating in the blood vessels of allografts , we assume that blood flow slowed down considerably in allografts and that endothelial cells and underlying extracellular matrix are in intensive and prolonged contact with products secreted locally . in the context of hypertension , tgm2 and other active transglutaminases were shown to contribute to the pathogenesis of vascular remodeling [ 4750 ] . 
 active transglutaminases were suggested to cross - link extracellular matrix proteins and to result in an increased rigidity of vascular walls , which precedes inward remodeling of arteries [ 4750 ] . in line with this idea , patients suffering from celiac disease , which typically form autoantibodies to tgm2 , are less likely to have a diagnosis of hypertension . it is not known if tgm2 plays a similar role in the pathogenesis of cav . 
 we performed animal experiments to investigate this hypothesis and chronically applied biologically meaningful concentrations of cystamine , an inhibitor of transglutaminase activity , for 4 weeks after transplantation . 
 however , our hypothesis was not supported . within 84 days after surgery , allografts developed first hallmarks of cai , such as ifta , cav , and renal dysfunction , irrespective of cystamine treatment . 
 we can , however , not exclude that extracellular transglutaminase activity is involved in both remodeling and re - remodeling of graft arteries as a protective effect of tgm2 was reported for experimental atherosclerosis [ 23 , 53 , 54 ] . 
 the experimental rat model is characterized by a minor mismatch in the mhc class-1 locus and does not include immunosuppression , which is not typical for clinical transplantation . in spite of these disadvantages , it is a favorable , experimental model to investigate an acute rejection episode preceding cai . 
 we investigate neither transglutaminase activity in the graft nor excretion of its products , and animal experiments were performed using a single dose of transglutaminase inhibitor , which was applied for just one month . 
 we ignore if treatment with other concentrations of cystamine or later time - points would lead to allograft protection . to evaluate the effect of cystamine , we focus on renal function and graft remodeling within 84 days after transplantation . 
 we do not know if , after longer periods of time , differences would have developed among the experimental groups . 
 in addition , cystamine is a weak and rather unspecific inhibitor of tgm2 as it also inhibits other members of the transglutaminase family as well as certain proteases . 
 more specific inhibitors for tgm2 are commercially available . finally , apart from its role in apoptosis , there is still a plethora of potential functions of tgm2 [ 11 , 15 ] , some of which are enumerated in the introduction , that are awaiting investigation in the context of acute and chronic allograft rejection . 
 only a part of these functions are inhibited by exogenous cystamine . in conclusion , this is the first study to demonstrate expression of tgm2 by monocytes activated in vivo during a reversible acute rejection episode . 
 potentially , the function of monocytic tgm2 is induction of apoptosis , which in turn might contribute to the reversion of acute rejection in this experimental model . 
 our data do not support but also do not falsify the hypothesis that tgm2 expressed by graft monocytes during reversible acute rejection contributes to an early induction of cai .","acute rejection is a major risk factor for chronic allograft injury ( cai ) . 
 blood leukocytes interacting with allograft endothelial cells during acute rejection were suggested to contribute to the still enigmatic pathogenesis of cai . 
 we hypothesize that tissue transglutaminase ( tgm2 ) , a multifunctional protein and established marker of m2 macrophages , is involved in acute and chronic graft rejection . 
 we focus on leukocytes accumulating in blood vessels of rat renal allografts ( fischer-344 to lewis ) , an established model for reversible acute rejection and cai . 
 monocytes in graft blood vessels overexpress tgm2 when acute rejection peaks on day 9 after transplantation . 
 concomitantly , caspase-3 is activated , suggesting that tgm2 expression is linked to apoptosis . 
 after resolution of acute rejection on day 42 , leukocytic tgm2 levels are lower and activated caspase-3 does not differ among isografts and allografts . 
 cystamine was applied for 4 weeks after transplantation to inhibit extracellular transglutaminase activity , which did , however , not reduce cai in the long run . in conclusion 
 , this is the first report on tgm2 expression by monocytes in vivo . 
 tgm2 may be involved in leukocytic apoptosis and thus in reversion of acute rejection . however 
 , our data do not support a role of extracellular transglutaminase activity as a factor triggering cai during self - limiting acute rejection .",pubmed
"endothelial cells ( huvec passage 46 ; clonetics , walkersville , md ) were grown to confluence , then stimulated with plasminogen activation inhibitor-1 ( pai-1 , 10 ng / ml ; american diagnostica inc . , 
 supernatant was collected 3 hours after stimulation and centrifuged ( 145 g , 8 minutes ) to remove cell debris . 
 the supernatant was subsequently ultracentrifuged ( 100,000 g , 6 minutes , 4 degrees ) and pelleted . 
 emps were re - suspended in phosphate buffered saline ( pbs ) and stored at 80c . 
 flow cytometric analysis was used to characterize and quantify emps based on cd31- and annexin v - positivity occurring in a size gate less than 1 m . 
 this size gate was set on forward versus side scatter using latex standard beads measuring 0.84 m ( spherotech fp-0856 - 2 ) . 
 enumeration was completed against a known concentration of 7.6 m polystyrene beads placed within the sample ( spherotech pps-4 ) . 
 the flow rate used for analysis was 12 l / min ( figs . 1 and 2 ) . 
 7.6 m latex beads are shown in gate p1 and allowed for enumeration of emps . within gate 
 p2 , events positive for annexin v ( p3 ) and cd31 ( p4 ) were enumerated against p1 to quantify emp levels . 
 the same settings were used to quantify emps in perfusate samples . as a separate and confirmatory measure , transmission electron microscopy ( tem ) 
 . a 25-l drop of emp in solution was pipetted onto a formvar plastic coated grid ( ted pella , inc . ) and allowed to air dry for 1 hour . 
 the microparticle grid was examined and photographed by tem with a jeol 100 cx microscope ( fig . 
 3 ) . emp size distribution as measured by tem ( 10,000magnification ) accompanied by a representative micrograph . 
 emps ( 30010 ) were introduced to an iv bag ( model patient ) containing 300 ml of 0.9 ns ( model patient ) and manually agitated for 3 minutes . 
 the iv bag was connected to a primed cvvh circuit containing 170 ml of 0.9 ns and mixed ( ~65010 emp / ml final mixed perfusate concentration ) as shown in fig . 
 , the perfusate was circulated through the cvvh filter ( nxstage purema , 200 m pore size , 35 m wall thickness , rate 250 ml / hour ) for a period of 70 minutes . 
 0.5 ml aliquots were removed for flow cytometric analysis at 510 minute intervals for 70 minutes of dialysis . 
 emp levels were recorded and expressed as a fraction of starting concentration after mixing occurred . 
 three runs were completed and the fraction of emps versus time was plotted with standard error of the mean ( sem ) . at the end of 4 hours filtration , 
 . a 300 ml iv bag of 0.9 ns served as the model patient and was spiked with emps . 
 the circuit contained 170 ml 0.9 ns and ran counter - current against 1 l of 0.9 ns dialysate . after mixing , 
 0.5 ml aliquots were drawn from the  patient  for analysis as dialysis was completed via the filter . 
 endothelial cells ( huvec passage 46 ; clonetics , walkersville , md ) were grown to confluence , then stimulated with plasminogen activation inhibitor-1 ( pai-1 , 10 ng / ml ; american diagnostica inc . , 
 supernatant was collected 3 hours after stimulation and centrifuged ( 145 g , 8 minutes ) to remove cell debris . 
 the supernatant was subsequently ultracentrifuged ( 100,000 g , 6 minutes , 4 degrees ) and pelleted . 
 emps were re - suspended in phosphate buffered saline ( pbs ) and stored at 80c . 
 flow cytometric analysis was used to characterize and quantify emps based on cd31- and annexin v - positivity occurring in a size gate less than 1 m . 
 this size gate was set on forward versus side scatter using latex standard beads measuring 0.84 m ( spherotech fp-0856 - 2 ) . 
 enumeration was completed against a known concentration of 7.6 m polystyrene beads placed within the sample ( spherotech pps-4 ) . 
 the flow rate used for analysis was 12 l / min ( figs . 1 and 2 ) . 
 7.6 m latex beads are shown in gate p1 and allowed for enumeration of emps . within gate 
 p2 , events positive for annexin v ( p3 ) and cd31 ( p4 ) were enumerated against p1 to quantify emp levels . 
 the same settings were used to quantify emps in perfusate samples . as a separate and confirmatory measure , transmission electron microscopy ( tem ) 
 . a 25-l drop of emp in solution was pipetted onto a formvar plastic coated grid ( ted pella , inc . ) and allowed to air dry for 1 hour . 
 the microparticle grid was examined and photographed by tem with a jeol 100 cx microscope ( fig . 
 3 ) . emp size distribution as measured by tem ( 10,000magnification ) accompanied by a representative micrograph . 
 emps ( 30010 ) were introduced to an iv bag ( model patient ) containing 300 ml of 0.9 ns ( model patient ) and manually agitated for 3 minutes . 
 the iv bag was connected to a primed cvvh circuit containing 170 ml of 0.9 ns and mixed ( ~65010 emp / ml final mixed perfusate concentration ) as shown in fig . 
 , the perfusate was circulated through the cvvh filter ( nxstage purema , 200 m pore size , 35 m wall thickness , rate 250 ml / hour ) for a period of 70 minutes . 
 0.5 ml aliquots were removed for flow cytometric analysis at 510 minute intervals for 70 minutes of dialysis . 
 emp levels were recorded and expressed as a fraction of starting concentration after mixing occurred . 
 three runs were completed and the fraction of emps versus time was plotted with standard error of the mean ( sem ) . at the end of 4 hours filtration , 
 a 300 ml iv bag of 0.9 ns served as the model patient and was spiked with emps . 
 the circuit contained 170 ml 0.9 ns and ran counter - current against 1 l of 0.9 ns dialysate . after mixing , 
 0.5 ml aliquots were drawn from the  patient  for analysis as dialysis was completed via the filter . 
 the decline in emp levels due to dialysis is rapid with 50% of clearance being seen in the first 30 minutes . within 40 minutes , 
 the circulating emp levels were reduced to 29% of starting concentration and this level stayed relatively constant over the duration of the study . to determine the fate of the cleared emps , we measured emp levels in the dialysate . 
 the dialysate contained 5.7% of the original emp concentration ( ~65010 emp / ml ) at the end of 4 hours of uninterrupted dialysis . 
 emps levels expressed as a fraction of initial concentration by time and averaged over 3 experiments . 
 the data presented here demonstrate that counter - current dialysis using a standard clinical cvvh filter and under clinical conditions removes emps from a modelled patient 's circulation . 
 given our previous evidence of emp - induced ali , these findings support the idea of cvvh as a meaningful therapeutic intervention in ali due to clearance of emps from the patient circulation . 
 the experimental conditions achieve the same plasma level of emps as seen in human clinical disease ( 110 per ml ) . 
 while this work demonstrates that a standard 200-m cvvh circuit and filter entraps emps , there are significant limitations to the model presented . 
 the observed emp clearance is likely a saturable phenomenon , which would explain the relatively constant emp concentration from 40 to 70 minutes and low dialysate emp levels . 
 the relative volume of human circulation ( ~5 l ) to cvvh circuit ( ~200 ml ) is much higher in the clinical condition as compared to our model ( 300 and 170 ml , respectively ) . while this would not affect a steady state of dialysis , a saturable filter emp - binding 
 additionally , emps may continue to be generated in a patient and a non - saturable clearance method would be required to achieve a lower steady state emp concentration . 
 convection and pressure gradient forces were not modelled , as these interventions require replacement fluid and this would confound emp measurement . 
 however , if emp charge and size are amenable , forced hindered diffusion via the application of convective and pressure forces coupled with replacement by emp - free fluid would likely sustain clearance . in conjunction with particle trapping by the filter 
 , the first order kinetics of emp clearance could be prolonged at a minimum . additionally , emp interactions in vivo with plasma oncotic elements ( circulating cells , albumin , etc . ) 
 we do not feel that such elements have a major impact upon the steady state of emps in vivo , since characterized emps in human disease states using flow cytometry have identified the 0.510 per ml threshold injury value using flow cytometry of submicron events occurring in platelet - free plasma ( 54 ) . 
 thus , the absence of these elements in our model is representative of the steady state of unbound plasma emps present in human disease . 
 the low dialysate emp level and plateauing circulation emp level beyond 40 minutes suggest that emp sequestration occurs within the cvvh filter or tubing and that this is a saturable phenomenon . 
 frequent changing of the cvvh filter is a logical step that may overcome this limitation . 
 additionally , more efficient and selective emps clearance can be realized by conjugating endothelium - specific antibodies to commercially available specialty cvvh filters ( aethlon adapt ) . 
 this approach would provide more selective binding sites and higher affinity for circulating emps . by combining a selective filter with convection and pressure gradients , emp clearance could likely be substantially prolonged . 
 clinical data generally support the protective role of cvvh in ali management . a clinical randomized trial and several animal trials 
 demonstrate that the achievement of negative fluid balance is associated with improved outcomes from ali ( 6265)(67,68 ) . 
 in addition to its salutary effects on emp levels , early cvvh may prevent or minimize volume overload that is commonly observed with the resuscitation phase of critical illness , particularly in persons suffering concomitant acute kidney injury ( aki ) . 
 thus , cvvh could be used as an effective tool targeting both the molecular machinery and iatrogenic components of ali . as a translatable 
 next step , we plan to assay patients pre- and post - dialysis to observe emp concentration effects . 
 ultimately , the application of dialysis to rodent models of emp - induced ali would examine efficacy in on - going diseases and emp thresholds associated with worse injuries . 
 while the utility of ultrafiltration can be tested in an animal model , diffusion appears to be enough to remove emps due to their small size : standard dialysis alone was effective in this in vitro model . 
 the authors have not received any funding or benefits from industry or elsewhere to conduct this study .","backgroundendothelium - derived microparticles ( emps ) are submicron vesicles released from the plasma membrane of endothelial cells in response to injury , apoptosis or activation . 
 we have previously demonstrated emp - induced acute lung injury ( ali ) in animal models and endothelial barrier dysfunction in vitro . 
 current treatment options for ali are limited and consist of supportive therapies . 
 we hypothesize that standard clinical continuous venovenous hemofiltration ( cvvh ) reduces serum emp levels and may be adapted as a potential therapeutic intervention.materials and methodsemps were generated from plasminogen activation inhibitor-1 ( pai-1)-stimulated human umbilical vein endothelial cells ( huvecs ) . 
 flow cytometric analysis was used to characterize emps as cd31- and annexin v - positive events in a submicron size gate . 
 enumeration was completed against a known concentration of latex beads . 
 ultimately , a concentration of ~650,000 emp / ml perfusate fluid ( total 470 ml ) was circulated through a standard cvvh filter ( pore size 200 m , flow rate 250 ml / hr ) for a period of 70 minutes . 
 0.5 ml aliquots were removed at 5- to 10-minute intervals for flow cytometric analysis . 
 emp concentration in the dialysate was measured at the end of 4 hours to better understand the fate of emps.resultsa progressive decrease in circulating emp concentration was noted using standard cvvh at 250 ml / hr ( a clinical standard rate ) from a 470 ml volume modelling a patient 's circulation . 
 a 50% reduction was noted within the first 30 minutes . 
 emps entering the dialysate after 4 hours were 5.7% of the emp original concentration.conclusionthese data demonstrate that standard cvvh can remove emps from circulation in a circuit modelling a patient . 
 an animal model of hemofiltration with induction of emp release is required to test the therapeutic potential of this finding and potential of application in early treatment of ali .",pubmed
"patients with frequent angina ( 1 episode per week ) report greater physical limitations and decreased quality of life compared with patients with minimal angina.1 the goals of chronic stable angina management are not only to control symptoms , but also to reduce the risk of adverse clinical outcomes.26 as symptom control impacts the patient s quality of life , maximum consideration should be given to the array of available management options that aim to reduce angina symptoms . for many patients 
 , this would include a physical activity program aimed at improving exercise tolerance in accordance with current secondary prevention guidelines . 
 several studies have shown that optimal medical therapy , along with aggressive risk factor modification , is as effective as revascularization in reducing the risk of adverse coronary events.79 optimal medical therapy includes anti - anginal or anti - ischemic as well as disease- modifying therapy.8,10 the anti - ischemic agents include short- and long - acting nitrates , beta - blockers , and calcium channel blockers.3,4,11 these agents reduce angina symptoms and prolong exercise duration and/ or time to st - segment depression.3,11 frequently , a combination of drugs is necessary for optimal symptom control.3 nitrates work primarily by venodilation and afford rapid relief of angina.12 a number of placebo - controlled studies have shown that the sublingual application of nitroglycerin increases exercise duration in patients with stable angina.1317 published data also suggest that residual and recurrent angina is frequently experienced by patients who have already undergone either a percutaneous or surgical revascularization procedure , and these patients require continued medical therapy with multiple anti - anginal agents , including short- and long - acting nitrates , for control of their symptoms.8,18,19 increased physical exercise is also important in patients with stable angina to reduce the risk of cardiovascular events , and current recommendations call for the use of sublingual nitroglycerin as part of optimal medical therapy.5,20 this randomized , double - blind , placebo - controlled , crossover , dose - ranging multicenter study in patients with chronic stable angina evaluated the effect of a sublingual nitroglycerin spray formulation on time to onset of angina during exercise , total exercise duration , and time to exercise - induced myocardial ischemia , as evidenced by electrocardiographic st - segment depression . 
 brief results were previously published in german.21 the goal of the present article is to more fully describe the study methodology , patient characteristics , and detailed results in the context of current optimal medical therapy . 
 this study was carried out at four centers in the united states and conducted in accordance with the international conference on harmonisation guidelines for good clinical practice and food and drug administration guidelines . 
 all patients provided written informed consent at the screening visit , prior to starting any study - related procedures . 
 adults ( 18 years of age ) with coronary artery disease as documented by a 50% reduction in arterial lumen diameter in 1 or more major coronary arteries or their primary branches , a previously documented myocardial infarction , or reversible perfusion defect on stress thallium-201 scintigraphy and a diagnosis of chronic stable exertional angina that had been made at least 2 months prior to screening were eligible to be screened . in the 2 months 
 prior to study entry , patients were required to have been on a stable medical treatment regimen for their angina . 
 patients with unstable angina , myocardial infarction , coronary angioplasty , or cardiac surgery within 3 months of screening were excluded . 
 additional exclusion criteria included a history of congestive heart failure ( nyha iii or iv ) , significant disease of the cardiac conduction system , uncontrolled hypertension or hypotension , any physical conditions known to affect st - segment morphology , or severe organ damage or disease of any system . 
 patients were not permitted to receive concomitant therapy with long - acting nitrates or digitalis preparations during the study . 
 in addition , participation in an investigational drug study within 1 month prior to the screening visit or while participating in the study was prohibited . 
 patients with clinically significant deviations from normal in either the physical exam or lab parameters and patients with peripheral arterial disease that limited their exercise capacity were also excluded . according to the approved protocol , each center was to enroll a minimum of 12 patients to ensure evaluable outcomes for 40 patients at the end of study . following an initial screening visit , eligible patients returned for a baseline exercise tolerance test ( ettb ) according to a standard bruce protocol.22 of note 
 , exercise testing is associated with minimal complications ; death and myocardial infarction occur at a rate of 1 per 2,500 tests performed.15,23 patients who developed angina within 39 minutes of commencing exercise were eligible to continue in the study . following a 90-minute rest period , these patients were assessed for nitrate response . 
 patients who were able to exercise for 60 seconds longer in this ett ( ettres ) than in the ettb were eligible to continue in the study . at their next visit , patients completed a confirmatory ett ( ettconf ) to establish reproducibility ( within 20% ) of the time to development of moderate angina when compared to the ettb . in order to reduce baseline variability , 
 study - related sublingual nitroglycerin was discontinued 6 hours prior to testing , while other anti - anginal medications were discontinued approximately 24 hours prior to each visit . after baseline and confirmatory etts 
 the goal of this crossover study design with each patient serving as his / her own control was to minimize the impact of any training effect on the mean values for each treatment dose . 
 the crossover period consisted of 5 additional visits within 14 days , at which patients were administered placebo or sublingual nitroglycerin spray ( 0.2 mg , 0.4 mg , 0.8 mg , or 1.6 mg ; nitrolingual pumpspray , g. pohl - boskamp gmbh & co. kg , germany ) . in order to minimize the impact of any training effect , patients completed a control ett ( ettc ) and an investigational ett ( ettinv ) at each visit . 
 ettc studies were carried out at approximately the same time of day as the introductory ett studies ( ettb and ettconf ) . following a 90-minute rest period , 
 primary efficacy endpoints were time to onset of angina symptoms and time to development of moderate angina ( defined as the level of activity at which the patient would normally stop activity ) . 
 the secondary endpoint was time to 1-mm st - segment depression ( myocardial ischemia ) . to minimize the impact of any training effect , efficacy endpoints are described as the mean difference between ettinv and ettc at each visit . 
 the difference between the confirmation and baseline etts was then subtracted from this value to further reduce the impact of any training effect . 
 safety parameters included evaluation of all changes in physical examination , vital signs , ecg , and laboratory evaluations . 
 this study was carried out at four centers in the united states and conducted in accordance with the international conference on harmonisation guidelines for good clinical practice and food and drug administration guidelines . 
 all patients provided written informed consent at the screening visit , prior to starting any study - related procedures . 
 adults ( 18 years of age ) with coronary artery disease as documented by a 50% reduction in arterial lumen diameter in 1 or more major coronary arteries or their primary branches , a previously documented myocardial infarction , or reversible perfusion defect on stress thallium-201 scintigraphy and a diagnosis of chronic stable exertional angina that had been made at least 2 months prior to screening were eligible to be screened . in the 2 months 
 prior to study entry , patients were required to have been on a stable medical treatment regimen for their angina . 
 patients with unstable angina , myocardial infarction , coronary angioplasty , or cardiac surgery within 3 months of screening were excluded . 
 additional exclusion criteria included a history of congestive heart failure ( nyha iii or iv ) , significant disease of the cardiac conduction system , uncontrolled hypertension or hypotension , any physical conditions known to affect st - segment morphology , or severe organ damage or disease of any system . 
 patients were not permitted to receive concomitant therapy with long - acting nitrates or digitalis preparations during the study . 
 in addition , participation in an investigational drug study within 1 month prior to the screening visit or while participating in the study was prohibited . 
 patients with clinically significant deviations from normal in either the physical exam or lab parameters and patients with peripheral arterial disease that limited their exercise capacity were also excluded . 
 according to the approved protocol , each center was to enroll a minimum of 12 patients to ensure evaluable outcomes for 40 patients at the end of study . following an initial screening visit , 
 eligible patients returned for a baseline exercise tolerance test ( ettb ) according to a standard bruce protocol.22 of note , exercise testing is associated with minimal complications ; death and myocardial infarction occur at a rate of 1 per 2,500 tests performed.15,23 patients who developed angina within 39 minutes of commencing exercise were eligible to continue in the study . following a 90-minute rest period , these patients were assessed for nitrate response . 
 patients who were able to exercise for 60 seconds longer in this ett ( ettres ) than in the ettb were eligible to continue in the study . at their next visit 
 , patients completed a confirmatory ett ( ettconf ) to establish reproducibility ( within 20% ) of the time to development of moderate angina when compared to the ettb . in order to reduce baseline variability , 
 study - related sublingual nitroglycerin was discontinued 6 hours prior to testing , while other anti - anginal medications were discontinued approximately 24 hours prior to each visit . after baseline and confirmatory etts 
 the goal of this crossover study design with each patient serving as his / her own control was to minimize the impact of any training effect on the mean values for each treatment dose . 
 the crossover period consisted of 5 additional visits within 14 days , at which patients were administered placebo or sublingual nitroglycerin spray ( 0.2 mg , 0.4 mg , 0.8 mg , or 1.6 mg ; nitrolingual pumpspray , g. pohl - boskamp gmbh & co. kg , germany ) . in order to minimize the impact of any training effect , patients completed a control ett ( ettc ) and an investigational ett ( ettinv ) at each visit . 
 ettc studies were carried out at approximately the same time of day as the introductory ett studies ( ettb and ettconf ) . following a 90-minute rest period , 
 primary efficacy endpoints were time to onset of angina symptoms and time to development of moderate angina ( defined as the level of activity at which the patient would normally stop activity ) . 
 the secondary endpoint was time to 1-mm st - segment depression ( myocardial ischemia ) . to minimize the impact of any training effect , efficacy endpoints are described as the mean difference between ettinv and ettc at each visit . 
 the difference between the confirmation and baseline etts was then subtracted from this value to further reduce the impact of any training effect . 
 safety parameters included evaluation of all changes in physical examination , vital signs , ecg , and laboratory evaluations . 
 of the 61 patients assessed for eligibility , 10 patients did not meet the protocol criteria and were therefore excluded . 
 a total of 51 patients performed the baseline exercise test and qualified for continuation . following a 90-minute rest period 
 , patients underwent a subsequent stress test after receiving 0.4 mg of open - label sublingual nitroglycerin spray . 
 all 51 patients showed an improvement in exercise duration of 60 seconds or greater ( range 1.024.7 minutes ) . 
 a total of 51 patients between the ages of 34 and 77 years continued in the double - blind phase and were randomized to 1 of 5 treatment sequences . 
 two patients did not complete the study ( 1 patient was lost to follow - up and 1 patient discontinued prematurely due to ventricular tachycardia / fibrillation requiring cardioversion after the control exercise test at visit 5 ; this event was not considered to be treatment related ) . 
 overall patient characteristics including angina and cardiovascular event history , along with concomitant medications , are described in table 1 . a dose - dependent increase in time to onset of moderate angina 
 the time to onset of angina was significantly higher in all treatment groups ( 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg ) than in the placebo group ( table 2 , fig . 
 the increase in time to onset of angina and to onset of moderate angina was most pronounced in the 1.6-mg group , with dose linearity between 0.4- and 1.6-mg doses of nitroglycerin spray . 
 the occurrence of a minimum 1.0-mm st - segment depression was later in all treatment groups ( 0.2 mg , 0.4 mg , 0.8 mg , 1.6 mg ) compared with the placebo group ( table 2 , fig . 
 of 51 patients , 12 ( 24% ) reported adverse events at the qualification visit ( open - label sublingual nitroglycerin response test ) . 
 adverse events considered related to treatment were reported for 2 ( 4% ) , 4 ( 8% ) , 8 ( 16% ) , 9 ( 18% ) , and 13 ( 26% ) patients receiving placebo and 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray , respectively . 
 the most frequently reported adverse events of this nature were headache and dizziness , all of which were mild to moderate in severity . 
 the incidence of treatment - related adverse events was dose dependent , with the incidence of headache increasing from 0 with placebo to 3 ( 6% ) , 5 ( 10% ) , 6 ( 12% ) , and 8 ( 16% ) after 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray , respectively . 
 no serious adverse events related to treatment were reported . per protocol , patients with systolic blood pressure ( sbp ) less than 100 mmhg were excluded from the study . 
 symptomatic hypotension was not reported as an adverse event , however sbp decreased to a level of 100 mmhg or less in 1 ( 2% ) , 0 ( 0% ) , 1 ( 2% ) , 0 ( 0% ) , and 3 ( 5% ) patients after receiving placebo and 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray in the double - blind phase . 
 of the 61 patients assessed for eligibility , 10 patients did not meet the protocol criteria and were therefore excluded . 
 a total of 51 patients performed the baseline exercise test and qualified for continuation . following a 90-minute rest period 
 , patients underwent a subsequent stress test after receiving 0.4 mg of open - label sublingual nitroglycerin spray . 
 all 51 patients showed an improvement in exercise duration of 60 seconds or greater ( range 1.024.7 minutes ) . 
 a total of 51 patients between the ages of 34 and 77 years continued in the double - blind phase and were randomized to 1 of 5 treatment sequences . 
 two patients did not complete the study ( 1 patient was lost to follow - up and 1 patient discontinued prematurely due to ventricular tachycardia / fibrillation requiring cardioversion after the control exercise test at visit 5 ; this event was not considered to be treatment related ) . 
 overall patient characteristics including angina and cardiovascular event history , along with concomitant medications , are described in table 1 . 
 a dose - dependent increase in time to onset of moderate angina was seen following administration of nitroglycerin spray ( table 2 , fig . 
 the time to onset of angina was significantly higher in all treatment groups ( 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg ) than in the placebo group ( table 2 , fig . 
 the increase in time to onset of angina and to onset of moderate angina was most pronounced in the 1.6-mg group , with dose linearity between 0.4- and 1.6-mg doses of nitroglycerin spray . 
 the occurrence of a minimum 1.0-mm st - segment depression was later in all treatment groups ( 0.2 mg , 0.4 mg , 0.8 mg , 1.6 mg ) compared with the placebo group ( table 2 , fig . 
 overall , nitroglycerin spray was generally well tolerated . of 51 patients , 12 ( 24% ) 
 reported adverse events at the qualification visit ( open - label sublingual nitroglycerin response test ) . 
 adverse events considered related to treatment were reported for 2 ( 4% ) , 4 ( 8% ) , 8 ( 16% ) , 9 ( 18% ) , and 13 ( 26% ) patients receiving placebo and 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray , respectively . 
 the most frequently reported adverse events of this nature were headache and dizziness , all of which were mild to moderate in severity . 
 the incidence of treatment - related adverse events was dose dependent , with the incidence of headache increasing from 0 with placebo to 3 ( 6% ) , 5 ( 10% ) , 6 ( 12% ) , and 8 ( 16% ) after 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray , respectively . 
 no serious adverse events related to treatment were reported . per protocol , patients with systolic blood pressure ( sbp ) less than 100 mmhg were excluded from the study . 
 symptomatic hypotension was not reported as an adverse event , however sbp decreased to a level of 100 mmhg or less in 1 ( 2% ) , 0 ( 0% ) , 1 ( 2% ) , 0 ( 0% ) , and 3 ( 5% ) patients after receiving placebo and 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray in the double - blind phase . 
 lifestyle modifications , including supervised exercise therapy , play an important role in the success of optimal medical therapy.2427 however , patients with stable angina generally have impaired exercise tolerance and reduced daily physical activity.28 sublingual nitroglycerin increases physical exercise tolerance which , in turn , helps to increase the amount of exertion possible prior to the onset of angina.12,13 sublingual forms of nitroglycerin are readily absorbed through mucous membranes , and their effect is prompt , reliable , and more effective than other forms of nitroglycerin , including ointments , transdermal patches , and sustained - release preparations.11,29 the results of this study are in line with prior findings and demonstrate a significant improvement in exercise tolerance with sublingual nitroglycerin spray in patients with stable angina . 
 although sublingual nitroglycerin spray was generally well tolerated , an increase of treatment - related adverse events was observed with higher dose levels ( 26% of patients with 1.6 mg nitroglycerin spray ) . 
 one important issue that is not specifically addressed in this study is the clinical relevance of the nearly 1-minute improvement in exercise duration with sublingual nitroglycerin spray over placebo . 
 it must be recognized that this improvement was experienced by patients who were exercising at a higher work load ( higher speed and incline during the treadmill test ) after receiving the study medication . 
 as most real - world patients have a much slower walking pace , this finding indicates that prophylactic nitroglycerin spray might allow much greater increases in walking times during normal daily activities . 
 although this outcome was not formally evaluated in the present study or discussed in previous publications to our knowledge , patients have often reported a marked increase in angina - free walking time and exercise duration after taking prophylactic sublingual nitroglycerin . 
 since this study of sublingual nitroglycerin spray was conducted , there have been significant changes in optimal medical therapy for stable coronary artery disease . 
 this is particularly apparent in the greater use of beta - blockers compared with calcium channel blockers and long - acting nitrates to treat angina , and the routine use of daily aspirin and a statin to reduce serious cardiovascular outcomes.3,8 although beta - blockers are currently considered a standard treatment for stable angina , patients receiving these medications may continue to experience angina.30 however , patients with a history of beta - blocker use who are appropriately supplemented with sublingual nitroglycerin or long - acting nitrates show significant improvements in angina - free exercise duration and total walking time compared with placebo.3,5,29 the american college of cardiology and american heart association guidelines for the management of patients with chronic stable angina emphasize the initial use of medical therapy.5,20,31 these guidelines include the recommendation for all patients to undergo a risk assessment with a physical activity history and/or an exercise test to guide prognosis and prescription . 
 ett is associated with a very low rate of serious adverse events such as death ( 0.5 per 10,000 tests ) , myocardial infarction ( 3.58 per 10,000 tests ) , or serious arrhythmias ( 4.78 per 10,000 tests).15 guideline - compliant medical therapy improves clinical outcome in many patients with stable angina , as evidenced by an observational study in which increasingly guideline - compliant therapy was associated with a reduction in death and myocardial infarction in patients with stable angina during 1 year of follow - up.32 indeed , implementation of a quality improvement program in utah to enhance the prescription of appropriate discharge medications among patients with cardiovascular disease was associated with improvements in cardiovascular readmission rates and reductions in mortality.33 a number of studies have compared the effect of optimal medical therapy versus revascularization on symptoms and outcomes in patients with coronary artery disease and stable angina . in general , these studies have shown that optimal medical therapy is effective in controlling symptoms and , along with risk factor modification , can reduce the risk of adverse cardiac events.8,34 moreover , these studies have shown that there is no significant difference in serious adverse clinical events , such as myocardial infarction and death , between patients who have received medical therapy and those who have undergone revascularization.2,5,8 the clinical outcomes utilizing revascularization and aggressive drug evaluation ( courage ) trial , which compared the efficacy of percutaneous coronary intervention ( pci ) plus optimal medical therapy versus optimal medical therapy alone , demonstrated similar rates of death and nonfatal myocardial infarction in both patient groups over a follow - up period of 55 months.8 moreover , both groups demonstrated a substantial reduction in angina symptoms , with 74% and 72% of patients in the pci and optimal medical therapy groups , respectively , angina - free after 5 years of follow - up.8 further analysis of data from the courage study found that both groups of patients had marked improvements in health status , including reductions in angina symptoms , and improved quality of life . 
 although the pci group demonstrated significantly greater health benefits in the short term , these benefits disappeared after longer - term follow - up , and by 36 months there was no significant difference in health status between the 2 groups.9 the bypass angioplasty revascularization investigation 2 diabetes trial in patients with diabetes , coronary artery disease , and classic angina compared the effects of revascularization with medical therapy alone on clinical outcomes . during the 5-year follow - up 
 , no difference was found between the medical therapy and revascularization groups on the risk of all - cause death , myocardial infarction , or stroke.34 furthermore , a meta - analysis of 11 randomized trials comparing pci with conservative treatment in patients with stable coronary artery disease found no significant difference between the 2 treatment strategies with regard to overall mortality , cardiac death , or myocardial infarction.7 current recommendations state that patients with stable angina may defer surgical intervention without additional risk to allow for determination of response to optimal medical therapy.5,11 despite the published outcome data and current guideline recommendations , many patients are still referred for revascularization , mainly due to a belief that these procedures reduce the risk of myocardial infarction or death more effectively than optimal medical therapy alone.2,3,8,35 additional studies assessing the long - term benefits of exercise in the context of medical therapy compared with revascularization outcomes could help to further inform the clinical decision making process . 
 in this study , sublingual nitroglycerin spray was shown to increase time to onset of moderate angina during exercise . 
 this improvement in physical exercise tolerance may be of great clinical relevance , as the prophylactic use of sublingual nitroglycerin spray allows patients to face exertion with confidence and permits exercise for a longer amount of time . 
 these results support the use of sublingual nitroglycerin spray as nitrate therapy in patients with stable angina who are being managed with optimal medical therapy . 
 this approach may also be beneficial for patients who have persistent angina post - revascularization . 
 further studies are needed to evaluate the long - term beneficial effects of using routine prophylactic nitroglycerin spray in patients with stable angina to increase exercise duration and improve quality of life and well - being .","backgroundsublingual nitroglycerin increases exercise duration in patients with stable angina . 
 brief results from this study were published previously in german . here , we more fully describe the study methodology , patient characteristics , and detailed results.methodsthis double - blind , crossover study enrolled 51 patients with stable angina . 
 patients were randomized to 1 of 5 treatment sequences and were administered placebo or nitroglycerin spray ( 0.2 mg , 0.4 mg , 0.8 mg , or 1.6 mg ) . 
 patients carried out 1 control exercise tolerance test ( ett ) and 1 investigational ett at each visit.resultsdose-dependent increases in time to onset of angina , time to onset of moderate angina , and the occurrence of a minimum 1.0-mm st - segment depression were seen following administration of nitroglycerin spray.conclusionsthese results support the use of sublingual nitroglycerin spray in patients with stable angina who are being managed with medical therapy and in patients who have persistent angina post - revascularization .",pubmed
"the residual and resistant microorganisms have been recognized as the basic reason for failures in root canal treatments . 
 fungi , chemoorganotrophic eukaryotic microorganisms , may play a key part in periradicular diseases . while they have been found sporadically in initial root canal infections , 
 failure of some endodontic treatments may therefore be explained by the presence of candida albicans in the oral flora . 
 it is reported that in 20% of endodontic treatment failures , c. albicans is present . 
 candida species , which have a tendency to invade dentin , are regarded as dentinophilic microorganisms . 
 the presence of c. albicans in endodontic treatment failures may be the result of coronal leakage after , or contamination during , the endodontic treatment . 
 several factors , such as an ability to survive despite a lack of nutrients or resistance to intracanalicular treatment materials , may result in c. albicans remaining in the root canal system . c. albicans has been found in some failed endodontic treatments ; a phenomenon that might be explained by the invasive nature of this fungus or its resistance to some intracanalicular medications , e.g. , calcium hydroxide . 
 if repeated treatment has either failed or is unfeasible , the only alternative is a root - end resection . 
 apical sealing , avoiding contamination , and reducing the number of microorganisms , including bacteria and fungi , influence the success rate of such treatment . therefore , the antibacterial and antifungal properties of root - end filling materials are important to the outcome of these procedures . 
 mineral trioxide aggregate ( mta ) was introduced in 1993 and , after different examinations , approved , in 1998 , by the us food and drug administration as an endodontic biomaterial , used in root - end filling , vital pulp capping , apexification , and root or furcal perforation repair.[58 ] mta is marketed as gray and white . however , because of the tooth - discoloring properties of gray mta , white mta has been pushed to replace it . 
 mta has a number of advantages , including excellent sealing ability , biocompatibility , high alkalinity , radiopacity , antimicrobial effects , and resistance to fluid and blood contamination.[911 ] in spite of having disadvantages , such as delayed setting time , poor handling properties , and high price , mta , because of its physical and chemical properties , has become the gold standard of root - end filling materials . 
 mta - angelus particles are not as homogenous as proroot mta . in comparison to proroot mta , 
 mta - angelus shows several advantages , such as good marginal adaptation , good sealing , sealing ability in mineralized tissues with complete closure , and has been reported to be inflammation - free in most cases.[1517 ] mta - angelus has an excellent antibacterial effect and a strong fungicidal effect against c. albicans . 
 while both the white and gray mta - angelus have arsenic in their structures , white mta - angelus has the benefit of containing arsenic levels below the limit set by the iso 9917 - 1 standard . 
 a limited number of researches have compared the antifungal effects of proroot mta and mta - angelus . 
 however , such studies have been conducted using agar diffusion test ( adt ) , a method that does not seem appropriate because of low solubility and diffusibility of mta . 
 the aim of this in vitro study was to evaluate and compare the antifungal properties of fresh and set proroot mta and mta - angelus , in concentrations of 50 mg / ml and 100 mg / ml , against c. albicans at 1- , 24- , and 48-hour time points . 
 the null hypothesis of this study was that there is no difference in the in vitro antifungal properties of these two materials . 
 the antifungal activity of proroot mta ( dentsply tulsa dental , tulsa , usa ) as well as mta - angelus ( angelus , londrina , brazil ) was evaluated against c. albicans . 
 the culture of sabouraud dextrose agar ( merck , darmstadt , germany ) was prepared according to the manufacturer 's instructions with a definite ratio of powder to liquid . 
 samples of live c. albicans , ( atcc 10231 ) provided by scientific research organization , ( tehran , iran ) were subcultured on the sabouraud dextrose agar plates . the agar plates , containing the fungus , were then maintained at 37c . 
 times and conditions for the culture of c. albicans in this study were determined following the nccls standards ( national committee on clinical laboratory standards ) . 
 a total of 50 culture wells were divided into four experimental groups ( freshly mixed mta , freshly mixed mta - angelus , 24-h set mta , 24-h set mta - angelus ) and two control groups ( positive control , negative control ) . 
 the root canal filling materials were prepared according to their instructions and then mixed with the appropriate volume of sterile water to reach the concentrations of 50mg / ml or 100mg / ml . for the set groups , the mixture was prepared and left for 24 hours . 
 each group consisted of 10 wells , and the control groups consisted of five wells each . 
 suspensions were then made by the addition of several fungal colonies from the surface of the sabouraud dextrose agar plates to sabouraud dextrose broth media . 
 a final concentration of 10 cfu / ml ( colony forming unit / milliliter ) was achieved , as recommended by the nccls , via microscopic cell counting . 
 wells with 1 ml of c. albicans suspension mixed and 1 ml of sabouraud dextrose broth media without root end - filling materials served as positive control and wells with 1 ml of sabouraud dextrose broth media without c. albicans served as negative control . 
 all wells were incubated at 37c and evaluated for 1 , 24 , and 48 hours by an independent observer by assessing the colonies of c. albicans in each well . 
 this assessment was based on the turbidity of the suspension in the tubes . at each time point , 0.02 ml of each suspension was cultured on sabouraud dextrose agar plate to confirm c. albicans growth . 
 after the culture of 0.02ml of the treated suspension on the agar plates , the fungal growth was assessed at 1- , 24- , and 48-hour time points . while fungal growth was not observed in any of the negative control groups , 
 after 1 hour , both the mta and mta - angelus were incapable of killing c. albicans . 
 the results were the same for both freshly mixed and set samples at both concentrations ( 50 and 100 mg / ml ) . increasing the incubation time to 24 hours and 
 both materials were fungicidal for c. albicans at both concentrations of 50 and 100 mg / ml , and in both states of fresh and set [ table 1 ] . 
 comparison of proroot mta and mta - angelus in concentration of 50 mg / ml and 100 mg / ml at 1 , 24 , and 48-h time intervals there was no significant difference between using the two materials at either of the concentrations or either of the 1-hour , 24-hour , or 48-hour time points ( p<0.05 ) . 
 in this study , the antifungal properties of proroot mta and mta - angelus against c. albicans in two concentrations and in fresh and set situations were compared by using tube - dilution test . because the two materials elicited the same reaction against c. albicans in different conditions , the null hypothesis that there is no difference in the in vitro properties of these two materials has to be accepted . the most common method for assessing the antifungal or antibacterial activity of root - end filling materials 
 however , several parameters , such as lack of standardization of inoculum density , adequate culture medium , agar viscosity , plate storage condition , size and number of specimens per plate , time and temperature of incubation , and reading point of the inhibition zones , can affect the results of the adt . 
 the method used in this in vitro study was the tube - dilution susceptibility test ; an efficient method for evaluating antifungal characteristics of materials . in this technique , direct contact between c. albicans and the experimental materials was allowed , allowing a more realistic interaction . 
 because of possible changes in the chemical structure of the products , root - end filling materials should be tested in two situations ; one where the material is used immediately after mixing , and another , where it is used after having been allowed to reach its final chemical composition , or set state . in this study 
 , proroot mta and mta - angelus were tested in freshly mixed and set states , to mimic clinical use of the materials . after setting , 
 consequently , the perceived difference in antifungal patterns of the materials may be a result of the differences in the degree of setting . 
 however , in our study , the results were similar in the two different states for all tested materials . based on nccls standards , 
 the results of this study showed that c. albicans was not affected by the freshly mixed and set , proroot mta and mta - angelus , at 1 hour . 
 however , these materials were effective in eliminating c. albicans by the 24-h time point . 
 some studies have reported the inhibitory effect of proroot mta and mta - angelus on c. albicans by determining the diameter of the inhibition zones in millimeters , and have resulted conclusions similar to this study . 
 the presence of calcium hydroxide , produced by a hydration reaction from the mixing of mta with water , and an increase in ph , as a result of the release of hydroxyl ions , is probably the mechanism of the antimicrobial activity of mta - angelus . 
 they suggested that the presence of calcium hydroxide is capable of , initially , reducing and , ultimately , eliminating the fungus . 
 it is important to mention that the results presented in this study were obtained under in vitro conditions , and may not accurately describe in vivo activity . 
 further studies are necessary to investigate the effect of these materials on bacteria and fungi in clinical application . 
 this study shows that mta - angelus has an effective antifungal property comparable to proroot mta at concentrations of 50 and 100 mg / ml .","background : fungi may play a key part in periradicular diseases . the aim of this study was to evaluate and compare the antifungal properties of two root - end filling materials , proroot mineral trioxide aggregate ( mta ) and mta - angelus , against candida albicans using tube - dilution test.materials and methods : the antifungal properties of proroot mta and mta - angelus against c. albicans was assessed at 1 , 24 , and 48 hours following administration of two concentrations of the antifungal agents ( 50 and 100 mg / ml ) . 
 a total of 50 culture wells were divided into four experimental groups ( freshly mixed mta , freshly mixed mta - angelus , 24-h set mta , and 24-h set 
 mta - angelus ) and two control groups . 
 each well was prepared for one specific agent with a specific concentration . 
 for the set groups , the mixture was prepared and left for 24 hours . 
 one milliliter of suspension of fungal colonies with concentration of 104 cfu / ml was then added to the mixtures in each well . 
 all wells were incubated at 37c and assessed at 1 , 24 , and 48 hours . 
 this observation was based on the turbidity of the suspension in the tubes . at each time point , 0.02 ml of each suspension was cultured on a sabouraud dextrose agar plate to confirm c. albicans growth . 
 the results were analyzed using kruskal - wallis test.results:although all fresh and set samples were incapable of killing c. albicans at 1 hour , they demonstrated fungicidal ability on agar plates at 24 and 48-hour time points.conclusion:mta-angelus proved to be an effective antifungal agent compared to proroot mta at concentrations of 50 mg / ml and 100 mg / ml .",pubmed
"deficits of motor and sensory functions after stroke on the side contralateral to the 
 damaged hemisphere are often evident1 , 
 whereas the ipsilateral side may be primarily regarded as normal or unaffected . however , 
 there is increasing evidence of the presence of subtle motor deficits in motor performance 
 on the ipsilateral side as well2 , 3 . 
 ipsilateral motor deficits emerge during 
 the acute phase and demonstrate chronic persistence3,4,5 ; the reasons for ipsilateral motor deficits are still unclear . 
 clinical assessment tools may not be sufficient for differentiating ipsilateral motor 
 deficits ; however , deficits in dexterous motor and coordination function on the ipsilateral 
 side have been identified in laboratory testing2 , 
 6,7,8 . 
 however , kinematic deficits in the ipsilateral upper limb in performance of various specific 
 motor tasks requiring dexterity and coordination , such as a tracking task , a goal - direction 
 movement , and a tapping task , have been found in recent studies2 , 6 , 8 , 11 . on the basis of these 
 observations , several possible mechanisms for ipsilateral motor deficits 
 have been 
 suggested , such as disrupted counterbalance of each hemisphere , dysfunction of the uncrossed 
 corticospinal track , or the different roles of both sides in hemispheric functions6 , 7 , 11,12,13,14,15 . 
 as mentioned above , patients with brain damage suffer from ipsilateral motor deficits in 
 performance of the ipsilateral upper limb . 
 until now , most studies of ipsilateral deficits 
 in stroke patients have concentrated on the motor , rather than sensory deficits . 
 therefore , the purpose of 
 the current study was to investigate the presence of motor and sensory deficits in the 
 ipsilateral upper limb , and to examine the correlation between the two variables in patients 
 with stroke using a tracking and reposition sense test . 
 fifty hemiparetic stroke patients ( 25 patients with right brain injury and 25 patients with 
 left brain injury ) referred to a local rehabilitation hospital were consecutively recruited 
 in the order of their registration . 
 the inclusion criteria were ; first ever stroke confirmed 
 by medical history and brain mri ; right handed individual verified by the edinburg 
 handedness inventory ; no symptoms of unilateral neglect or hemianopsia ; no cognitive problem 
 ( mini - mental state examination>24 points ) ; no apraxic behavior ( ideomotor apraxia score 
 developed by ambosoni et al . 
 ( > 11 points)16 ; and no musculoskeletal dysfunction in the unaffected upper limb . 
 we recruited 40 sex- and age - matched normal control subjects . to control the known effects 
 of hand asymmetry , the accuracy and proprioceptive tests were performed by the 20 control 
 subjects using their dominant right hand , and the remaining subjects used their non - dominant 
 left hand . 
 all subjects gave their written informed consent prior to participation , and this 
 study was approved by the local ethics committee . 
 tracking and joint position sense tests were conducted for the hand ipsilateral to the 
 damaged hemisphere of the patients , and with the corresponding hand of the same side of the 
 control subjects . 
 all subjects were seated in front of a table , with the forearm comfortably 
 supported and the elbow flexed at 90. a plastic frame with an embedded potentiometer was 
 used to measure the accuracy of movement and proprioceptive sense in the metacarpophalangeal 
 ( mp ) joints . 
 the potentiometer detected flexion / extension motion of the mp joint , and 
 transmitted the analog signal to a computer with analog - to - digital data acquisition 
 software , that sampled the signal at a frequency of 200  hz . in the tracking task , the subject was instructed to track the red target sine wave 
 displayed for 15 seconds on the computer screen as accurately as possible . 
 the response sine 
 wave made by the subject was displayed as a black solid line , which tracked up as the mp 
 joint was extended , and tracked down as the mp joint was flexed . 
 accuracy of the motor performance was 
 analyzed by an accuracy index ( ai ) , which was normalized to the range of motion of the mp 
 joint of each individual subject , and takes into account the differences between subjects in 
 the excursion of the target and response waves17.ai = 100(p  e)/p where e is the root mean square ( rms ) error between the target line and the response line , 
 and p is the size of the subject s target pattern , calculated as the rms difference between 
 the sine wave and the midline dividing the upper and lower phases of the sine wave . 
 the 
 degree of p is determined by the scale of the vertical axis of the range of subject s mp 
 joint motion . 
 prior to the evaluation , three practice trials were provided after one demonstration , using 
 sine waves which were different from the sine waves used in the actual test to prevent a 
 learning effect . 
 the joint position sense was evaluated on the mp joint ipsilateral to the 
 damaged brain hemisphere of the patients , and the joint on the corresponding side of the 
 control subjects . 
 in addition , the same experimental apparatus and environment used for the 
 performance of the tracking task were used . 
 the subjects were instructed to actively 
 reproduce the position of the mp joint which was passively positioned by the examiner . 
 three 
 different passively - positioned angles were randomly presented , in terms of 50% , 70% , and 90% 
 flexion of the total range of motion of the mp joint . 
 the mean value of three trials of the 
 joint reposition errors between the passively - positioned angles and the actively - positioned 
 angles was calculated . 
 the  test was performed to analyze the differences in sex distribution between 
 the patient and control groups . 
 the independent t - test was performed to determine the 
 significance of differences in age and accuracy of the tracking task / joint position sense . 
 in addition , correlation between the ai and joint position sense was investigated using 
 pearson s correlation coefficient . 
 chicago , il , usa ) was used for the 
 statistical analysis of all data , and statistical significance was accepted for p values 
 < 0.05 . 
 no significant differences were observed between the two 
 groups in terms of distribution of sex and age . 
 the meanssd of the accuracy index and 
 reposition error score of both groups are shown in table 2 . in terms of motor function , 
 the stroke group showed a lower accuracy 
 index in the mp joint than the control group . 
 a higher score of reposition errors in the 
 joint reposition test in relation to sensory function was observed in the stroke group , 
 compared to the control group . 
 the results of the statistical analysis indicate that both 
 measures in the stroke group were significantly different from the control group 
 ( p<0.05 ) . 
 ipsilateral sensory deficits showed significant correlation with motor deficits 
 ( r= 0.549 ) ( p<0.001 ) ( table 2 ) . 
 in the current study , we attempted to assess motor function using a tracking task for 
 visuomotor coordination , and proprioceptive sense using a joint reposition test for the 
 ipsilateral upper limb . 
 our findings reveal a lower accuracy index in the tracking task , and 
 higher error scores in the joint reposition test in the stroke group , compared to sex- and 
 age - matched normal subjects . 
 in addition , there was a negative correlation between the motor 
 and sensory deficits , indicating stroke patients would have difficulty in performing 
 complicated motor tasks requiring delicate sensoriomotor functions using pure integrity of 
 movement accuracy and proprioceptive sense . 
 our present results are in accordance with those of several previous studies2 , 8 , 
 suggesting the presence of motor deficits in upper limbs ipsilateral to the damaged 
 hemisphere in the visuomotor tracking task . 
 a possible mechanism for the motor dysfunction 
 in the ipsilateral hemisphere of stroke patients has been suggested by previous studies , 
 which reported bilateral hemisphere activation when normal subjects executed a unilateral 
 upper limb task18,19,20,21 . 
 if functional integrity of both the right and left brain cortex is 
 necessary for normal motor control of the upper limb , it is expected that the ipsilateral 
 upper limb would be affected after stroke . in the present study , 
 ipsilateral sensory 
 deficits related to proprioceptive sense were also observed . according to our findings 
 , 
 ipsilateral sensory deficits may be connected with bilateral hemisphere activation during 
 performance of motor tasks . 
 the primary sensory cortex ( s1 ) conveys efferent projection to 
 the posterior parietal cortex ( brodmann s area 5 and 7 ) , which is connected bilaterally 
 through the corpus callsosum . 
 therefore , as suggested by our results , it is possible that 
 disturbance of transcallosal transfer after unilateral brain damage may lead to ipsilateral 
 sensory deficits . 
 in addition , there is a close relationship between sensory and motor 
 function , because the posterior parietal cortex is connected with the frontal motor areas . 
 thus , the posterior parietal cortex would have an effect on the initial movement and sensory 
 feedback during performance of a complex motor task22 . on this basis , 
 the correlation shown in our study between 
 ipsilateral motor deficits and sensory deficits can be explained . 
 these findings imply that interest in the ipsilateral side of stroke patients should focus 
 on ipsilateral sensory deficits as well as ipsilateral motor deficits . 
 motor deficits of the 
 ipsilateral limbs of individuals with stroke have been reported in many studies ; besides , 
 our study showed sensory deficits related to proprioceptive sense . on the basis of these 
 results 
 , we think that the difficultly stroke patients experience in task performance using 
 the ipsilateral upper limb may be affected by both motor and sensory deficits . 
 studies on 
 recovery of motor deficits on the ipsilateral side after stroke are in progress . 
 jung et 
 al.14 reported that motor deficits in 
 the ipsilateral upper limb show maximal recovery within one month after onset of stroke , but 
 the deficits do not completely recover . 
 thus , it will be necessary to study the recovery of 
 ipsilateral sensory deficits after stroke onset , and we will be investigating this . 
 we 
 acknowledge that our study had some limitations , in that the effects of specific lesion 
 location and the extent of the damage were not identified . 
 therefore , future studies will be 
 required in order to determine more detailed mechanisms of other movement and sensory 
 deficits , other than proprioceptive sense , in the ipsilateral upper limb of patients with 
 unilateral brain injury .","[ purpose ] previous studies have reported on motor deficits in the ipsilateral upper 
 limbs ( ul ) of a damaged brain hemisphere in motor tasks . however , little is known about 
 sensory deficits on the ipsilateral side . 
 therefore , we investigated whether both motor 
 and sensory function of the ipsilateral ul are affected in patients with stroke . 
 [ subjects 
 and methods ] fifty patients with unilateral stroke and 40 age- and sex- matched normal 
 subjects participated in this study . 
 subjects were evaluated on performance of a tracking 
 task for motor function , and by the joint reposition test for integrity of proprioceptive 
 sense in the ipsilateral ul . [ result ] the comparison of the stroke group and the control 
 group showed significant differences in performance of the tracking task and the joint 
 reposition test . 
 the accuracy index for the tracking task showed significant correlation 
 with the error score for the joint reposition test in the stroke group . 
 [ conclusion ] these 
 results suggest that the ipsilateral ul of stroke patients has impairment in sensory 
 function which is related to proprioceptive sense , along with motor deficits . 
 therefore , 
 we think that the difficulty stroke patients experience with motor tasks for the 
 ipsilateral ul is induced by diminished integrity of sensorimotor function due to both 
 sensory and motor deficits .",pubmed
"the specific targeting of nanoparticles to target cells in an organ of interest has been a long - term goal of many laboratories interested in using nanoparticles to mediate the functional delivery of biopharmaceutical agents ( including nucleic acids ) to defined target cells . 
 one of the main problems in using targeting ligands in conjunction with nanoparticles is the competition between specific - binding mediated cell entry and non - specific enhanced cell uptake mechanisms ( andreu et al , 2008 ) . 
 previously , we have observed that non - specific effects can be completely overwhelming ( waterhouse et al , 2005 ) , an observation that has led to a series of further studies ( wang et al , 2009 ) intended to determine and characterize those factors that might influence non - specific enhanced cell uptake mechanisms such as nanoparticle size and heterogeneity , surface hydrophilicity and charge , not to forget other factors such as ligand conformation , points of attachment and surface density . 
 recently , we described how ph - triggered , pegylated sirna nanoparticles ( also known as ph - triggered sirna - abc nanoparticles according to our recently described abcd nanoparticle structural paradigm ( kostarelos and miller , 2005 ; thanou et al , 2007 ; miller , 2008a ; miller , 2008b ) . 
 these nanoparticles with a surface - covering of 5mol% polyethylene glycol 2000 ( peg ) were able to mediate the passive targeting of sirna to liver cells in vivo , thereby providing proof of concept for an effective rnai therapeutic approach to the treatment of hepatitis b virus infections ( sifectplus nanoparticles ) ( carmona et al , 2009 ) . 
 in addition , pegylated sirna nanoparticles enabled for long - term circulation ( ltc sirna - abc nanoparticles ; lesirna nanoparticles ) were shown to do the same to tumour cells in vivo , thereby providing proof of concept for an effective rnai therapeutic approach to cancer treatment ( kenny et al , 2011 ) . in the case of 
 the ph - triggered , pegylated sirna nanoparticles , the peg coating required for the biological stabilization of these nanoparticles was introduced by a purpose - designed post - coupling methodology . 
 here , we report how this methodology has now been adapted to introduce integrin - targeting peptide motifs onto liposome and nanoparticle surfaces and so enable integrin receptor - mediated cellular uptake of agents to cells . there have been some spectacular data obtained in recent years using v6 integrin - receptor binding motifs for receptor - mediated delivery of different imaging agents to cells in vivo ( dicara et al , 2007 ; hausner et al , 2007 ; dicara et al , 2008 ; hausner et al , 2009 ) . 
 we have described previously the use of integrin - receptor targeting peptides to promote functional gene delivery to cells ( harbottle et al , 1998 ; cooper et al , 1999 ) . here , we now report on the use of an alternative cyclic pentapeptide construct that makes use of a single d - tyrosyl amino acid residue to present the arginine - glycine - aspartate ( rgd ) amino acid residue triad in an "" active "" conformation capable of mediating intracellular delivery through v3/5 receptor binding and internalization ( chen et al , 2004 ) . 
 arginine - glycine - glutamate ( rge ) controls are also reported in order to provide the means to demonstrate pure integrin - receptor mediated cellular uptake by rgd presenting imaging nanoparticles . 
 dioleoyl l--phosphatidylethanolamine ( dope ) 1 , dimyristoyl l--phosphatidylcholine ( dmpc ) 2 ( sigma ) , dope - lissamine - rhodamine b ( dope - rhoda ) 3 were obtained from avanti polar lipids ( usa ) ( figure 1 ) . 
 general synthetic procedures were performed as described previously ( carmona et al , 2009 ; mvel et al , 2010 ) . 
 syntheses of n - cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine ( cdan ) 4 and cholesteryl - aminoxy ( ca ) lipid 5 were performed as described previously ( keller et al , 2003 ; oliver et al , 2004 ; carmona et al , 2009 ) ( figure 1 ) . 
 the synthesis of other compounds necessary for our experiments were performed as described ( scheme 1 ) . 
 hplc purification of final products required the use of vydac c-4 reversed phase preparative column with 1ml / min flow rate : mobile phases as follows used trifluoroacetic acid ( tfa ) ; a : h2o ( 0.1% , v / v , tfa ) ; b : mecn ( 0.1% , v / v , tfa ) ; c : meoh ( 0.1% , v / v , tfa ) . program set at : 0 - 15.0min ( 100% , a ) , 15.1 - 25.0min ( 0 - 100% , b ) , 25.1 - 45min ( 100% , c ) , 45.1 - 55min ( 100% , a ) . 
 elution parameters : rgd - peg - cho 13a rt 15.2min , rge - peg - cho 13b rt 14.8min , prnhco - peg - cho 14 rt 15.8min , ca lipid 5 rt 23.8min , rgd - peg - ca 15a product rt 24.1min , rge - peg - ca 15b product rt 23.8min , and prnhco - peg - ca 16 product rt 26.3min ; mass spectrometry : m / z ( esi ) 1878 ( m - h , rgd - peg - cho 13a ) , 1917 ( mna , rge - peg - cho 13b ) , 1326 ( mh , prnhco - peg - cho 14 ) , 2406 ( mh , rgd - peg - ca 15a ) , 2443 ( mna , rge - peg - ca 15b ) , 1869 ( mna , prnhco - peg - ca 16 ) ( figure 1 ) . 
 main lipids were used to prepare liposomes cl1 and cl2 and hence 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles . 
 syntheses of -terminally modified - peg - chos : i ) a ) side chain protected , fluorenylmethyloxycarbonyl ( fmoc ) amino acid residue n - terminal coupling of l - arg(npbf ) , l - lys(boc ) , d - tyr(otbu ) and finally l - asp(otbu ) [ or l - glu(otbu ) ] to glycyl-2-chlorotrityl resin 6 using 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) with di - isopropylethylamine ( dipea ) to mediate residue coupling and 2% ( v / v ) each of 1,8-diazabicyclo[5.4.0]undec-7-ene ( dbu ) and piperidine in dimethylformamide ( dmf ) for fmoc deprotection between rounds of peptide bond formation : b ) resin release with 20% ( v / v ) each of acetic acid and trifluoroethanol ( tfe ) in ch2cl2 , 31 - 78% ; ii ) a ) 3eqv diphenylphosphoryl azide ( dppa ) in dry dmf , 5 eqv nahco3 , 0c  ambient temperature , ph 8.5 , 74 - 80% : 
 b ) 2.5% ( v / v ) each h2o and triisopropylsilane ( tis ) in trifluoroacetic acid ( tfa ) , 80 - 100% ; iii ) a ) ho - c2h4-oh , al2o3 , ccl4 , reflux , 27% : b ) pentafluorophenol ( pfpoh ) , n , n'-dicyclohexylcarbodiimide ( dcc ) , etoac , 71% ; iv ) a ) amino - peg - propionic acid , 4 eqv triethylamine ( tea ) , chcl3 , reflux , 84% : b ) 1.2 eqv pfpoh , 1.2 eqv dcc , etoac , 76% ; v ) 0.2 m na2hpo4 , 0.1 m naoh , 69 - 100% ; vi ) 10% ( v / v ) tfa in h2o , 100% ; vii ) a ) propylamine ( prnh2 ) , 4 eqv tea , chcl3 , reflux , 100% : b ) 10% ( v / v ) tfa in h2o , 51% ( v / v ) . 
 designated lipids were dissolved in chloroform at 5mg / ml and then appropriate aliquots were combined in a presilanized round - bottom flask ( 5ml ) . in each case , the organic solvent was evaporated to dryness to form an even thin lipid film that was further purged with a stream of argon gas to remove residual traces of the organic solvent . 
 the lipid film was hydrated using double distilled h2o ( ddh2o ) to give a multilamellar liposome suspension that was subsequently subjected to sonication in a water bath at 40c for 30min ( sonomatic water bath , langford ultrasonics , 33 khz ultrasound frequency ) . 
 post - sonication , small unilamellar vesicles ( 50 - 80 nm ) ( b - component , cl1 or cl2 ) were diluted to 1.5mg / ml and incubated at room temperature for 15min . 
 thereafter , appropriate aliquots of -terminally modified - peg - cho 13a , 13b or 14 ( 1mg / ml in water , cd molecules ) were introduced for post - coupling such that their final composition was between 1 and 10mol% of total lipid . 
 the ph of solution was monitored by ph boy ( camlab ltd , cambridgeshire , uk ) and adjusted to ph 4 if required by addition of small aliquots of aqueous solutions of naoh ( 0.99 m ) or hcl ( 0.99 m ) . on completion of the reaction ( approximately 16 - 24hr ) ( as judged by hplc analysis ) , the solution ph was accurately readjusted to ph 7 resulting in 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles , respectively , ready for use . 
 1 - 5mol% pegylated abcd2 nanoparticles were prepared by combining 1 - 5mol% pegylated bcd2 nanoparticles with various volumes of aqueous 4 mm hepes , ph 7.0 - 7.4 , sirna solution ( 50m a - component ) under heavy vortex conditions ( final [ sirna ] typically 100g / ml ; 7m ) using a lipid : sirna ratio of 12:1 ( w / w ) that correlates with a lipid - nucleic acid n / p charge ratio of approximately 2 . 
 lipid compositions of two liposome formulations prepared in double distilled water with final diameters of 50 - 80 nm . for lipid structures see figure 1 . 
 these experiments were performed on a facs calibur instrument ( bd biosciences ) . in each set of indicated experiments 1 , 2 or 5mol% pegylated abcd2 nano - particles ( 14060 nm ) 
 were prepared with sirna ( [ sirna ] 80pmoles / well ) and uptake studies performed with huvec cells previously grown to 80% confluent at 37c , 10% co2 . 
 dioleoyl l--phosphatidylethanolamine ( dope ) 1 , dimyristoyl l--phosphatidylcholine ( dmpc ) 2 ( sigma ) , dope - lissamine - rhodamine b ( dope - rhoda ) 3 were obtained from avanti polar lipids ( usa ) ( figure 1 ) . 
 general synthetic procedures were performed as described previously ( carmona et al , 2009 ; mvel et al , 2010 ) . 
 syntheses of n - cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine ( cdan ) 4 and cholesteryl - aminoxy ( ca ) lipid 5 were performed as described previously ( keller et al , 2003 ; oliver et al , 2004 ; carmona et al , 2009 ) ( figure 1 ) . 
 the synthesis of other compounds necessary for our experiments were performed as described ( scheme 1 ) . 
 hplc purification of final products required the use of vydac c-4 reversed phase preparative column with 1ml / min flow rate : mobile phases as follows used trifluoroacetic acid ( tfa ) ; a : h2o ( 0.1% , v / v , tfa ) ; b : mecn ( 0.1% , v / v , tfa ) ; c : meoh ( 0.1% , v / v , tfa ) . program set at : 0 - 15.0min ( 100% , a ) , 15.1 - 25.0min ( 0 - 100% , b ) , 25.1 - 45min ( 100% , c ) , 45.1 - 55min ( 100% , a ) . 
 elution parameters : rgd - peg - cho 13a rt 15.2min , rge - peg - cho 13b rt 14.8min , prnhco - peg - cho 14 rt 15.8min , ca lipid 5 rt 23.8min , rgd - peg - ca 15a product rt 24.1min , rge - peg - ca 15b product rt 23.8min , and prnhco - peg - ca 16 product rt 26.3min ; mass spectrometry : m / z ( esi ) 1878 ( m - h , rgd - peg - cho 13a ) , 1917 ( mna , rge - peg - cho 13b ) , 1326 ( mh , prnhco - peg - cho 14 ) , 2406 ( mh , rgd - peg - ca 15a ) , 2443 ( mna , rge - peg - ca 15b ) , 1869 ( mna , prnhco - peg - ca 16 ) ( figure 1 ) . 
 main lipids were used to prepare liposomes cl1 and cl2 and hence 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles . 
 syntheses of -terminally modified - peg - chos : i ) a ) side chain protected , fluorenylmethyloxycarbonyl ( fmoc ) amino acid residue n - terminal coupling of l - arg(npbf ) , l - lys(boc ) , d - tyr(otbu ) and finally l - asp(otbu ) [ or l - glu(otbu ) ] to glycyl-2-chlorotrityl resin 6 using 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) with di - isopropylethylamine ( dipea ) to mediate residue coupling and 2% ( v / v ) each of 1,8-diazabicyclo[5.4.0]undec-7-ene ( dbu ) and piperidine in dimethylformamide ( dmf ) for fmoc deprotection between rounds of peptide bond formation : b ) resin release with 20% ( v / v ) each of acetic acid and trifluoroethanol ( tfe ) in ch2cl2 , 31 - 78% ; ii ) a ) 3eqv diphenylphosphoryl azide ( dppa ) in dry dmf , 5 eqv nahco3 , 0c  ambient temperature , ph 8.5 , 74 - 80% : 
 b ) 2.5% ( v / v ) each h2o and triisopropylsilane ( tis ) in trifluoroacetic acid ( tfa ) , 80 - 100% ; iii ) a ) ho - c2h4-oh , al2o3 , ccl4 , reflux , 27% : b ) pentafluorophenol ( pfpoh ) , n , n'-dicyclohexylcarbodiimide ( dcc ) , etoac , 71% ; iv ) a ) amino - peg - propionic acid , 4 eqv triethylamine ( tea ) , chcl3 , reflux , 84% : b ) 1.2 eqv pfpoh , 1.2 eqv dcc , etoac , 76% ; v ) 0.2 m na2hpo4 , 0.1 m naoh , 69 - 100% ; vi ) 10% ( v / v ) tfa in h2o , 100% ; vii ) a ) propylamine ( prnh2 ) , 4 eqv tea , chcl3 , reflux , 100% : b ) 10% ( v / v ) tfa in h2o , 51% ( v / v ) . 
 designated lipids were dissolved in chloroform at 5mg / ml and then appropriate aliquots were combined in a presilanized round - bottom flask ( 5ml ) . in each case , the organic solvent was evaporated to dryness to form an even thin lipid film that was further purged with a stream of argon gas to remove residual traces of the organic solvent . 
 the lipid film was hydrated using double distilled h2o ( ddh2o ) to give a multilamellar liposome suspension that was subsequently subjected to sonication in a water bath at 40c for 30min ( sonomatic water bath , langford ultrasonics , 33 khz ultrasound frequency ) . 
 post - sonication , small unilamellar vesicles ( 50 - 80 nm ) ( b - component , cl1 or cl2 ) were diluted to 1.5mg / ml and incubated at room temperature for 15min . 
 thereafter , appropriate aliquots of -terminally modified - peg - cho 13a , 13b or 14 ( 1mg / ml in water , cd molecules ) were introduced for post - coupling such that their final composition was between 1 and 10mol% of total lipid . 
 the ph of solution was monitored by ph boy ( camlab ltd , cambridgeshire , uk ) and adjusted to ph 4 if required by addition of small aliquots of aqueous solutions of naoh ( 0.99 m ) or hcl ( 0.99 m ) . on completion of the reaction ( approximately 16 - 24hr ) ( as judged by hplc analysis ) , the solution ph was accurately readjusted to ph 7 resulting in 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles , respectively , ready for use . 
 1 - 5mol% pegylated abcd2 nanoparticles were prepared by combining 1 - 5mol% pegylated bcd2 nanoparticles with various volumes of aqueous 4 mm hepes , ph 7.0 - 7.4 , sirna solution ( 50m a - component ) under heavy vortex conditions ( final [ sirna ] typically 100g / ml ; 7m ) using a lipid : sirna ratio of 12:1 ( w / w ) that correlates with a lipid - nucleic acid n / p charge ratio of approximately 2 . 
 lipid compositions of two liposome formulations prepared in double distilled water with final diameters of 50 - 80 nm . for lipid structures see figure 1 . 
 these experiments were performed on a facs calibur instrument ( bd biosciences ) . in each set of indicated experiments 1 , 2 or 5mol% pegylated abcd2 nano - particles ( 14060 nm ) 
 were prepared with sirna ( [ sirna ] 80pmoles / well ) and uptake studies performed with huvec cells previously grown to 80% confluent at 37c , 10% co2 . 
 post - coupling represents an optimal way to attach a biological targeting ligand of interest to a nanoparticle surface with controlled mol% attachment and ligand orientation ( scheme 2 ) . 
 our approach to the development of a post - coupling methodology has been to learn from the chemistry employed to create ph - triggered , pegylated sirna nanoparticles ( as mentioned in the introduction ) in order to design - hybrid targeting ligands ( l ) that comprise a peptide targeting moiety conjugated to a peg extension chain enabled for postcoupling to a nanoparticle surface ( scheme 2 ) . 
 rgd peptides and corresponding rge control peptides were prepared as follows ( scheme 1 ) . starting with glycyl-2-chlorotrityl solid - phase peptide resin 6 , 
 the desired pentapeptide rgd - targeting moiety was prepared in an open chain , protected form 7 by solid - peptide synthesis , then subject to cyclization and deprotection to give cyclic pentapeptide 8a . 
 a control cyclic - rge pentapeptide 8b ( with aspartate [ d ] replaced by glutamate [ e ] ) was prepared in an equivalent manner . 
 thereafter , the aldehyde functionality of 4-carboxybenzaldehyde 9 was acetal protected and the carboxy function was activated for coupling by means of pentafluorophenol ( pfpoh ) esterification giving 10 . 
 reagent 10 was then coupled to amino - peg - propionic acid and the free carboxylate activated once again for coupling by means of pfpoh esterification giving heterofunctional peg moiety 11 . 
 the coupling of 11 to 8a or 8b resulted in rgd acetal 12a and rge acetal 12b respectively . 
 routine acetal deprotection yielded the desired products rgd - peg - cho 13a and control rge - peg - cho 13b ( scheme 1 ) . 
 an alternative control aldehyde was obtained through combination of heterofunctional peg moiety 11 with propylamine followed by acetal deprotection resulting in the non - peptide control product prnhco - peg - cho 14 ( scheme 1 ) . 
 all three -terminally modified - peg - cho compounds prepared as described above were used in subsequent experiments described below . using all three -terminally modified - peg - cho compounds in turn , coupling reactions were performed individually with neutral cholesteryl - aminoxy lipid ( ca ) 5 ( figure 1 ) and then with neutral liposomes ( equivalent in composition to cl1 , table 1 ) . 
 reactions were monitored by hplc and mass spectrometry in water at ph4 and shown to reach completion after 16 - 24h stirring at ambient temperature giving 15a , 15b , or 16 as appropriate . 
 liposome - coupling experiments were then performed under similar conditions in order to couple rgd - peg - cho 13a ( or control aldehydes rge - peg - cho 13b and prnhco - peg - cho 14 ) to alternative liposome formulations cl1 or cl2 ( table 1 , figure 1 ) , making use of ca lipid 5 once again in order to present the aminoxy functional groups required for conjugation of liposome surfaces with each -terminally modified - peg - cho compound in turn ( scheme 2 ) . upon conclusion of these liposome - coupling reactions , 
 ph was raised to 7 in order to stabilize the resulting oxime bonds and the resulting pegylated liposomes . 
 resulting ligand - pegylated liposomes ( approx 100 nm in diameter ) were described as belonging to either bcd1 or bcd2 nanoparticle families depending upon whether the parent liposome formulation used in preparation was cl1 or cl2 , respectively . 
 initially bcd1 family members rgd - bcd1 , rge - bcd1 and prnhco - bcd1 were prepared with 1mol% of dope - rhoda 3 for fluorescence microscopy cell uptake studies . 
 the results demonstrate that only 10mol% pegylated rgd - bcd1 nanoparticles were capable of delivering measurable and observable levels of fluorescent - label to integrin - rich human umbilical vein endothelial cells ( huvec ) ( figure 2a ) . 
 this is very interesting in view of the physical properties of bcd1 nanoparticles , namely approx 100 nm in diameter , 10mol% pegylation with peg , and a -potential of essentially zero mv . 
 the lack of charge originates from the fact that the majority lipid components needed to prepare cl1 liposomes were neutral while the minority ca lipid 5 component possesses an aminoxy functional group unprotonated at ph 7 due to its pka value of about 4.5 ( compared with the pka values of more typical amine functional groups that are found around 10 ) ( castro , 1990 ) . 
 we would argue that such nanoparticle physical properties could be an ideal starting point from which to optimize receptor specific cellular uptake of nanoparticles relative to non - specific enhanced cell uptake . 
 subsequently , bcd2 family members rgd - bcd2 , rge - bcd2 and prnhco - bcd2 were prepared with 1.0 mol% of dope - rhoda 3 and up to 5mol% of each -terminally modified - peg - cho for fluorescence - activated cell sorting ( facs ) studies . 
 in addition these cationic liposome systems were further combined with a non - functional nucleic acid ( small interfering rna [ sirna ] ) ( a component ) in order to neutralize the positive charge and convert the bcd2 family members in to a varieties of corresponding low charge abcd2 nanoparticles with lipid : sirna 
 the physical properties of these nanoparticles were diameters of approximately 140 nm , up to 5mol% pegylation with peg , and a -potential of less than 10mv . in these cases , 
 facs studies were performed with low charge 1- , 2- and 5mol% pegylated rgd - abcd2 , rge - abcd2 and prnhco - abcd2 nanoparticles . in each case , the discrimination between rgd presenting and control nanoparticles was still clearly present and was found to increase as a function of the mol% pegylation coverage with peg ( figure 2b ) . 
 the fact that the discrimination was not as substantial as observed above ( figure 2a ) appears to be due to the lower mol% levels of pegylation coverage and the presence of residual nanoparticle charge . 
 therefore , control of both pegylation levels ( plus other peg - structure related criteria ) and residual charges are essential in order to optimize receptor mediated cell uptake in preference to non - specific enhanced cell uptake mechanisms . 
 schematic illustration of nanoparticle formulations involving the inclusion of an aminoxy coupling lipid by a formulation procedure known as premodification followed by the post coupling of -terminally ligand ( l)-modified peg aldehydes ( l - peg - cho ) . 
 biological receptor mediated functional delivery of imaging agents is well established ( dicara et al , 2007 ; dicara et al , 2008 ; hausner et al . 2007 ; 
 hausner et al , 2009 ) , even of genes ( harbottle et al , 1998 ; cooper et al , 1999 ) . 
 however , receptor - mediated delivery can not be taken as a given following the covalent attachment of a biological targeting ligand to a nanoparticle ( waterhouse et al , 2005 ; andreu et al , 2008 ) . 
 therefore , in our view , receptor - mediated delivery phenomena should never be treated with the casual assumption that receptor - mediation is inevitable once an appropriate receptor - specific ligand has been mounted on a nanoparticle surface . 
 any given receptor - specific ligand should in fact be subject to a standard set of trial experiments to ensure that the biophysical properties of a nanoparticle platform and the presentation of the receptor - specific ligand are optimized for bonefide receptor - mediated delivery of the corresponding nanoparticle . in this paper 
 we describe just such a set of trial experiments ( figure 2 ) with sufficient controls to demonstrate clearly that specific receptor - mediated uptake by cells has been enabled over background . 
 accordingly , we would like to propose that both studies involving first the delivery of an imaging agent and then second a potential agent of pharmaceutical interest ( api ) , should be standard assays to perform and demonstrate before all else that a nanoparticle - attached ligand is truly a receptor - specific ligand in the context of the nanoparticle platform to which the ligand is covalently attached . if this is not shown , then nanoparticle biophysical properties , ligand attachment and orientation , and mol% ligand presentation should all be systematically altered until specific receptor - mediated cell uptake can be observed substantially over and above any non - specific enhanced cell uptake background ( kamaly et al , 2009 ; kamaly et al , 2010 ) . 
 fluorescence microscopy of huvec cells , previously grown to 80% confluent at 37c with 10% ( v / v ) co2 , treated with 10mol% pegylated bcd1 nanoparticles prepared from rgd - peg - cho 13a or rge - peg - cho 13b . 
 cells were in contact with nanoparticles for at least 4hr prior to mounting and observation of rhodamine fluorescence under a nikon 600 fluorescence microscope . 
 cell uptake was determined after 4hr using rhodamine fluorescence and detected using the fl-2 channel . 
 ab corresponds with simple sirna - lipoplex control nanoparticles , formulated from cl2 cationic liposomes and an equivalent final [ sirna ] . in our case here 
 , we can say with confidence that our post - coupling chemistry and methodology for the attachment of integrin - targeting rgd ligands has resulted in the successful formation of integrin - targeted imaging nanoparticles that can also mediate integrin - specific delivery of an api such as sirna to v3/5 integrin - receptor presenting cells . 
 therefore , this post - coupling chemistry and premodification - postcoupling methodology could be applicable to other nanoparticle platforms with equal success provided that the biophysical properties of the nanoparticle platform also conform to the following biophysical parameters : nanoparticle dimensions of approx 100 nm in diameter nanoparticle -potential values that converge on neutral ( 0 mv ) nanoparticle ligand surface coverage of approx 2 mol% ( or higher ) further research with this and other nanoparticle systems will now be needed to demonstrate if these three nanoparticle "" rules "" for receptor - mediated cell entry are indeed general rules or simply guidelines for receptor - mediated cellular uptake of nanoparticle systems by corresponding receptor expressing cells in vitro , ex vivo and/or in vivo . 
 the data described here represent the completion of a first study involving the preparation of ligand - mounted pegylated nanoparticles constructed by a bespoke pre - modification postcoupling methodology . 
 data suggest for the first time in our hands that this methodology may be used to ensure that receptor mediated cell uptake of attached nanoparticles can be "" engineered "" to dominate non - specific enhanced cell uptake mechanisms .","a key goal of our research is the targeted delivery of functional biopharmaceutical agents of interest , such as small interfering rna ( sirna ) , to selected cells by means of receptor - mediated nanoparticle technologies . 
 recently , we described how ph - triggered , pegylated sirna - nanoparticles ( ph triggered sirna - abc nanoparticles ) were able to mediate the passive targeting of sirna to liver cells in vivo . 
 in addition , pegylated sirna nanoparticles enabled for long - term circulation ( ltc sirna - abc nanoparticles , lesirna nanoparticles ) were shown to do the same to tumour cells in vivo . 
 further gains in the efficiency of sirna delivery are expected to require active targeting with nanoparticles targeted for delivery and cellular uptake by means of attached biological ligands . 
 here we report on the development of a new synthetic chemistry and a bioconjugation methodology that allows for the controlled formulation of pegylated nanoparticles which surface - present integrin - targeting peptides unambiguously and so enable integrin receptor - mediated cellular uptake . 
 furthermore , we present delivery data that provide a clear preliminary demonstration of physical principles that we propose should underpin successful , bonefide receptor - mediated targeted delivery of therapeutic and/or imaging agents to cells .",pubmed
"the millennium cohort is the largest prospective study using primary data collection ever undertaken by the department of defense ( dod ) . 
 the study began in 2001 with the objective to collect and evaluate data on health , behavioral risk factors , and occupational characteristics related to military service that may be associated with adverse health outcomes ( 14 ) . 
 this report includes participants from the first enrollment cycle that spanned 20012003 and comprised 77,047 participants ( 36% of those able to be contacted ) . 
 approximately 71% of those participants enrolled at baseline responded to follow - up surveys conducted in 2004 and 2007 . 
 the surveys were administered via paper and the internet and included questions on self - reported provider - diagnosed medical conditions , mental health symptoms , physical and functional status , alcohol and tobacco use , occupational status , military exposures , sleep patterns , and demographic information . 
 this study was approved by the naval health research center institutional review board , and informed consent was obtained from all study participants . 
 this research has been conducted in compliance with all applicable federal regulations governing the protection of human subjects in research ( protocol nhrc.2000.0007 ) . 
 of the 77,047 participants enrolled at baseline , follow - up surveys were completed by 55,021 participants at year 3 and 54,790 at year 6 . since incident diabetes was the outcome of interest , eligible participants included those who were followed until either the outcome was reported to have occurred at year 3 or 6 , or until their last completed follow - up if the outcome was not reported . 
 exclusions included participants reporting provider - diagnosed diabetes at baseline , missing outcome or exposure data , or if dod outpatient or inpatient medical encounter data contained codes for type 1 diabetes ( n = 72 ) ( icd-9 codes 250.x1 and 250.x3 ) . 
 the primary data sources were the self - administered millennium cohort surveys and dod electronic personnel files , managed by the defense manpower data center ( dmdc , seaside , ca ) . 
 for these analyses , electronic records from dmdc provided information on age , sex , race / ethnicity , education , pay grade , service branch , service component , military occupation , dates of separation , and deployment dates . 
 dmdc information was supplemented with self - reported data to reduce missing values when necessary . 
 three sleep variables were included in the analyses : 1 ) trouble sleeping , 2 ) sleep duration , and 3 ) sleep apnea . 
 trouble sleeping was assessed using the sleep items on the prime - md patient health questionnaire ( phq ) and the ptsd checklist  civilian version ( pcl - c ) ( 1720 ) . 
 the phq asks ,  over the last 4 weeks , how often have you experienced trouble falling asleep or staying asleep ?  the pcl - c asks ,  in the past month , have you had trouble falling asleep or staying asleep ?  participants with trouble sleeping were defined as those who responded 
 moderately  or above on the pcl - c sleep item or  several days  or longer on the phq sleep item . 
 sleep duration was reported as whole number of hours slept in an average 24-h period over the past month . 
 participants with sleep apnea were identified by self - report as being told by a doctor or other health professional they had sleep apnea . 
 ptsd was assessed using the pcl - c ( 17 ) , a 17-item self - report screening tool for ptsd symptoms during the past 30 days on a 5-point likert scale , ranging from 1 ( not at all ) to 5 ( extremely ) . 
 a positive screen for ptsd at baseline was defined as a report of a moderate or higher level of at least one intrusion symptom , three avoidance symptoms , and two hyperarousal symptoms ( criteria established by dsm - iv ) ( 21 ) . 
 major depressive disorder was assessed using nine items from the phq ( sensitivity = 0.93 ; specificity = 0.89 ) corresponding to the depression diagnosis based on the dsm - iv ( 19 ) . 
 a positive screen for depression at baseline was defined as 1 ) endorsement of depressed mood or anhedonia and 2 ) response of  more than half the days  or  nearly every day  to at least five items . other anxiety ( 6 items ) and panic ( 15 items ) symptoms 
 problem drinking was defined using items from the phq on risky behaviors related to alcohol use , such as driving under the influence more than one time over the past year , where endorsement of at least one item indicated problem drinking . 
 binge drinking was defined as consuming five or more drinks ( men ) or four or more drinks ( women ) in 1 day during the past year . 
 smokers were identified as those ever smoking 100 cigarettes in their lifetime , with those persisting in this habit defined as current smokers and those having quit as past smokers . in longitudinal models , service branch , service component , 
 pay grade , military occupation , birth year , race / ethnicity , and education were assessed at baseline . 
 smoking status , alcohol use , bmi , combat deployment , ptsd , depression , anxiety , panic , and all sleep variables were assessed at all available time points . 
 the outcome of interest was incident diagnosed diabetes self - reported on a follow - up survey among people without diabetes at the baseline assessment . at baseline , participants reported if they had ever been told by a doctor or health professional that they had diabetes or 
 at follow - up assessments , participants responded to the same question in regard to the previous 3 years . 
 univariate analyses assessed associations of incident diabetes with sleep , military , demographic , behavioral , and mental health characteristics while accounting for multiple periods of observation per subject . since repeated measurements were available with up to two follow - up assessments per person , generalized estimating equations ( gees ) were used to account for these multiple assessments while adjusting for fixed and time - varying covariates , to estimate adjusted odds of reporting incident diabetes ( 22 ) 
 . incident diabetes was determined at each follow - up assessment , and all covariates were evaluated using data from the previous assessment ( 22 ) . in all multivariable models , adjustments were performed for known type 2 diabetes risk factors captured in the study : age , sex , education , bmi , and race / ethnicity . 
 the final adjusted models were built using a stepwise backward reduction algorithm that retained all significant variables ( p < 0.10 ) ( table 1 ) while always keeping age , sex , race / ethnicity , education , and bmi in the model . 
 additionally , mental health variables were added to the final models to determine if this altered the relationship between sleep and diabetes . 
 first - order interaction terms between retained sleep variables and age , sex , and bmi were tested to determine whether the exposure - outcome associations varied by these characteristics 
 . possible presence of multicollinearity in multivariable models was considered if the variance inflation factor exceeded 4 . 
 best - fitting gee models were judged using the quasilikelihood information criterion , the measure developed for gee models analogous to the akaike information criterion . 
 statistical analyses were performed using sas statistical software , version 9.3 ( sas institute , inc . , 
 baseline demographic , military , and behavioral characteristics of millennium cohort participants by development of diabetes at follow - up 
 of the 77,047 participants enrolled at baseline , follow - up surveys were completed by 55,021 participants at year 3 and 54,790 at year 6 . since incident diabetes was the outcome of interest , eligible participants included those who were followed until either the outcome was reported to have occurred at year 3 or 6 , or until their last completed follow - up if the outcome was not reported . 
 exclusions included participants reporting provider - diagnosed diabetes at baseline , missing outcome or exposure data , or if dod outpatient or inpatient medical encounter data contained codes for type 1 diabetes ( n = 72 ) ( icd-9 codes 250.x1 and 250.x3 ) . 
 the primary data sources were the self - administered millennium cohort surveys and dod electronic personnel files , managed by the defense manpower data center ( dmdc , seaside , ca ) . 
 for these analyses , electronic records from dmdc provided information on age , sex , race / ethnicity , education , pay grade , service branch , service component , military occupation , dates of separation , and deployment dates . 
 dmdc information was supplemented with self - reported data to reduce missing values when necessary . 
 three sleep variables were included in the analyses : 1 ) trouble sleeping , 2 ) sleep duration , and 3 ) sleep apnea . 
 trouble sleeping was assessed using the sleep items on the prime - md patient health questionnaire ( phq ) and the ptsd checklist  civilian version ( pcl - c ) ( 1720 ) . 
 the phq asks ,  over the last 4 weeks , how often have you experienced trouble falling asleep or staying asleep ?  the pcl - c asks ,  in the past month , have you had trouble falling asleep or staying asleep ?  participants with trouble sleeping were defined as those who responded 
 moderately  or above on the pcl - c sleep item or  several days  or longer on the phq sleep item . 
 sleep duration was reported as whole number of hours slept in an average 24-h period over the past month . 
 participants with sleep apnea were identified by self - report as being told by a doctor or other health professional they had sleep apnea . 
 ptsd was assessed using the pcl - c ( 17 ) , a 17-item self - report screening tool for ptsd symptoms during the past 30 days on a 5-point likert scale , ranging from 1 ( not at all ) to 5 ( extremely ) . 
 a positive screen for ptsd at baseline was defined as a report of a moderate or higher level of at least one intrusion symptom , three avoidance symptoms , and two hyperarousal symptoms ( criteria established by dsm - iv ) ( 21 ) . 
 major depressive disorder was assessed using nine items from the phq ( sensitivity = 0.93 ; specificity = 0.89 ) corresponding to the depression diagnosis based on the dsm - iv ( 19 ) . 
 a positive screen for depression at baseline was defined as 1 ) endorsement of depressed mood or anhedonia and 2 ) response of  more than half the days  or  nearly every day  to at least five items . other anxiety ( 6 items ) and panic ( 15 items ) symptoms 
 problem drinking was defined using items from the phq on risky behaviors related to alcohol use , such as driving under the influence more than one time over the past year , where endorsement of at least one item indicated problem drinking . 
 binge drinking was defined as consuming five or more drinks ( men ) or four or more drinks ( women ) in 1 day during the past year . 
 smokers were identified as those ever smoking 100 cigarettes in their lifetime , with those persisting in this habit defined as current smokers and those having quit as past smokers . in longitudinal models , service branch , service component , 
 pay grade , military occupation , birth year , race / ethnicity , and education were assessed at baseline . 
 smoking status , alcohol use , bmi , combat deployment , ptsd , depression , anxiety , panic , and all sleep variables were assessed at all available time points . 
 the outcome of interest was incident diagnosed diabetes self - reported on a follow - up survey among people without diabetes at the baseline assessment . at baseline , participants reported if they had ever been told by a doctor or health professional that they had diabetes or 
 at follow - up assessments , participants responded to the same question in regard to the previous 3 years . 
 univariate analyses assessed associations of incident diabetes with sleep , military , demographic , behavioral , and mental health characteristics while accounting for multiple periods of observation per subject . since repeated measurements were available with up to two follow - up assessments per person , generalized estimating equations ( gees ) were used to account for these multiple assessments while adjusting for fixed and time - varying covariates , to estimate adjusted odds of reporting incident diabetes ( 22 ) 
 . incident diabetes was determined at each follow - up assessment , and all covariates were evaluated using data from the previous assessment ( 22 ) . in all multivariable models , adjustments were performed for known type 2 diabetes risk factors captured in the study : age , sex , education , bmi , and race / ethnicity . 
 the final adjusted models were built using a stepwise backward reduction algorithm that retained all significant variables ( p < 0.10 ) ( table 1 ) while always keeping age , sex , race / ethnicity , education , and bmi in the model . 
 additionally , mental health variables were added to the final models to determine if this altered the relationship between sleep and diabetes . 
 first - order interaction terms between retained sleep variables and age , sex , and bmi were tested to determine whether the exposure - outcome associations varied by these characteristics 
 . possible presence of multicollinearity in multivariable models was considered if the variance inflation factor exceeded 4 . 
 best - fitting gee models were judged using the quasilikelihood information criterion , the measure developed for gee models analogous to the akaike information criterion . 
 statistical analyses were performed using sas statistical software , version 9.3 ( sas institute , inc . , 
 baseline demographic , military , and behavioral characteristics of millennium cohort participants by development of diabetes at follow - up 
 a total of 47,093 participants were included in this analysis and followed for up to 6 years . 
 incident diabetes was assessed at each 3-year period separately , with 13,621 participants followed for one 3-year period , and 33,472 participants followed for two 3-year periods . 
 a total of 383 cases occurred during years 03 and 488 cases occurred during years 36 of follow - up . 
 the 488 cases that did not occur until years 36 are also present in the first column of table 1 as not having diabetes from year 03 . at baseline , 
 participants overall were on average young ( 34.9 years of age , sd 9.0 years ) , slightly overweight ( bmi 26.0 kg / m , sd 3.3 ) , 25.6% female , and more likely than not to be white , non - hispanic race , less than college educated , army service branch , and on active duty ( table 1 ) . 
 overall , 871 participants newly self - reported diabetes , for a cumulative annual incidence of 3.6/1,000 person - years . 
 participants who newly self - reported diabetes during follow - up were significantly more likely at baseline to have reported trouble sleeping , sleep duration 5 h , and sleep apnea ( table 1 ) . 
 sleep duration > 8 h was reported by similar proportions of participants with and without newly self - reported diabetes . 
 participants with new - onset diabetes were on average older , had a higher bmi , and were more likely of nonwhite race . 
 characteristics associated with new - onset diabetes included no deployment , having separated from the military since baseline , army or navy / coast guard service branch , reserves / national guard service component , enlisted pay grade , and administration / supply occupation . 
 binge or problem drinking was less likely to have been reported in people reporting new - onset diabetes , and current and former smoking frequency was similar in groups with and without new - onset diabetes . 
 all mental health symptoms occurred significantly more frequently among those who newly reported diabetes during follow - up . 
 factors associated with a higher odds of incident diabetes in models adjusted for sex and known type 2 diabetes risk factors ( age , bmi , education , and race / ethnicity ) included trouble sleeping , sleep duration 5 h compared with 7 h , and sleep apnea ( table 2 ) 
 . several military service characteristics remained significantly related to the outcome , including no deployment , enlisted pay grade , administration / supply occupation , and having separated from the military since baseline ( table 2 ) . 
 binge or problem drinking remained significantly associated , and current smoking emerged as a risk factor for new - onset diabetes after adjustment . screening positive for depression , panic , other anxiety disorder , and ptsd were all significantly associated with higher odds of new - onset diabetes in these adjusted models ( table 2 ) . 
 relative odds of incident self - reported diabetes by each baseline characteristic of interest in the multivariable model ( table 3 ) , sleep characteristics remained significantly and independently associated with higher diabetes odds . 
 the sleep variables that entered the final model included reported trouble sleeping and sleep apnea , which were associated with significant increases in the odds of diabetes of 1.21 to 1.78 , respectively . 
 these associations were not only independent of each other but also the other covariates adjusted for in the model ( table 3 ) . 
 military service characteristics significantly associated with diabetes odds included deployment without combat experience , administration or supply occupations , pay grade , and having separated from the military since baseline . 
 a positive screen for ptsd was significantly associated with higher diabetes odds , independent of sleep characteristics and other covariates in the model , and panic disorder was of borderline significance . 
 tests for interactions between the sleep variables included in the models in table 3 and age , sex , and bmi were all nonsignificant ( p > 0.10 ) . 
 no variable had a variance inflation factor > 4 , indicating that collinearity was unlikely . 
 the model in table 3 was rerun with sleep duration substituted for trouble sleeping but with the other included covariates unchanged . 
 the quasilikelihood information criterion for the model containing trouble sleeping was slightly lower ( 8,640.9 ) than when sleep duration was included in the model ( 8,646.3 ) . 
 the sleep duration variable was insignificant overall in this model ( p = 0.1585 ) , although two sleep categories were significantly related to diabetes odds ( < 5-h odds ratio [ or ] 1.52 [ 95% ci 1.092.14 ] ; 5-h or 1.28 [ 1.011.62 ] ) . 
 several additional modifications of the multivariable model shown in table 3 were conducted . in the first modification , symptoms of depression and anxiety disorder 
 were included in the model to assess whether adjustment for these additional factors altered the association between sleep and diabetes odds . in the second modification , all 
 subjects reporting sleep apnea were removed from the model shown in table 3 . in each of these modifications , 
 the statistical significance of the associations between the sleep variables and diabetes odds remained unchanged , and the associations were of similar magnitude to those seen in table 3 ( data not shown ) . 
 longitudinal multivariable regression model of sleep characteristics and mental health symptoms as risk factors for incident self - reported diabetes in the millennium cohort 
 our study findings confirm that sleep characteristics are associated with subsequent new - onset self - reported diabetes . 
 these associations persisted after adjustment for multiple mental health conditions known to affect sleep duration and quality , including ptsd and depression ( 1113 ) . 
 therefore , it is unlikely that sleep simply serves as a surrogate marker for associated mental health conditions previously shown to predict higher diabetes risk ( 9,10 ) . additionally , participants reporting provider - diagnosed sleep apnea were at higher risk of developing self - reported diabetes during follow - up . 
 although sleep apnea is associated with higher bmi and both altered sleep quality and quantity , adjustment for these variables did not alter the significance of the higher diabetes risk associated with this condition ( 23 ) . 
 overall , these results suggest that the associations between type 2 diabetes risk and mental health conditions and sleep characteristics , if causal , may involve different pathways . whereas trouble sleeping and shorter sleep duration were associated with higher risk of diabetes in a model adjusted for known diabetes risk factors , longer sleep duration was not , in contrast to findings that others have reported ( 4 ) . 
 the reason for this discrepancy is unclear , but it may be due to the relatively younger age of our cohort compared with investigations reporting higher diabetes risk with longer sleep duration ( 24,25 ) . the independent role of mental health conditions associated with poor quality and quantity of sleep in predicting risk of diabetes has only been addressed in a limited manner in previous research . 
 the nurses health study investigators examined sleep as a predictor of incident diabetes in this cohort from 19861996 and adjusted for an assessment of depression based on the short form 36 health survey that was obtained in 1992 , approximately midway through the follow - up period ( 24 ) . 
 thus , it is unclear whether depression preceded or followed diabetes onset during the first 6 years of follow - up . 
 an analysis using the national health and nutrition examination survey i epidemiologic follow - up study adjusted for depression while examining the association between sleep and diabetes incidence , although no association was seen between depression and diabetes incidence in this cohort ( 25 ) . 
 lastly , a prospective study of swedish men assessed the presence of depression with a simple yes / no question ,  do you feel depressed ?  and did not provide information on the accuracy of this question in capturing this condition ( 26 ) . to our knowledge , our study is the first to consider mental health conditions other than depression as potential confounding factors and to measure their occurrence prior to the onset of diabetes . 
 previous prospective research on sleep characteristics and type 2 diabetes risk has not considered whether sleep duration or quality is related to this outcome independent from sleep apnea , a known cause of sleep disturbance . in our fully adjusted analysis , 
 both sleep apnea and trouble sleeping had independent associations with diabetes risk , suggesting that the associations between sleep characteristics and diabetes risk do not merely serve as markers for the presence of sleep - disordered breathing . 
 in addition to the known higher risk of obesity associated with sleep apnea , other factors associated with sleep - disordered breathing may be involved in higher diabetes risk and include abnormal sympathetic activity and release of proinflammatory mediators such as tumor necrosis factor- and interleukin-6 ( 27 ) . in support of the latter , we observed an independent association between sleep apnea and diabetes risk after adjustment for bmi . 
 this suggests that manifestations of sleep apnea other than the impact of greater general adiposity may be associated with diabetes risk , although it is possible that incomplete adjustment due to residual confounding occurred due to the inaccuracy of bmi in characterizing body composition . 
 the association between trouble sleeping or shorter sleep duration and diabetes risk independent of associated weight gain and sleep apnea is not well understood , but it may be related to both changes in insulin sensitivity , secretion , and glucose effectiveness . 
 one investigation reported significant or borderline - significant reductions in insulin sensitivity , insulin secretion , glucose effectiveness , and the disposition index immediately after a 5-day period of sleep deprivation ( 4 h of sleep per night ) among 11 healthy lean males in a controlled setting ( 28 ) . 
 all these changes have been associated with higher risk of the development of type 2 diabetes ( 29 ) . 
 several characteristics of military service were significantly associated with lower diabetes risk in multivariable models , including deployment , officer pay grade , and remaining in military service . 
 for most comparisons , military deployment with or without combat exposure was related to a lower risk for diabetes , possibly reflecting selection of people medically fit for deployment who are at lower risk for diabetes . 
 the mental health conditions independently associated with diabetes risk included those characterized by excessive stress , including ptsd and panic disorder . 
 a review of the literature on stress and diabetes risk concluded that both general emotional and job stress were repeatedly associated with a higher risk of developing diabetes in adults ( 30 ) . 
 overactivation of the hypothalamic - pituitary axis and abnormal stimulation of the sympathetic nervous system are thought to accompany excessive levels of stress , with accumulation of visceral fat and promotion of an inflammatory state with greater insulin resistance ( 31 ) . a previous analysis of the members of this cohort recruited in 2001 and followed for 3 years similarly demonstrated a higher diabetes risk associated with ptsd symptoms . 
 first , type 2 diabetes was determined using self - reported data , and it was not confirmed by medical record review . 
 however , because self - reported diabetes compared with physician diagnosis or medical record data have been shown to have a high sensitivity ( 7099% ; median 81% ) and specificity ( 9299% ) , there is less likelihood for misclassification ( 32 ) . 
 also , it is possible that individuals self - reporting type 1 diabetes were included in this analysis . 
 however , individuals with a type 1 diabetes diagnosis in their dod medical records were excluded , although this elimination process would miss those participants no longer receiving care from military treatment facilities after discharge or as reservists . in addition , positive screens for mental disorders were determined from self - report . also , we were unable to adjust for diet or family history , which are known risk factors for diabetes . 
 we expect these findings to generalize to younger populations who are sufficiently healthy to enter into the u.s . 
 armed forces , but it is unclear whether the results would apply to dissimilar populations . 
 self - report of clinician - diagnosed sleep apnea was obtained , and the sensitivity and specificity of this report compared with the medical record are not known . 
 medical encounter data were not used to confirm diagnoses of diabetes or sleep apnea in this cohort due to the potential for some of its members , particularly those in the reserves or national guard , to have received care in non - dod facilities . 
 inaccurate classification of sleep apnea and other covariates potentially may have limited our ability to adjust for confounding . 
 finally , self - reported sleep data were assessed during the prior 4 weeks , and sleep duration was rounded to the nearest whole hour , potentially biasing the estimate of actual sleep time ( 3335 ) . 
 this is the first study , to our knowledge , to examine baseline sleep characteristics in relation to incident diabetes while adjusting for several mental health conditions known to impair sleep and be associated with type 2 diabetes . 
 furthermore , this research was conducted in a large population - based study of active duty and reserve / national guard u.s . military personnel from all service branches . 
 additional strengths include the large sample size and up to 6-year duration of follow - up . 
 moreover , no association was found between reporting type 2 diabetes at baseline and nonresponse to the first follow - up questionnaire among the first panel of enrollees , suggesting that the development of diabetes was unlikely to have influenced the probability of responding to the follow - up survey ( 36 ) . 
 finally , previous evaluations of possible biases suggest the cohort is reasonably representative of military personnel in terms of baseline demographic and mental health characteristics , that participants report health and exposure data reliably , and that enrollment in the study is not influenced by health status ( 14,3740 ) . 
 we conclude that trouble sleeping and sleep apnea predict higher diabetes risk , independent of multiple diabetes risk factors and several mental health conditions . of note 
 , self - reported sleep apnea was also associated with a significantly higher risk for diabetes even after adjustment for other sleep characteristics , bmi , and mental health conditions . 
 substitution of sleep duration for trouble sleeping in multivariable modeling yielded nearly identical results , with significantly higher risk seen with < 6 h of sleep . 
 confirmation of these findings through further analyses may advance our understanding of diabetes pathophysiology and create new opportunities for prevention .","objectiveresearch has suggested that a higher risk of type 2 diabetes associated with sleep characteristics exists . 
 however , studies have not thoroughly assessed the potential confounding effects of mental health conditions associated with alterations in sleep.research design and methodswe prospectively assessed the association between sleep characteristics and self - reported incident diabetes among millennium cohort study participants prospectively followed over a 6-year time period . 
 surveys are administered approximately every 3 years and collect self - reported data on demographics , height , weight , lifestyle , features of military service , sleep , clinician - diagnosed diabetes , and mental health conditions assessed by the prime - md patient health questionnaire and the ptsd checklist  civilian version . 
 statistical methods for longitudinal data were used for data analysis.resultswe studied 47,093 participants ( mean 34.9 years of age ; mean bmi 26.0 kg / m2 ; 25.6% female ) . during 6 years of follow - up , 871 incident diabetes cases occurred ( annual incidence 3.6/1,000 person - years ) . in univariate analyses , 
 incident diabetes was significantly more likely among participants with self - reported trouble sleeping , sleep duration < 6 h , and sleep apnea . 
 participants reporting incident diabetes were also significantly older , of nonwhite race , of higher bmi , less likely to have been deployed , and more likely to have reported baseline symptoms of panic , anxiety , posttraumatic stress disorder , and depression . after adjusting for covariates , trouble sleeping ( odds ratio 1.21 [ 95% ci 1.031.42 ] ) and sleep apnea ( 1.78 [ 1.392.28 ] ) were significantly and independently related to incident diabetes.conclusionstrouble sleeping and sleep apnea predict diabetes risk independent of mental health conditions and other diabetes risk factors .",pubmed
"we retrospectively reviewed the radiological and clinical findings of 13 patients with a pathologically confirmed mesenchymal hamartoma of the liver . 
 the institutional review board approved the review of the radiological and clinical data for this study and waived the requirement for informed consent . 
 a computed hospital information system was used to identify all patients with a pathologically proved mesenchymal hamartoma during a 15-year period . the pathological diagnosis for a mesenchymal hamartoma 
 was based on overgrowth of the mesenchymal stroma and the proliferation of architecturally abnormal bile ducts with or without a cystic change , accompanied by periductal collaring of the stromal cells ( 1 , 9 ) . 
 three pediatric radiologists retrospectively reviewed the radiological features of the hepatic mesenchymal hamartomas by consensus . 
 ct scans were obtained with various third generation ct scanners at 80 - 120 ma , 100 - 120 kvp , and 5- to 10-mm thick sections . 
 ct scans were obtained after an intravenous bolus injection of contrast media and portal venous phase scans of the liver were available for all patients . 
 ct images of the tumor were evaluated for location , size , presence of septa within the cystic portion , ct attenuation of the tumor relative to the surrounding hepatic parenchyma ( low , iso , or high ) , pattern of contrast - enhancement ( homogeneous or heterogeneous enhancement of the solid portion , and septal enhancement of the cystic portion ) and presence of calcification on a pre - contrast ct scan . according to the extent of cystic component on the ct scans , 
 we classified each tumor into one of three types : 1 ) multiseptated cystic - a purely cystic tumor with multiple thin septa ; 2 ) mixed cystic and solid - a partially cystic tumor with irregular thick septa ; 3 ) solid - a purely solid tumor without a cystic portion . 
 the cystic portion was defined as an area of low attenuation that showed 0 - 40 hounsfield units ( hu ) of ct attenuation value . 
 we classified the septal thickness as follows : thin ( < 2 mm in thickness ) or thick ( > 2 mm in thickness ) . 
 ultrasonographic ( us ) scans of nine patients were available for review . among them , seven patients had a color or power doppler us examination . 
 us images were evaluated for the presence of capsule , internal content of the cystic portion ( echogenic debris or fluid - fluid level ) , septal thickness within the cystic portion , and the echogenicity of the solid portion . 
 the amount of mesenchymal stroma , the presence or absence of duct proliferation , the amount of hepatocytes , and the presence or absence of vascular proliferation was evaluated . 
 the amount of mesenchymal stroma and hepatocytes were evaluated semi - quantitatively : little in < 10% of the mesenchymal hamartoma , moderate in 10 - 50% of the mesenchymal hamartoma and abundant in > 50% of the mesenchymal hamartoma . 
 the age of the patients , size of the tumor and various histological findings were compared with the imaging features of the hepatic mesenchymal hamartomas among the three ct types using the kruskall - wallis test . 
 chicago , il ) . a two - tailed p - value of < 0.05 was considered to be statistically significant . 
 we retrospectively reviewed the radiological and clinical findings of 13 patients with a pathologically confirmed mesenchymal hamartoma of the liver . 
 the institutional review board approved the review of the radiological and clinical data for this study and waived the requirement for informed consent . 
 a computed hospital information system was used to identify all patients with a pathologically proved mesenchymal hamartoma during a 15-year period . the pathological diagnosis for a mesenchymal hamartoma 
 was based on overgrowth of the mesenchymal stroma and the proliferation of architecturally abnormal bile ducts with or without a cystic change , accompanied by periductal collaring of the stromal cells ( 1 , 9 ) . 
 three pediatric radiologists retrospectively reviewed the radiological features of the hepatic mesenchymal hamartomas by consensus . 
 ct scans were obtained with various third generation ct scanners at 80 - 120 ma , 100 - 120 kvp , and 5- to 10-mm thick sections . 
 ct scans were obtained after an intravenous bolus injection of contrast media and portal venous phase scans of the liver were available for all patients . 
 ct images of the tumor were evaluated for location , size , presence of septa within the cystic portion , ct attenuation of the tumor relative to the surrounding hepatic parenchyma ( low , iso , or high ) , pattern of contrast - enhancement ( homogeneous or heterogeneous enhancement of the solid portion , and septal enhancement of the cystic portion ) and presence of calcification on a pre - contrast ct scan . according to the extent of cystic component on the ct scans , 
 we classified each tumor into one of three types : 1 ) multiseptated cystic - a purely cystic tumor with multiple thin septa ; 2 ) mixed cystic and solid - a partially cystic tumor with irregular thick septa ; 3 ) solid - a purely solid tumor without a cystic portion . 
 the cystic portion was defined as an area of low attenuation that showed 0 - 40 hounsfield units ( hu ) of ct attenuation value . 
 we classified the septal thickness as follows : thin ( < 2 mm in thickness ) or thick ( > 2 mm in thickness ) . 
 ultrasonographic ( us ) scans of nine patients were available for review . among them , seven patients had a color or power doppler us examination . 
 us images were evaluated for the presence of capsule , internal content of the cystic portion ( echogenic debris or fluid - fluid level ) , septal thickness within the cystic portion , and the echogenicity of the solid portion . 
 the amount of mesenchymal stroma , the presence or absence of duct proliferation , the amount of hepatocytes , and the presence or absence of vascular proliferation was evaluated . 
 the amount of mesenchymal stroma and hepatocytes were evaluated semi - quantitatively : little in < 10% of the mesenchymal hamartoma , moderate in 10 - 50% of the mesenchymal hamartoma and abundant in > 50% of the mesenchymal hamartoma . 
 the age of the patients , size of the tumor and various histological findings were compared with the imaging features of the hepatic mesenchymal hamartomas among the three ct types using the kruskall - wallis test . 
 chicago , il ) . a two - tailed p - value of < 0.05 was considered to be statistically significant . 
 the subjects were seven boys and six girls , aged from 6 months to 7 years 6 months ( mean age : 3 years 2 months ) . 
 there was no significant difference of age ( p = 0.93 ) among the three ct types of tumors . 
 the patients presented with abdominal distension ( n = 8) , a palpable mass ( n = 3 ) or an incidentally detected tumor during us ( n = 2 ) for other clinical indications : a patient ( case 8) with peutz - jeghers syndrome , and a patient ( case 13 ) that had a kasai operation for biliary atresia at one month of age , respectively . the serum level of alpha - fetoprotein , total   and direct bilirubin level , alkaline phosphatase level , and level of transaminases ( aspartate transaminase and alanine transaminase ) were normal in all except two patients that had an elevated level of alpha - fetoprotein : the patient with biliary atresia ( case 13 ) and a 9-month - old girl ( case 5 ) . 
 the tumor occurred in the right lobe of the liver in nine patients and in the left lobe in four patients . 
 splenomegaly , ascites and retroperitoneal lymphadenopathy were noted in one patient that had biliary atresia . 
 the longest diameter of the tumors ranged from 1.8 to 20 cm ( mean 13.0 cm ) and was larger than 10 cm in 11 patients ( 85% ) . 
 there was no significant difference in the size of the tumors ( p = 0.79 ) among the three ct types of tumors . 
 1 ) , mixed solid and cystic in five patients ( 38% ) ( figs . 2 , 3 ) , and solid in four patients ( 31% ) ( figs . 4 , 5 ) . in four patients with a multiseptated cystic tumor , us ( n = 1 ) showed a well - defined tumor with variable cystic spaces and multiple thin septa in the liver ( fig . 
 all of these tumors showed fine enhancing septa within the cystic tumor on post - contrast ct ( fig . 
 neither calcification   nor a solid portion was detected in these patients . in five patients with a mixed cystic and solid tumor , us ( n 
 = 5 ) showed a well - defined tumor with variable - sized cystic spaces , irregularly thick septa , and a solid portion of variable extent with heterogeneous hyperechogenicity ( figs . 
 2a , 3a ) . in three patients , internal debris with fluid - fluid level was seen in the cystic portion of the tumor ( figs . 
 2a , 2c ) . on color and power doppler us ( n = 4 ) 
 a tiny nodular intratumoral calcification was seen in one patient ( case 8) ( fig . 
 = 3 ) showed a well - defined , isoechoic tumor ( n = 1 ) or slightly hyperechoic ( n = 2 ) ( fig . 
 color or power doppler us   ( n = 3 ) showed central ( fig . 
 these tumors showed low attenuation on a pre - contrast ct scan in all patients , except for one with a high attenuating lesion . at post - contrast ct , 
 4b , 5c ) . in the patient that had biliary atresia ( case 13 ) , the tumor appeared as a 1.8-cm sized enhancing nodule with a peripheral rim of low attenuation at post - contrast ct ( fig . 
 the mesenchymal hamartomas showed multiple variable sized cystic portions within the tumor in their gross morphology ( figs . 
 each mesenchymal hamartoma with a multiseptated cystic appearance contained various amounts of myxoid or collagenous stroma and showed proliferation of the malformed bile ducts with periductal collaring of the stromal cells ( fig . 
 all of the four patients with a multiseptated cystic tumor contained a small amount of hepatocytes ( < 10% ) on a semiquantitative evaluation . 
 the mesenchymal hamartomas with a mixed solid and cystic appearance also contained various amounts of myxoid or collagenous stroma and showed proliferation of the malformed bile ducts with periductal collaring of the stromal cells ( fig . 
 the amount of hepatocytes was small in three patients and moderate in two patients . in four patients with a solid mesenchymal hamartoma 
 , a moderate ( n = 3 ) amount or abundant amount ( n = 1 ) of hepatocytes was found in the cords , islands or lobular pattern . 
 the solid tumor also contained various amounts of mesenchymal stroma and proliferation of the small bile duct was seen ( fig . 
 4d ) . in one patient with an enhancing nodule with a peripheral rim of low attenuation ( case 13 ) , the enhancing portion revealed a large amount hepatocytes histologically with a rim of myxoid stroma . 
 the amount of hepatocytes was significantly different among the multiseptated cystic , mixed solid and cystic , and solid tumors ( p = 0.042 ) . 
 the other pathological findings such as the amount of mesenchymal stroma , the presence of a bile duct or vessel proliferation were not statistically significant among the three groups of the different imaging spectrum . 
 the subjects were seven boys and six girls , aged from 6 months to 7 years 6 months ( mean age : 3 years 2 months ) . 
 there was no significant difference of age ( p = 0.93 ) among the three ct types of tumors . 
 the patients presented with abdominal distension ( n = 8) , a palpable mass ( n = 3 ) or an incidentally detected tumor during us ( n = 2 ) for other clinical indications : a patient ( case 8) with peutz - jeghers syndrome , and a patient ( case 13 ) that had a kasai operation for biliary atresia at one month of age , respectively . the serum level of alpha - fetoprotein , total   and direct bilirubin level , alkaline phosphatase level , and level of transaminases ( aspartate transaminase and alanine transaminase ) were normal in all except two patients that had an elevated level of alpha - fetoprotein : the patient with biliary atresia ( case 13 ) and a 9-month - old girl ( case 5 ) . 
 the tumor occurred in the right lobe of the liver in nine patients and in the left lobe in four patients . 
 splenomegaly , ascites and retroperitoneal lymphadenopathy were noted in one patient that had biliary atresia . 
 the longest diameter of the tumors ranged from 1.8 to 20 cm ( mean 13.0 cm ) and was larger than 10 cm in 11 patients ( 85% ) . 
 there was no significant difference in the size of the tumors ( p = 0.79 ) among the three ct types of tumors . 
 1 ) , mixed solid and cystic in five patients ( 38% ) ( figs . 2 , 3 ) , and solid in four patients ( 31% ) ( figs . 4 , 5 ) . in four patients with a multiseptated cystic tumor , us ( n = 1 ) showed a well - defined tumor with variable cystic spaces and multiple thin septa in the liver ( fig . 
 all of these tumors showed fine enhancing septa within the cystic tumor on post - contrast ct ( fig . 
 neither calcification   nor a solid portion was detected in these patients . in five patients with a mixed cystic and solid tumor , us ( n 
 = 5 ) showed a well - defined tumor with variable - sized cystic spaces , irregularly thick septa , and a solid portion of variable extent with heterogeneous hyperechogenicity ( figs . 
 2a , 3a ) . in three patients , internal debris with fluid - fluid level was seen in the cystic portion of the tumor ( figs . 
 2a , 2c ) . on color and power doppler us ( n = 4 ) 
 a tiny nodular intratumoral calcification was seen in one patient ( case 8) ( fig . 
 = 3 ) showed a well - defined , isoechoic tumor ( n = 1 ) or slightly hyperechoic ( n = 2 ) ( fig . 
 color or power doppler us   ( n = 3 ) showed central ( fig . 
 these tumors showed low attenuation on a pre - contrast ct scan in all patients , except for one with a high attenuating lesion . at post - contrast 
 4b , 5c ) . in the patient that had biliary atresia ( case 13 ) , the tumor appeared as a 1.8-cm sized enhancing nodule with a peripheral rim of low attenuation at post - contrast ct ( fig . 
 the mesenchymal hamartomas showed multiple variable sized cystic portions within the tumor in their gross morphology ( figs . 
 each mesenchymal hamartoma with a multiseptated cystic appearance contained various amounts of myxoid or collagenous stroma and showed proliferation of the malformed bile ducts with periductal collaring of the stromal cells ( fig . 
 all of the four patients with a multiseptated cystic tumor contained a small amount of hepatocytes ( < 10% ) on a semiquantitative evaluation . 
 the mesenchymal hamartomas with a mixed solid and cystic appearance also contained various amounts of myxoid or collagenous stroma and showed proliferation of the malformed bile ducts with periductal collaring of the stromal cells ( fig . 
 the amount of hepatocytes was small in three patients and moderate in two patients . in four patients with a solid mesenchymal hamartoma 
 , a moderate ( n = 3 ) amount or abundant amount ( n = 1 ) of hepatocytes was found in the cords , islands or lobular pattern . 
 the solid tumor also contained various amounts of mesenchymal stroma and proliferation of the small bile duct was seen ( fig . 
 4d ) . in one patient with an enhancing nodule with a peripheral rim of low attenuation ( case 13 ) 
 , the enhancing portion revealed a large amount hepatocytes histologically with a rim of myxoid stroma . 
 the amount of hepatocytes was significantly different among the multiseptated cystic , mixed solid and cystic , and solid tumors ( p = 0.042 ) . 
 the other pathological findings such as the amount of mesenchymal stroma , the presence of a bile duct or vessel proliferation were not statistically significant among the three groups of the different imaging spectrum . 
 a mesenchymal hamartoma is the second most common benign hepatic tumor in children and accounts for 8% of all hepatic tumors in children ( 10 - 14 ) . 
 histologically , a mesenchymal hamartoma appears as a disordered arrangement of mesenchyme , bile ducts and hepatocytes ( 1 - 3 , 9 , 15 ) 
 . cords of normal - appearing hepatocytes are separated by zones of loose poorly cellular mesenchyme . 
 cystic degeneration of the mesenchyme with resultant fluid accumulation from obstruction and dilatation of the lymphatics and/or by entrapped bile ducts may lead to enlargement of the tumor ( 12 ) . 
 the margin between the liver and the tumor is distinct ; however , a true capsule is generally not present ( 2 ) . the level of serum alpha - fetoprotein , which is believed to be secreted by the proliferating hepatocytes within the tumor , can be elevated ( 13 , 14 ) . a multiseptated cystic appearance , which may be a typical finding of mesenchymal hamartoma , is rarely seen in other hepatic tumors in children . 
 intratumoral calcification , which can be frequently detected in hepatoblastomas ( over 50% ) or infantile hemangioendotheliomas ( up to 40% ) ( 11 ) , has been reported very rarely for a mesenchymal hamartoma . 
 ( 8) described the ct findings of a mesenchymal hamartoma containing central and peripheral calcifications in a 57-year - old woman . 
 ( 15 ) also described the ct findings of the multiseptated cystic form of a mesenchymal hamartoma with a septal calcification in a 10-year - old boy . in this series 
 although a mesenchymal hamartoma present most frequently as a multiseptated cystic or mixed solid and cystic tumor ( 1 - 5 ) , it can rarely occur as a solid tumor ( 6 - 9 ) . in this study , according to the extent of the cystic component on ct scans , we classified each tumor into one of three types for evaluating the imaging spectrum of this tumor . in this study , 
 the tumor was totally solid in 31% of the patients , and one case of a solid mesenchymal hamartoma ( case 11 ) showed features of a so - called "" mixed hamartoma . "" 
 the mixed hamartoma is a rare hamartomatous lesion composed of all cellular components of the normal liver , including a larger amount of hepatocytes , ductal proliferation , and hepatocellular - ductal transition . 
 the mixed hamartoma and mesenchymal hamartoma may be in one disease spectrum , since there is some overlapping in the histological features of both hamartomas ( 16 , 17 ) . in children with a solid mesenchymal hamartoma 
 , radiological differential diagnosis should include various hepatic tumors such as a hepatoblastoma , a hepatocellular carcinoma , an infantile hemangioendothelioma , a hepatic adenoma , or a focal nodular hyperplasia . 
 heterogeneous enhancement and absence of a tumor capsule in a mesenchymal hamartoma can be helpful in the differential diagnosis from other tumors . as in one of our patients that had biliary atresia and a solid mesenchymal hamartoma ( case 13 ) , lack 
 ( 18 ) described a mesenchymal hamartoma in a 5-month - old girl with biliary atresia , which was found incidentally during an exploratory laparotomy for obstructive jaundice . 
 lack ( 18 ) showed an interest in this case because one of the theories for development of a mesenchymal hamartoma is biliary obstruction ( 19 ) . in conclusion , 
 a mesenchymal hamartoma of the liver in children can show a wide spectrum of radiological features from a multiseptated cystic tumor to a mixed solid and cystic tumor , and even a solid tumor .","objectivea hepatic mesenchymal hamartoma is an uncommon benign tumor in children and little is known about the spectrum of its radiological features . 
 the purpose of this study is to describe the spectrum of radiological features of a hepatic mesenchymal hamartoma in children.materials and methodsthirteen children with a pathologically confirmed hepatic mesenchymal hamartoma ( m : f = 7:6 ; mean age , 3 years 2 months ) were included in our study . ultrasonography ( us ) was performed in nine patients including color and power doppler us ( n = 7 ) . 
 ct scans were performed in all patients . 
 we evaluated the imaging findings of the hepatic mesenchymal hamartomas and the corresponding pathological features.resultseach patient had a single tumor ( mean diameter : 13 cm [ 1.8 - 20 cm ] ) . 
 on ct and/or us , four patients ( 31% ) had a "" multiseptated cystic tumor "" , five patients ( 38% ) had a "" mixed solid and cystic tumor "" , and four patients ( 31% ) had a "" solid tumor . "" 
 the septa of the cystic portion were thin in the multiseptated cystic tumors and irregularly thick in the mixed solid and cystic tumors as seen on us . on a post - contrast ct scan , solid portions or thick septa of the tumors showed heterogeneous enhancement . 
 the amount of hepatocytes was significantly different among the three tumor groups according to the imaging spectrum ( p = 0.042).conclusiona hepatic mesenchymal hamartoma in children can show a wide spectrum of radiological features , from a multiseptated cystic tumor to a mixed solid and cystic tumor , and even a solid tumor .",pubmed
"rheumatoid arthritis ( ra ) is a disabling and progressive chronic autoimmune disease that carries a significant burden.1 although ra affects people of all ages , its prevalence increases with increasing age and it is more common in women than in men.2,3 a prevalence of 0.5%1% and a mean annual incidence ( ie , rate of new cases ) of 0.02%0.05% have been estimated in studies carried out in northern europe and in north america.4 the prevalence of ra in southern europe has been estimated to be lower than that in northern europe , with rates of 0.45% and 0.66% reported in the two areas , respectively.5 the annual incidence of ra is also lower in southern than in northern european countries.6 estimates of prevalence are often very variable as a result of several factors , including the time period when the study was undertaken ; the age distribution of the country reporting the estimates ; differences in the health care organizations in the reporting countries ; and the source of information.6 similarly , changes in the epidemiology of ra are difficult to predict ; while some studies might suggest a decline in incidence among countries with high rates of ra,5 other investigators suggest that the incidence is expected to increase in europe because of the growing proportion of older people.7,8 ra is associated with a significant economic burden for patients , families , and health care systems.9 moreover , an increase in the costs associated with ra is anticipated as a result of the aging population.7 there is a great amount of variability in estimates of the cost of ra across countries ; these estimates are affected by methods of calculation , reimbursement policies , and availability ( and time of availability ) of drugs and social services . 
 differences in country - specific treatment guidelines on when to introduce newer costly drug classes ( ie , biological agents ) for patients with severe active and erosive disease also contribute to the variation in costs across the european union ( eu).6 however , the macroeconomic condition and the treatment guidelines remain the most important determinants that drive choice of therapy.6 italy has the highest proportion of people aged 65 years or older in the eu and it has been estimated that , by 2029 , 25.7% of the population in italy will be 65 years.7 thus , the health care costs associated with age - related diseases like ra are likely to increase in italy 
 . the introduction of newer costly therapies may further contribute to the expected increase in the economic burden of ra in italy . 
 this review aims to evaluate the economic impact of ra in italy in the past two decades and to compare the costs of ra before and after the introduction of biological agents . 
 in addition , the review will attempt to predict the future costs of ra in italy , and to compare these costs with those in other european countries . while this is not designed as a 
 systematic review  as such , it does follow a similar procedure for identification of publications of relevance . 
 comprehensive electronic searches of pubmed / medline were performed using the search terms ( in any field ) : prevalence or cost and rheumatoid arthritis and italy , with no language restriction . 
 the search identified 414 papers ; abstracts of these publications were assessed for their relevance . in total , six papers reporting data on the prevalence of ra in italy1015 and four papers reporting economic data1619 were initially considered as adequate for the objectives of this review . 
 in addition , further data were extracted from the websites of italian or european public health agencies and from italian journals to add to the previously identified published literature on the health care costs of ra in italy . 
 this led to the inclusion of a further two articles6,20 that add important information on the past and current costs of ra in italy , as well as on estimates of future costs . 
 although the small population samples and limited coverage of the studies do not allow a firm estimate of the national prevalence of the disease , the studies do give a rough idea of the likely prevalence and also highlight potential geographical variations . a study10 undertaken in 19911992 in chiavari , a small town located on the ligurian coast , found that ra had a prevalence of 0.33% ( 95% confidence interval [ ci ] 0.130.53 ) in the general population , 0.13% ( 95% ci 00.31 ) in men , and 0.51% ( 95% ci 0.180.84 ) in women . 
 this study included 3,294 individuals ( 73.9% of those contacted ) , aged 16 years , identified from four general practices . 
 the mean age of subjects was 48.3 years , which may account for the relatively low prevalence reported in this study . a higher prevalence of 0.46% ( 95% ci 0.330.59 ) was reported in another study11 conducted from april 2004 to june 2004 in 2,155 subjects aged 18 years in the marche region . 
 similar estimates were reported from a survey conducted in 20022003 in tempio pausania ( northern sardinia ) that involved 30,264 subjects aged 18 years;12 prevalence was estimated to be 0.46% in the general population , 0.73% in women , and 0.19% in men . 
 another study carried out in the province of pisa in the years 2006 and 200714 evaluated the prevalence of ra , the reliability of the ra diagnosis and prevalence estimated by general practitioners ( gps ) , and the economic impact of the disease in a total of 26,709 subjects aged over 18 years . 
 each gp completed a questionnaire on their patients ; patients considered by gps to have ra were then invited to attend an appointment at a specialist rheumatology center for confirmation or rejection of the diagnosis made . 
 the estimated prevalence of correctly diagnosed ra was 0.51% ( 95% ci 0.440.57 ) and the reliability of gps when making a diagnosis of ra was 69% overall , although there was a high degree of heterogeneity among gps . 
 benucci et al13 evaluated the incidence of early ra ( new cases ) in a suburban area of florence between september 2005 and august 2006 and suggested a rate of 0.98 ( 95% ci 0.641.32 ) in the overall population , and 1.42 ( 95% ci 0.851.99 ) for females and 0.51 ( 95% ci 0.160.87 ) for males . 
 the mean age of newly diagnosed subjects was 47.7 years in females and 54.9 years in males . 
 the incidence rate observed in this study was higher than that observed in other european countries , possibly due to tight adherence to diagnostic criteria for early ra and to the use of the criteria of the american college of rheumatology ( acr)21 as a method of assessment . 
 the most recent treatment - driven estimation of the prevalence and incidence of ra in italy , performed in a cohort of 2,268,514 males and 2,446,769 females , and based on a diagnosis made according to prescription of at least three ra - specific drugs ( corticosteroids , disease - modifying anti - rheumatic drugs , and biological agents ) , was calculated for the year 2011.15 patients were classified as follows : those on specific drugs were classed as having active ra ; those who had never had more than four prescriptions in the past were classed as unlikely ra ; and those previously on chronic treatment but who had discontinued therapy for > 1 year were classed as having ra in remission . 
 a diagnosis of ra was made by a qualified specialist in a total of 22,801 of these subjects ( 0.48% ) , with a prevalence of active ra , ra in remission , and confirmed ra ( active + remission ra ) of 0.32% , 0.09% , and 0.41% , respectively . 
 the yearly incidence of active ra per 100,000 subjects was 48 ( 95% ci 4057 ) for females and 20 ( 95% ci 1030 ) for males . 
 the female : male ratio for both prevalence and incidence was ~3.5:1 before the fifth decade of life and tended to decline to about 2:1 in subjects aged > 70 years . 
 a modeling analysis of the prevalence of ra in 27 countries in the eu ( plus iceland , norway , and switzerland ) conducted in 20086 estimated that the overall prevalence of ra in italy was 0.49% and was exactly the same as the calculated average prevalence in europe . in this analysis , 
 italy was ranked as eighth in terms of estimated prevalence of ra and was one of four european countries ( together with the uk , sweden , and germany ) in which subjects aged 65 years contributed more than 50% of the diagnosed cases of ra . 
 the data also showed that italy and germany were the european countries with the highest proportion of people aged 65 years . 
 taken together , these data6 suggest that the prevalence of ra will further increase in europe and in italy in line with projected increases in the elderly population . 
 few studies have estimated the costs associated with ra in italy before the availability of biological agents . 
 a retrospective , prevalence - based , multicenter , cost - of - illness study , undertaken in 1998 in ten rheumatology institutes located in northern , central , and southern italy,19 evaluated the direct ( hospitalizations , specialist visits , treatments , diagnostics , and nonmedical costs ) , indirect ( productivity loss and informal care ) , and intangible ( deterioration in health - related quality of life [ hrqol ] of patients , their families , and their friends ) costs associated with ra in 200 patients aged 1865 years . 
 participants were categorized into four classes of severity according to the acr ra functional status index in place at the time of the study.22 the results showed that indirect costs accounted for the largest part of the disease management costs and that social costs ( direct plus indirect costs ) increased as ra worsened . the calculated average annual cost for each patient varied significantly depending on disease severity and correlated with the health assessment questionnaire disability index23 and acr criteria.22 total costs ranged from  3,718.3 for patients in functional class i to  22,946.0 for patients in class iv . both the direct costs ( respectively ,  1,643.4 ,  2,910.2 ,  4,236.5 , and  5,696.8 per patient from class i to class iv ) and the indirect costs (  2,074.9 ,  9,566.4 ,  12,183.1 , and  17,249.2 per patient , respectively ) over the 12 months of observation increased significantly ( p<0.0005 ) across the four functional classes . 
 the modeling study conducted in 20086 used a specifically developed model to estimate the total cost of ra in europe . 
 costs , extracted from the studies identified in a comprehensive literature review , were separated into different categories : medical costs , drugs , nonmedical costs , informal care costs , and indirect costs , and inflated to 2008 values . 
 the estimated costs per patient were then combined with country- and age - specific prevalence data to obtain a figure for the total cost of ra in each country . 
 the mean estimated annual cost per patient in europe with ra was  12,902 , with a clear difference between western europe ( mean  14,997 ) and central / eastern europe ( mean  3,752 ) . 
 the total mean annual cost of the disease across all european countries was estimated at about  25.1 billion . 
 the estimated mean annual cost per patient in the study countries ranged from about  2,000 to  21,000 , the estimated mean annual cost per patient in italy was  11,546 in line with the mean for europe as a whole . in italy , mean costs were estimated at  4,552 for direct costs 
 ( excluding biological agents ) ,  731 for biological agents ,  3,290 for informal care , and  2,972 for indirect costs . 
 this distribution indicates that , at the time of this modeling study ( 2008 ) , direct costs in europe overall represented 43% of the total expenditure . 
 notably , at the time of the analysis , italy and germany had the lowest use of biologics in western europe due to tight budget control policies . 
 for example , in italy , biologics could only be prescribed in the hospital setting and use of such drugs was limited to a small percentage of the total expenditure.6 despite individual differences in costs between countries , costs outside the health care sector , such as informal care and productivity loss , represented the greater part of the costs of ra in all countries.6 the structure of the costs of ra has changed over the years , with a substantial increase in the direct costs of the disease largely as a result of the availability of newer , more costly biological agents . for example , in france , the direct annual costs of ra were estimated to be  4,000 in 2000 ( before the introduction of biological agents)24 and about  12,000 in a 2005 study in which 20% of patients were receiving these agents.25 although the introduction of biological agents has contributed to increased direct medical costs,26,27 their use is associated with marked improvements in disease activity , joint damage , and loss of productivity.18 furthermore , data from the consortium of rheumatology researchers of north america registry have shown that the use of biological agents is associated with a reduction in the risk of cardiovascular events in patients with ra.28 in a health economic analysis carried out in the province of pisa in the years 2006 and 2007,14 the mean annual cost per patient with ra was estimated to be  5,878 ( median  6,434 ; interquartile range  6697,052 ) , with high variability , mainly depending on the degree of disability . in this analysis , more than 90% of the overall annual cost per patient was due to the medical and nonmedical components of direct costs . 
 a more recently published literature review16 evaluated the mean annual social costs per patient and the total social cost of ra in the last decade in italy . 
 data from each study ( table 2 ) , when further analyzed , showed that the mean annual social cost ( ie , overall direct plus indirect costs ) of ra was  13,595 per adult patient ( 2012 values ) . based on prevalence data 
 , it was estimated that ra resulted in a total social cost of  3.5 billion in italy per year . 
 direct medical costs accounted for 21% of the total costs , while the remaining 79% were nonmedical costs ( direct nonmedical costs and indirect costs ) . 
 nonmedical and indirect costs were mainly sustained by individual households for informal care ( 40% of the total cost ) , and by the economy ( due to absence from work of patients and caregivers ; 31% of the total cost ) . 
 based on these data , it is evident that assessment of the economic burden of ra based solely on the evaluation of direct medical costs gives a limited view of the total economic burden of the disease . 
 italy has the second highest percentage of population 65 years of age in the world,29 therefore , an increase in the costs of ra in italy is expected due to the aging population . since mean costs per patient increase with increasing functional disability , reliable health - economic evaluations should estimate the long - term consequences of the changing course of the disease and the effects of preventing or delaying the development of severe disability.30 short - term analyses may be misleading as they tend to neglect the importance of modifications in the course of the disease as a result of treatment intervention and its positive impact on the deterioration of hrqol . 
 a systematic literature review18 on the economic consequences of , and pharmacoeconomic issues relating to , ra , based on a total of 127 published articles , has shown that disease severity , functional disability , age , and socioeconomic status are the most relevant predictors of cost increase in ra . 
 a published editorial20 has estimated the economic impact of ra in italy over the next 30 years using cost - of - illness modeling . 
 this analysis assumed that the costs of ra varied as the disease evolved , and classified disease by stage according to the steinbrocker radiological classification.31 using inferential statistical methods , the probability of evolution toward a more severe stage of the disease in the absence of treatment was estimated . 
 a total of 411,692 patients with ra were classified according to the four stages of the disease and their progression over the next 30 years was simulated . 
 the results showed that , in the next 30 years , the annual cost of ra will increase from  3.3 billion ( for 2009 ) to a peak of  4 billion ( for 2029 ) , including both direct and indirect costs . 
 cost estimates will vary between regions , with higher costs estimated in lombardy (  430500 million ) , lazio (  300330 million ) , sicily (  238281 million ) , and emilia - romagna (  131187 million ) . 
 this suggests that costs will be only partially correlated with the number of inhabitants in different italian regions and will possibly depend on differing regional health care policies . 
 expenditure will not greatly differ between workers ( from  320 million in 2009 to  420 million in 2029 ) and homemakers ( from  318 million in 2009 to  421 million in 2029 ) . 
 the disease was also estimated to have a significant impact on costs due to loss of productivity that are supported by the italian national health care system ( nhs ) , which are predicted to increase from 0.67% of total social security spending in 2009 to 0.7% of this spending in 2029 . 
 in addition , ra was predicted to account for a total of 1% of all nhs costs in 2029 . 
 these data indicate that the costs associated with untreated ra would make a significant contribution to nhs and societal costs in the next decades , thus confirming that delaying early diagnosis and intervention will result in increased overall costs for the community . 
 the development of biosimilar biologics may alter the landscape of biologics and their associated costs in the future . 
 physicians attitudes toward the interchangeability of drugs , indications from regulatory authorities , and national recommendations will have a major impact on the use of biosimilars in the management of ra . while , a statement position from the italian society of rheumatology has raised concerns about the true equivalence of biosimilars in rheumatic diseases and hence on the potential for interchangeability of drugs , studies in ra currently support the similarity of biosimilars and their originators.32 furthermore , as biologics require a more sophisticated manufacturing process than that of existing conventional medications , tight regulation will be required to avoid any possible additional safety risks to make these agents truly cost - effective.33 
 although the small population samples and limited coverage of the studies do not allow a firm estimate of the national prevalence of the disease , the studies do give a rough idea of the likely prevalence and also highlight potential geographical variations . a study10 undertaken in 19911992 in chiavari , a small town located on the ligurian coast , found that ra had a prevalence of 
 0.33% ( 95% confidence interval [ ci ] 0.130.53 ) in the general population , 0.13% ( 95% ci 00.31 ) in men , and 0.51% ( 95% ci 0.180.84 ) in women . 
 this study included 3,294 individuals ( 73.9% of those contacted ) , aged 16 years , identified from four general practices . 
 the mean age of subjects was 48.3 years , which may account for the relatively low prevalence reported in this study . a higher prevalence of 0.46% ( 95% ci 0.330.59 ) was reported in another study11 conducted from april 2004 to june 2004 in 2,155 subjects aged 18 years in the marche region . 
 similar estimates were reported from a survey conducted in 20022003 in tempio pausania ( northern sardinia ) that involved 30,264 subjects aged 18 years;12 prevalence was estimated to be 0.46% in the general population , 0.73% in women , and 0.19% in men . 
 another study carried out in the province of pisa in the years 2006 and 200714 evaluated the prevalence of ra , the reliability of the ra diagnosis and prevalence estimated by general practitioners ( gps ) , and the economic impact of the disease in a total of 26,709 subjects aged over 18 years . 
 each gp completed a questionnaire on their patients ; patients considered by gps to have ra were then invited to attend an appointment at a specialist rheumatology center for confirmation or rejection of the diagnosis made . 
 the estimated prevalence of correctly diagnosed ra was 0.51% ( 95% ci 0.440.57 ) and the reliability of gps when making a diagnosis of ra was 69% overall , although there was a high degree of heterogeneity among gps . 
 benucci et al13 evaluated the incidence of early ra ( new cases ) in a suburban area of florence between september 2005 and august 2006 and suggested a rate of 0.98 ( 95% ci 0.641.32 ) in the overall population , and 1.42 ( 95% ci 0.851.99 ) for females and 0.51 ( 95% ci 0.160.87 ) for males . 
 the mean age of newly diagnosed subjects was 47.7 years in females and 54.9 years in males . 
 the incidence rate observed in this study was higher than that observed in other european countries , possibly due to tight adherence to diagnostic criteria for early ra and to the use of the criteria of the american college of rheumatology ( acr)21 as a method of assessment . 
 the most recent treatment - driven estimation of the prevalence and incidence of ra in italy , performed in a cohort of 2,268,514 males and 2,446,769 females , and based on a diagnosis made according to prescription of at least three ra - specific drugs ( corticosteroids , disease - modifying anti - rheumatic drugs , and biological agents ) , was calculated for the year 2011.15 patients were classified as follows : those on specific drugs were classed as having active ra ; those who had never had more than four prescriptions in the past were classed as unlikely ra ; and those previously on chronic treatment but who had discontinued therapy for > 1 year were classed as having ra in remission . 
 a diagnosis of ra was made by a qualified specialist in a total of 22,801 of these subjects ( 0.48% ) , with a prevalence of active ra , ra in remission , and confirmed ra ( active + remission ra ) of 0.32% , 0.09% , and 0.41% , respectively . 
 the yearly incidence of active ra per 100,000 subjects was 48 ( 95% ci 4057 ) for females and 20 ( 95% ci 1030 ) for males . 
 the female : male ratio for both prevalence and incidence was ~3.5:1 before the fifth decade of life and tended to decline to about 2:1 in subjects aged > 70 years . a modeling analysis of the prevalence of ra in 27 countries in the eu ( plus iceland , norway , and switzerland ) conducted in 20086 estimated that the overall prevalence of ra in italy was 0.49% and was exactly the same as the calculated average prevalence in europe . in this analysis , 
 italy was ranked as eighth in terms of estimated prevalence of ra and was one of four european countries ( together with the uk , sweden , and germany ) in which subjects aged 65 years contributed more than 50% of the diagnosed cases of ra . 
 the data also showed that italy and germany were the european countries with the highest proportion of people aged 65 years . 
 taken together , these data6 suggest that the prevalence of ra will further increase in europe and in italy in line with projected increases in the elderly population . 
 few studies have estimated the costs associated with ra in italy before the availability of biological agents . a retrospective , prevalence - based , multicenter , cost - of - illness study , undertaken in 1998 in ten rheumatology institutes located in northern , central , and southern italy,19 evaluated the direct ( hospitalizations , specialist visits , treatments , diagnostics , and nonmedical costs ) , indirect ( productivity loss and informal care ) , and intangible ( deterioration in health - related quality of life [ hrqol ] of patients , their families , and their friends ) costs associated with ra in 200 patients aged 1865 years . 
 participants were categorized into four classes of severity according to the acr ra functional status index in place at the time of the study.22 the results showed that indirect costs accounted for the largest part of the disease management costs and that social costs ( direct plus indirect costs ) increased as ra worsened . the calculated average annual cost for each patient varied significantly depending on disease severity and correlated with the health assessment questionnaire disability index23 and acr criteria.22 total costs ranged from  3,718.3 for patients in functional class i to  22,946.0 for patients in class iv . both the direct costs ( respectively ,  1,643.4 ,  2,910.2 ,  4,236.5 , and  5,696.8 per patient from class i to class iv ) and the indirect costs (  2,074.9 ,  9,566.4 ,  12,183.1 , and  17,249.2 per patient , respectively ) over the 12 months of observation increased significantly ( p<0.0005 ) across the four functional classes . 
 the modeling study conducted in 20086 used a specifically developed model to estimate the total cost of ra in europe . 
 costs , extracted from the studies identified in a comprehensive literature review , were separated into different categories : medical costs , drugs , nonmedical costs , informal care costs , and indirect costs , and inflated to 2008 values . 
 the estimated costs per patient were then combined with country- and age - specific prevalence data to obtain a figure for the total cost of ra in each country . 
 the mean estimated annual cost per patient in europe with ra was  12,902 , with a clear difference between western europe ( mean  14,997 ) and central / eastern europe ( mean  3,752 ) . 
 the total mean annual cost of the disease across all european countries was estimated at about  25.1 billion . 
 the estimated mean annual cost per patient in the study countries ranged from about  2,000 to  21,000 , the estimated mean annual cost per patient in italy was  11,546 in line with the mean for europe as a whole . in italy , 
 ( excluding biological agents ) ,  731 for biological agents ,  3,290 for informal care , and  2,972 for indirect costs . 
 this distribution indicates that , at the time of this modeling study ( 2008 ) , direct costs in europe overall represented 43% of the total expenditure . 
 notably , at the time of the analysis , italy and germany had the lowest use of biologics in western europe due to tight budget control policies . 
 for example , in italy , biologics could only be prescribed in the hospital setting and use of such drugs was limited to a small percentage of the total expenditure.6 despite individual differences in costs between countries , costs outside the health care sector , such as informal care and productivity loss , represented the greater part of the costs of ra in all countries.6 the structure of the costs of ra has changed over the years , with a substantial increase in the direct costs of the disease largely as a result of the availability of newer , more costly biological agents . for example , in france , the direct annual costs of ra were estimated to be  4,000 in 2000 ( before the introduction of biological agents)24 and about  12,000 in a 2005 study in which 20% of patients were receiving these agents.25 although the introduction of biological agents has contributed to increased direct medical costs,26,27 their use is associated with marked improvements in disease activity , joint damage , and loss of productivity.18 furthermore , data from the consortium of rheumatology researchers of north america registry have shown that the use of biological agents is associated with a reduction in the risk of cardiovascular events in patients with ra.28 in a health economic analysis carried out in the province of pisa in the years 2006 and 2007,14 the mean annual cost per patient with ra was estimated to be  5,878 ( median  6,434 ; interquartile range  6697,052 ) , with high variability , mainly depending on the degree of disability . in this analysis , 
 more than 90% of the overall annual cost per patient was due to the medical and nonmedical components of direct costs . 
 a more recently published literature review16 evaluated the mean annual social costs per patient and the total social cost of ra in the last decade in italy . 
 data from each study ( table 2 ) , when further analyzed , showed that the mean annual social cost ( ie , overall direct plus indirect costs ) of ra was  13,595 per adult patient ( 2012 values ) . based on prevalence data 
 , it was estimated that ra resulted in a total social cost of  3.5 billion in italy per year . 
 direct medical costs accounted for 21% of the total costs , while the remaining 79% were nonmedical costs ( direct nonmedical costs and indirect costs ) . 
 nonmedical and indirect costs were mainly sustained by individual households for informal care ( 40% of the total cost ) , and by the economy ( due to absence from work of patients and caregivers ; 31% of the total cost ) . based on these data , 
 it is evident that assessment of the economic burden of ra based solely on the evaluation of direct medical costs gives a limited view of the total economic burden of the disease . 
 italy has the second highest percentage of population 65 years of age in the world,29 therefore , an increase in the costs of ra in italy is expected due to the aging population . since mean costs per patient increase with increasing functional disability , reliable health - economic evaluations should estimate the long - term consequences of the changing course of the disease and the effects of preventing or delaying the development of severe disability.30 short - term analyses may be misleading as they tend to neglect the importance of modifications in the course of the disease as a result of treatment intervention and its positive impact on the deterioration of hrqol . 
 a systematic literature review18 on the economic consequences of , and pharmacoeconomic issues relating to , ra , based on a total of 127 published articles , has shown that disease severity , functional disability , age , and socioeconomic status are the most relevant predictors of cost increase in ra . 
 a published editorial20 has estimated the economic impact of ra in italy over the next 30 years using cost - of - illness modeling . 
 this analysis assumed that the costs of ra varied as the disease evolved , and classified disease by stage according to the steinbrocker radiological classification.31 using inferential statistical methods , the probability of evolution toward a more severe stage of the disease in the absence of treatment was estimated . 
 a total of 411,692 patients with ra were classified according to the four stages of the disease and their progression over the next 30 years was simulated . 
 the results showed that , in the next 30 years , the annual cost of ra will increase from  3.3 billion ( for 2009 ) to a peak of  4 billion ( for 2029 ) , including both direct and indirect costs . 
 cost estimates will vary between regions , with higher costs estimated in lombardy (  430500 million ) , lazio (  300330 million ) , sicily (  238281 million ) , and emilia - romagna (  131187 million ) . 
 this suggests that costs will be only partially correlated with the number of inhabitants in different italian regions and will possibly depend on differing regional health care policies . 
 expenditure will not greatly differ between workers ( from  320 million in 2009 to  420 million in 2029 ) and homemakers ( from  318 million in 2009 to  421 million in 2029 ) . 
 the disease was also estimated to have a significant impact on costs due to loss of productivity that are supported by the italian national health care system ( nhs ) , which are predicted to increase from 0.67% of total social security spending in 2009 to 0.7% of this spending in 2029 . 
 in addition , ra was predicted to account for a total of 1% of all nhs costs in 2029 . 
 these data indicate that the costs associated with untreated ra would make a significant contribution to nhs and societal costs in the next decades , thus confirming that delaying early diagnosis and intervention will result in increased overall costs for the community . 
 the development of biosimilar biologics may alter the landscape of biologics and their associated costs in the future . 
 physicians attitudes toward the interchangeability of drugs , indications from regulatory authorities , and national recommendations will have a major impact on the use of biosimilars in the management of ra . 
 while , a statement position from the italian society of rheumatology has raised concerns about the true equivalence of biosimilars in rheumatic diseases and hence on the potential for interchangeability of drugs , studies in ra currently support the similarity of biosimilars and their originators.32 furthermore , as biologics require a more sophisticated manufacturing process than that of existing conventional medications , tight regulation will be required to avoid any possible additional safety risks to make these agents truly cost - effective.33 
 few studies have estimated the costs associated with ra in italy before the availability of biological agents . a retrospective , prevalence - based , multicenter , cost - of - illness study , undertaken in 1998 in ten rheumatology institutes located in northern , central , and southern italy,19 evaluated the direct ( hospitalizations , specialist visits , treatments , diagnostics , and nonmedical costs ) , indirect ( productivity loss and informal care ) , and intangible ( deterioration in health - related quality of life [ hrqol ] of patients , their families , and their friends ) costs associated with ra in 200 patients aged 1865 years . 
 participants were categorized into four classes of severity according to the acr ra functional status index in place at the time of the study.22 the results showed that indirect costs accounted for the largest part of the disease management costs and that social costs ( direct plus indirect costs ) increased as ra worsened . the calculated average annual cost for each patient varied significantly depending on disease severity and correlated with the health assessment questionnaire disability index23 and acr criteria.22 total costs ranged from  3,718.3 for patients in functional class i to  22,946.0 for patients in class iv . both the direct costs ( respectively ,  1,643.4 ,  2,910.2 ,  4,236.5 , and  5,696.8 per patient from class i to class iv ) and the indirect costs (  2,074.9 ,  9,566.4 ,  12,183.1 , and  17,249.2 per patient , respectively ) over the 12 months of observation increased significantly ( p<0.0005 ) across the four functional classes . 
 the modeling study conducted in 20086 used a specifically developed model to estimate the total cost of ra in europe . 
 costs , extracted from the studies identified in a comprehensive literature review , were separated into different categories : medical costs , drugs , nonmedical costs , informal care costs , and indirect costs , and inflated to 2008 values . 
 the estimated costs per patient were then combined with country- and age - specific prevalence data to obtain a figure for the total cost of ra in each country . 
 the mean estimated annual cost per patient in europe with ra was  12,902 , with a clear difference between western europe ( mean  14,997 ) and central / eastern europe ( mean  3,752 ) . 
 the total mean annual cost of the disease across all european countries was estimated at about  25.1 billion . 
 the estimated mean annual cost per patient in the study countries ranged from about  2,000 to  21,000 , the estimated mean annual cost per patient in italy was  11,546 in line with the mean for europe as a whole . in italy , 
 ( excluding biological agents ) ,  731 for biological agents ,  3,290 for informal care , and  2,972 for indirect costs . 
 this distribution indicates that , at the time of this modeling study ( 2008 ) , direct costs in europe overall represented 43% of the total expenditure . 
 notably , at the time of the analysis , italy and germany had the lowest use of biologics in western europe due to tight budget control policies . 
 for example , in italy , biologics could only be prescribed in the hospital setting and use of such drugs was limited to a small percentage of the total expenditure.6 despite individual differences in costs between countries , costs outside the health care sector , such as informal care and productivity loss , represented the greater part of the costs of ra in all countries.6 
 the structure of the costs of ra has changed over the years , with a substantial increase in the direct costs of the disease largely as a result of the availability of newer , more costly biological agents . 
 for example , in france , the direct annual costs of ra were estimated to be  4,000 in 2000 ( before the introduction of biological agents)24 and about  12,000 in a 2005 study in which 20% of patients were receiving these agents.25 although the introduction of biological agents has contributed to increased direct medical costs,26,27 their use is associated with marked improvements in disease activity , joint damage , and loss of productivity.18 furthermore , data from the consortium of rheumatology researchers of north america registry have shown that the use of biological agents is associated with a reduction in the risk of cardiovascular events in patients with ra.28 in a health economic analysis carried out in the province of pisa in the years 2006 and 2007,14 the mean annual cost per patient with ra was estimated to be  5,878 ( median  6,434 ; interquartile range  6697,052 ) , with high variability , mainly depending on the degree of disability . in this analysis , more than 90% of the overall annual cost per patient was due to the medical and nonmedical components of direct costs . 
 a more recently published literature review16 evaluated the mean annual social costs per patient and the total social cost of ra in the last decade in italy . 
 data from each study ( table 2 ) , when further analyzed , showed that the mean annual social cost ( ie , overall direct plus indirect costs ) of ra was  13,595 per adult patient ( 2012 values ) . based on prevalence data 
 , it was estimated that ra resulted in a total social cost of  3.5 billion in italy per year . 
 direct medical costs accounted for 21% of the total costs , while the remaining 79% were nonmedical costs ( direct nonmedical costs and indirect costs ) . 
 nonmedical and indirect costs were mainly sustained by individual households for informal care ( 40% of the total cost ) , and by the economy ( due to absence from work of patients and caregivers ; 31% of the total cost ) . based on these data , 
 it is evident that assessment of the economic burden of ra based solely on the evaluation of direct medical costs gives a limited view of the total economic burden of the disease . 
 italy has the second highest percentage of population 65 years of age in the world,29 therefore , an increase in the costs of ra in italy is expected due to the aging population . since mean costs per patient increase with increasing functional disability 
 , reliable health - economic evaluations should estimate the long - term consequences of the changing course of the disease and the effects of preventing or delaying the development of severe disability.30 short - term analyses may be misleading as they tend to neglect the importance of modifications in the course of the disease as a result of treatment intervention and its positive impact on the deterioration of hrqol . 
 a systematic literature review18 on the economic consequences of , and pharmacoeconomic issues relating to , ra , based on a total of 127 published articles , has shown that disease severity , functional disability , age , and socioeconomic status are the most relevant predictors of cost increase in ra . 
 a published editorial20 has estimated the economic impact of ra in italy over the next 30 years using cost - of - illness modeling . 
 this analysis assumed that the costs of ra varied as the disease evolved , and classified disease by stage according to the steinbrocker radiological classification.31 using inferential statistical methods , the probability of evolution toward a more severe stage of the disease in the absence of treatment was estimated . 
 a total of 411,692 patients with ra were classified according to the four stages of the disease and their progression over the next 30 years was simulated . 
 the results showed that , in the next 30 years , the annual cost of ra will increase from  3.3 billion ( for 2009 ) to a peak of  4 billion ( for 2029 ) , including both direct and indirect costs . 
 cost estimates will vary between regions , with higher costs estimated in lombardy (  430500 million ) , lazio (  300330 million ) , sicily (  238281 million ) , and emilia - romagna (  131187 million ) . 
 this suggests that costs will be only partially correlated with the number of inhabitants in different italian regions and will possibly depend on differing regional health care policies . 
 expenditure will not greatly differ between workers ( from  320 million in 2009 to  420 million in 2029 ) and homemakers ( from  318 million in 2009 to  421 million in 2029 ) . 
 the disease was also estimated to have a significant impact on costs due to loss of productivity that are supported by the italian national health care system ( nhs ) , which are predicted to increase from 0.67% of total social security spending in 2009 to 0.7% of this spending in 2029 . 
 in addition , ra was predicted to account for a total of 1% of all nhs costs in 2029 . 
 these data indicate that the costs associated with untreated ra would make a significant contribution to nhs and societal costs in the next decades , thus confirming that delaying early diagnosis and intervention will result in increased overall costs for the community . 
 the development of biosimilar biologics may alter the landscape of biologics and their associated costs in the future . 
 physicians attitudes toward the interchangeability of drugs , indications from regulatory authorities , and national recommendations will have a major impact on the use of biosimilars in the management of ra . while , a statement position from the italian society of rheumatology has raised concerns about the true equivalence of biosimilars in rheumatic diseases and hence on the potential for interchangeability of drugs , studies in ra currently support the similarity of biosimilars and their originators.32 furthermore , as biologics require a more sophisticated manufacturing process than that of existing conventional medications , tight regulation will be required to avoid any possible additional safety risks to make these agents truly cost - effective.33 
 the studies conducted to date have indicated that the prevalence of ra in italy is ~0.5% . 
 although most of these studies have been undertaken in small communities or at a regional level , and are therefore poorly representative of the national situation , the calculated prevalence has not changed over the years . 
 the largest of the studies on the prevalence and incidence of ra in italy,15 which involved more than 4.5 million people , has clearly indicated that both the prevalence and incidence of ra progressively increase in both men and women up to the age of 80 years . as italy 
 is undergoing a process of demographic transition , in which higher life expectancy will lead to a dramatic increase in the number of elderly people in the near future,7 it is expected that ra will impose a heavy socioeconomic burden on the nhs in coming years . 
 it is well established that costs associated with ra are likely to rise as the disease progresses , accompanied by a substantial reduction in hrqol.17 as in other disabling diseases , much of the cost of ra occurs outside the health care system , because it is associated with loss of work capability and the need for informal care in the later stages of the disease.25 the severity and the progression of ra have a great influence on the costs of the disease . 
 disease progression is associated with increasing functional impairment and reduced hrqol , which may lead to work absenteeism and inability and hence increased indirect costs , which are estimated to be twice as high as direct costs when the disease progresses.34,35 the introduction of biological drugs for the treatment of ra highlights the role of economics in the considerations for patient access to innovative but expensive treatments . 
 inclusion in the reimbursement system is a very important condition for access to innovative treatments . 
 cost - effectiveness is a further criterion to fulfill , in addition to a favorable risk  benefit profile , for marketing approval by the european medicines agency and national medicines agencies . 
 health - economic analyses conducted on a cross - sectional basis or over a short - term period reflect differences in the costs of drugs and are less sensitive to the long - term evolution of the disease in terms of its impact on social costs and loss of productivity . 
 therefore , a reliable estimate of the cost - effectiveness of a drug class should not be based solely on the net costs of the compounds , but should also take into consideration the effects of such drugs on the progression of the disease , as rapid / severe progression can lead to huge increases in indirect social costs and loss of productivity . 
 in fact , the use of biologics in the early stage of ra may reduce other health care costs by slowing the evolution of the disease . the extent to which the high direct costs incurred from early use of biologics may be balanced in part by other health care cost offsets due to the reduction in the need for surgery or other interventions beyond the fourth year of the disease , remains to be elucidated.36 although cost  benefit and cost - effectiveness analyses are recognized as standard techniques for pharmacoeconomic evaluations , other methods , such as the cost  utility analysis , are better tailored to evaluate the health improvement attributable to a treatment , in which health improvement is generally measured in quality - adjusted life - years ( qalys ) gained.37 in particular , the qaly combines life expectancy and hrqol by weighing life - years with a quality index named utility . 
 as hrqol is among the most important outcomes in ra , in which it is difficult to simply study morbidity and mortality , interest has been focused on how well the different treatment programs improve patients physical functioning , social functioning , and psychological well - being . 
 accurate pharmacoeconomic estimates may drive the use of new and more expensive therapies in the treatment of ra and help health care providers and budget holders to choose the most effective strategies for patients . 
 analyses should not be focused only on the effects of a therapy on the onset of disability and the progression of joint destruction , but also on the adverse event profile of the drug and on its efficacy and safety over the long term , which are generally not investigated in randomized clinical trials . 
 pharmacoeconomic analysis of the existing therapies for ra should be based on the awareness that treatment will prevent progression to the next level of disease severity or that the disease will take longer to progress , thus avoiding or delaying the high costs associated with more severe stages of the disease.38 this will help attenuate the impact on indirect and social costs due to disease progression and , most importantly , on the intangible costs due to the progression of the disease and associated comorbidities over the years . indeed , there is a high prevalence of comorbidities and their associated risk factors among ra patients , as shown in a recent international observational study conducted in 17 countries worldwide.39 the study has shown a high intercountry variability in the prevalence of comorbidities in ra and suggests that the diagnosis and management of such comorbidities is far from optimal . 
 depression ( 15% of patients on average across countries ) was the most commonly observed comorbidity , followed by asthma ( 6.6% ) , gastrointestinal ulcer ( 10.8% ) , ischemic cardiovascular disease ( 6.0% ) , and solid tumors ( 4.5% ) . 
 italy had the highest prevalence of hepatitis b ( 9.0% ) and c ( 6.6% ) , rate of patients with > 40 skin nevi ( 12% ) and of framingham score > 20% ( 59% ) . 
 although the high prevalence of some of these comorbidities may be related to the medications used ( eg , corticosteroids ) or to traditional risk factors ( eg , tobacco smoking ) , some others ( eg , depression ) may be a direct consequence of systemic inflammation and its associated symptoms and effect on hrqol.40 a task force of the acr and the european league against rheumatism has recently highlighted the importance of comorbidities in the management of ra and in the treatment decision - making strategies.41 while the use of biologic response modifiers is not associated with an increased risk of malignancy,42 improvements in disease activity following 1-year treatment with biologic agents is associated with important gains in terms of qalys and hrqol,43 suggesting that successful treatment of ra can result in lower levels of depression . 
 the recent published editorial20 on the 30-year costs of untreated ra in italy , which has shown that actual costs may rise in the coming years if the disease is not managed , highlights that costly interventions aimed at sparing future expenses are of particular relevance in countries like italy , where ra and associated comorbidities will make a significant contribution to the economic burden for the nhs , given the increased proportion of elderly people expected in years to come . 
 further research is needed to develop estimates of the economic impact of different therapeutic approaches in patients with ra in italy . 
 in particular , accurate pharmacoeconomic analyses would be valuable to help guide treatment decisions , ensuring effective spending without compromising the quality of care delivered .","this literature review examines available evidence on the current and past costs associated with rheumatoid arthritis ( ra ) in italy , together with the future health - economic prospects for the disease . 
 studies have been conducted to date on the prevalence , or the associated costs , of ra in italy . 
 although future changes in the incidence of ra are a matter of debate , the impact of ra on health care costs is expected to grow in coming decades in line with projected increases in life expectancy and in the proportion of elderly people in italy . 
 it has been estimated that the indirect ( productivity loss and informal care ) and intangible ( deterioration in health - related quality of life ) costs of the disease will contribute to an increase in national health service expenditure , which will correspond to 1% of the total health care costs of the nation in the near future . 
 the introduction of biological agents for the treatment of rheumatic diseases has resulted in an increase in the direct costs of ra ; however , economic analyses that exclude indirect costs will underestimate the full economic impact of ra 
 . the effectiveness of innovative therapies in preventing disease progression and functional impairment may , over time , attenuate the cost impact of ra in terms of hospitalizations and work absenteeism . 
 further research is needed to develop estimates of the economic impact of different therapeutic approaches in patients with ra in italy , in order to provide tools that can drive the choice of the most cost - effective therapeutic option while maintaining high - quality care .",pubmed
"in prosthodontic practice , limited mouth opening in patients is a common occurrence . prosthetic rehabilitation of patient with restricted mouth opening due to any causative factors , presents difficulties at all stages , right from the preliminary impressions to the insertion of prosthesis . a maximal oral opening smaller than the size of a complete denture can make prosthetic treatment challenging . 
 limited oral opening itself is not a disease but manifest as a consequence of certain conditions namely surgical treatments of orofacial carcinoma , cleft lip , trauma , burns , plummer vinson syndrome , scleroderma , trismus , rheumatism , temporomandibular joint dysfunction syndrome , oral submucous fibrosis , and any damage to masticatory muscles . 
 a 71-year - old male patient reported to the department of prosthodontics , rama dental college and hospital kanpur ( up ) , india , with the chief complaint of missing teeth and wanted replacement of the missing teeth by means of fabrication of complete denture . on examination , it was found that patient had oral submucous fibrosis with heavy vertical fibrous bands and homogenous leukoplakia on both sides of buccal mucosa which lead to reduced mouth opening of 2.5 cm . 
 patient was also diagnosed with bilateral angular cheilitis which often bleed on opening mouth wide . 
 treatment plan included sectional maxillary and mandibular final impression procedures , followed by fabricating maxillary hinged and mandibular sectional and hinged complete denture . 
 primary impression for maxillary arch was taken using smallest size edentulous stock tray with impression compound ( y dents ; mdm corp . ) , and after proper disinfection ( 1:213 iodophore ) , primary cast was poured in type ii dental plaster ( dentico ; neelkanth mine chem.).two autopolymerizing acrylic resin special trays ( trevelon ; dentsply india pvt . ltd . ) 
 both trays were trimmed one side short of maxillary tuberosity.border molding was done with each tray using low fusing compound ( dpi ; pinnacle , india ) and the final impression was made with light body polyvinyl siloxane addition silicon impression material ( examix nds ; gc company).one sectional impression tray was poured with dental stone ( denstone , grade iii ) . over that cast second sectional impression tray was assembled and the remaining part of the cast was poured.similarly , mandibular primary impression was taken using smallest size stock tray and after disinfection , primary cast was poured . 
 a conventional special tray was fabricated over the primary cast and that was later sectioned from the midline into two halves . 
 one hole was drilled in the center of the tray handle and a brass dowel pin was passed through it , from one end to the other end of the hole . 
 similarly , two holes were drilled over the premolar region on both sides of the tray and the two brass dowel pins were placed vertically in those holes and were connected by acrylic plate which was detachable.border molding and final impression were made individually with the each half of the tray and the tray was joined extra - orally with the help of dowel pin and acrylic plate . 
 the master cast was poured with dental stone.after obtaining maxillary and mandibular master casts , denture bases were fabricated and then were sectioned from the midline . 
 after sectioning , the two ends of the denture bases were rejoined from the midline using press buttons ( snap fasteners ; needles ind ) . 
 wax rims ( link , modeling wax , mdm corp . ) were prepared over the denture bases . 
 the wax rim was not prepared in the mandibular anterior region because of the presence of the press buttons over the anterior segment of the ridge . 
 jaw relation was recorded.then the teeth arrangement was done [ figure 1 ] ( lactodent ; pyrax enterprises ) . 
 patient was called for try-in.after try - in , the press buttons were removed from the maxillary denture base and the gap between the two parts of the denture base was closed by incorporating wax between them and the processing of denture was done in a conventional manner . 
 similarly , the press buttons were also removed from the anterior segment of the mandibular denture base and the anterior teeth were arranged . 
 an index was made for the anterior teeth with the help of putty addition polyvinyl siloxane impression material ( exaflex , gc company ) . 
 the anterior teeth were then removed and placed back in putty index , rest of the denture was processed with heat cure acrylic resin ( trevlon , dentsply ) , without anterior teeth in a conventional manner so that the hinge assembly can be placed later in the anterior teeth region.after processing , the dentures were retrieved , finished , and polished and then again sectioned from the midline with the help of thin disk bur.an orthodontic mandibular molar tube with 1-mm stainless steel wire was used to form a hinge assembly for the maxillary and mandibular sectional dentures so that the dentures can collapse at the midline.for maxillary denture , two mandibular molar tubes ( orthotechno wire and co. ) were used with 1-mm stainless steel wire which was passing through the slot of the tube . 
 the maxillary denture was placed back over the cast and these assemblies were incorporated between the two sections of the denture in the center of the palatal surface using self - cure acrylic resin and the rest of the gap created during sectioning of the maxillary denture was also filled with self - cure acrylic resin . 
 proper care was taken to avoid the entrapment of acrylic material inside the hinge , which can block the hinge movement of the dentures.for mandibular denture , one orthodontic mandibular molar tube with 1-mm stainless steel wire was used to form a hinge assembly so that the denture can collapse at the midline . 
 this assembly was incorporated between the two sections of the denture using self - cure acrylic resin.the mandibular hinge denture was placed back over the master cast and the index of the mandibular denture was made with irreversible hydrocollo i d ( alginate , zelgan 2002 , dentsply ) and the cast was poured with dental stone.over that cast , mandibular anterior teeth were arranged back in their correct position with the help of previously made putty index . 
 two clasps were adapted over the adjacent teeth to retain the acrylic removable partial denture [ figure 2 ] . 
 wax - up was done and the acrylic removable partial denture was processed in a conventional acrylization manner.maxillary hinged denture and the mandibular sectional ( separate anterior removable segment ) and hinged denture [ figure 3 ] were placed in patient 's mouth.patient was educated about the insertion and removal of dentures . 
 instructions were given to maintain the denture.patient was recalled for the follow - up visits to check for the maintenance and patient 's adaptability with the dentures . 
 first follow - up was done after 24 h of denture insertion followed by 1 week to check for the comfort and any tissue reaction . 
 initially , patient felt difficulty while placing and removal of the dentures from the oral cavity but with time patient got used to it . 
 figure 1(a ) maxillary and ( b ) mandibular sectional denture bases with occlusion rims and teeth arrangement 
 figure 2(a ) maxillary denture sectioned from the midline incorporating two hinge assemblies in the palatal region of the maxillary denture ; ( b ) mandibular acrylized denture with hinge assembly in anterior region . the index of the denture made and the cast obtained , anterior teeth arranged back in position with putty index to make anterior removable partial denture 
 figure 3(a ) collapsed maxillary hinged and ( b ) mandibular sectional and hinged complete denture with anterior removable partial denture 
  primary impression for maxillary arch was taken using smallest size edentulous stock tray with impression compound ( y dents ; mdm corp . ) , and after proper disinfection ( 1:213 iodophore ) , primary cast was poured in type ii dental plaster ( dentico ; neelkanth mine chem . ) . two autopolymerizing acrylic resin special trays ( trevelon ; dentsply india pvt . ltd . ) 
 border molding was done with each tray using low fusing compound ( dpi ; pinnacle , india ) and the final impression was made with light body polyvinyl siloxane addition silicon impression material ( examix nds ; gc company ) . 
 one sectional impression tray was poured with dental stone ( denstone , grade iii ) . over that cast second sectional impression tray 
 similarly , mandibular primary impression was taken using smallest size stock tray and after disinfection , primary cast was poured . 
 a conventional special tray was fabricated over the primary cast and that was later sectioned from the midline into two halves . 
 one hole was drilled in the center of the tray handle and a brass dowel pin was passed through it , from one end to the other end of the hole . 
 similarly , two holes were drilled over the premolar region on both sides of the tray and the two brass dowel pins were placed vertically in those holes and were connected by acrylic plate which was detachable . 
 border molding and final impression were made individually with the each half of the tray and the tray was joined extra - orally with the help of dowel pin and acrylic plate . 
 after sectioning , the two ends of the denture bases were rejoined from the midline using press buttons ( snap fasteners ; needles ind ) . 
 wax rims ( link , modeling wax , mdm corp . ) were prepared over the denture bases . 
 the wax rim was not prepared in the mandibular anterior region because of the presence of the press buttons over the anterior segment of the ridge . 
 then the teeth arrangement was done [ figure 1 ] ( lactodent ; pyrax enterprises ) . 
 after try - in , the press buttons were removed from the maxillary denture base and the gap between the two parts of the denture base was closed by incorporating wax between them and the processing of denture was done in a conventional manner . 
 similarly , the press buttons were also removed from the anterior segment of the mandibular denture base and the anterior teeth were arranged . 
 an index was made for the anterior teeth with the help of putty addition polyvinyl siloxane impression material ( exaflex , gc company ) . 
 the anterior teeth were then removed and placed back in putty index , rest of the denture was processed with heat cure acrylic resin ( trevlon , dentsply ) , without anterior teeth in a conventional manner so that the hinge assembly can be placed later in the anterior teeth region . after processing , 
 the dentures were retrieved , finished , and polished and then again sectioned from the midline with the help of thin disk bur . 
 an orthodontic mandibular molar tube with 1-mm stainless steel wire was used to form a hinge assembly for the maxillary and mandibular sectional dentures so that the dentures can collapse at the midline . 
 for maxillary denture , two mandibular molar tubes ( orthotechno wire and co. ) were used with 1-mm stainless steel wire which was passing through the slot of the tube . 
 the maxillary denture was placed back over the cast and these assemblies were incorporated between the two sections of the denture in the center of the palatal surface using self - cure acrylic resin and the rest of the gap created during sectioning of the maxillary denture was also filled with self - cure acrylic resin . 
 proper care was taken to avoid the entrapment of acrylic material inside the hinge , which can block the hinge movement of the dentures . 
 for mandibular denture , one orthodontic mandibular molar tube with 1-mm stainless steel wire was used to form a hinge assembly so that the denture can collapse at the midline . 
 this assembly was incorporated between the two sections of the denture using self - cure acrylic resin . 
 the mandibular hinge denture was placed back over the master cast and the index of the mandibular denture was made with irreversible hydrocollo i d ( alginate , zelgan 2002 , dentsply ) and the cast was poured with dental stone . over that cast , mandibular anterior teeth were arranged back in their correct position with the help of previously made putty index . 
 two clasps were adapted over the adjacent teeth to retain the acrylic removable partial denture [ figure 2 ] . 
 wax - up was done and the acrylic removable partial denture was processed in a conventional acrylization manner . 
 maxillary hinged denture and the mandibular sectional ( separate anterior removable segment ) and hinged denture [ figure 3 ] were placed in patient 's mouth . 
 patient was recalled for the follow - up visits to check for the maintenance and patient 's adaptability with the dentures . 
 first follow - up was done after 24 h of denture insertion followed by 1 week to check for the comfort and any tissue reaction . 
 initially , patient felt difficulty while placing and removal of the dentures from the oral cavity but with time patient got used to it . 
 ( a ) maxillary and ( b ) mandibular sectional denture bases with occlusion rims and teeth arrangement ( a ) maxillary denture sectioned from the midline incorporating two hinge assemblies in the palatal region of the maxillary denture ; ( b ) mandibular acrylized denture with hinge assembly in anterior region . the index of the denture made and the cast obtained , anterior teeth arranged back in position with putty index to make anterior removable partial denture ( a ) collapsed maxillary hinged and ( b ) mandibular sectional and hinged complete denture with anterior removable partial denture 
 several techniques have been described in the literature for the fabrication of sectional and hinged tray / complete dentures , utilizing various mechanisms for connecting each of the components . 
 pubmed database for the last two decades was reviewed using key words : microstomia , hinged and sectional dentures and different techniques for patients with microstomia are found to be documented [ table 1 ] . 
 different techniques and denture modifications for microstomia other management techniques described in the literature for patients with restricted mouth opening are surgery , use of dynamic opening devices , and modification of denture design . in cases where microstomia is not manageable by surgeries or dynamic opening devices , modified impression procedure and prosthesis design facilitate prosthetic rehabilitation . 
 the denture aids for a better function , health , esthetics , and overall well - being of the patient and thus a rewarding service can be provided by the prosthodontist . 
 the reduction in maximal oral opening ( microstomia ) less than the normal ( 35 - 40 mm ) hinders conventional prosthetic treatment of edentulous patients . 
 it is often difficult to apply conventional clinical procedures to construct dentures for patients who demonstrate limited mouth opening . 
 however , with careful treatment planning and prudent designing , the use of either sectional impression techniques and/or sectional dentures , many of the apparent clinical difficulties can be overcome . here , 
 the sectional impression procedures and the prosthesis designs are described which aid in fabrication of prosthesis for a patient with the limited mouth opening which aids for a better function , health , esthetics , and overall well - being of the patient . 
 simplified sectional tray design and ease of fabrication are the major advantages of this technique . 
 this technique can be accomplished in any dental laboratory , without using complicated machinery or attachment devices for sectioning or assembling the trays / prosthesis together . 
 the press buttons and mandibular molar bands are easily available and are easy to maintain . 
 this technique shares disadvantages common to all sectional tray / prosthesis designs , such as additional time , labor , and materials . 
 however , to determine the long - term success of this technique , periodic recall , maintenance , and further improvements in design are needed .","restricted mouth opening is a definite prosthodontic hindrance to carry out treatment successfully . 
 restricted mouth opening can be due to many reasons such as microstomia , oral submucous fibrosis , some genetic disorder , and as a result of some surgical treatment . in the past , 
 various techniques for prosthetic rehabilitation of limited oral opening have been tried such as surgeries , use of dynamic opening devices , magnetic devices , and modification of denture design . here 
 we present ; a simplified technique and simple design for fabrication of maxillary hinged and mandibular hinged and sectional complete denture for a patient with restricted mouth opening due to oral submucous fibrosis .",pubmed
"hepatocellular carcinoma ( hcc ) is the sixth most common malignancy worldwide and the third most common cause of global cancer related mortality [ 1 , 2 ] . 
 hcc burden disproportionately impacts developing countries and males ; as of 2008 , 85% of cases occurred in africa and asia , with worldwide male : female sex ratio of 2.4 . 
 risk factors for the development of hcc include chronic liver inflammation from hepatitis b and c infection , autoimmune hepatitis , excessive alcohol use , nonalcoholic steatohepatitis , primary biliary cirrhosis , environmental carcinogens such as aflatoxin b , and genetic metabolic disease ( such as hemochromatosis and alpha-1 antitrypsin deficiency ) . 
 prognostic and therapeutic options are dependent upon the severity of underlying liver disease , and median overall survival ( os ) for metastatic or locally advanced disease is estimated at 58 months . 
 hcc is relatively refractory to cytotoxic chemotherapy , likely due to overexpression of multidrug - resistant genes , protein products such as heat shock 70   and p - glycoprotein , and p53 mutations . 
 presently , systemic therapeutic options in the locally advanced or metastatic setting are limited to sorafenib , an oral multikinase inhibitor targeting raf kinase , vascular endothelial growth factor ( vegf ) , and platelet - derived growth factor ( pdgf ) receptor tyrosine kinase signaling .   although the transition from normal hepatocyte to hcc is not fully understood , hepatocarcinogenesis is a complex multistep process driven by accumulation of heterogeneous molecular alterations from initial hepatocyte injury to metastatic invasion . 
 dysplastic nodules subsequently progress to early hcc through cumulative genetic alterations , while advanced hcc often involves intrahepatic metastasis and portal vein invasion . 
 molecular alterations implicated in hcc development include mutations in oncogenes and tumor suppressor genes ( p53 and p16 ) , epigenetic alterations , chromosomal changes , and aberrant activation of signaling cascades necessary for proliferation , angiogenesis , invasion and metastasis , and survival . 
 pathogenesis of early and advanced hcc may be modulated through different mechanisms ; for example , p53 mutations , p16 gene silencing , and aberrant akt signaling are more frequently observed in advanced hcc [ 46 ] . 
 the molecular pathogenesis of hcc is multifactorial and is reliant upon dysregulation of multiple pathways including wnt / b - catenin , mitogen - activated protein kinase ( mapk ) , phosphatidylinositol-3 ( pi3k)/akt / mammalian target of rapamycin ( mtor ) , vegf , pdgf , insulin - like growth factor ( igf ) , epidermal growth factor ( egf ) , tgf - beta , and hepatocyte growth factor [ 6 , 7 ] . 
 the hepatocyte growth factor ( hgf ) and its transmembrane tyrosine kinase receptor , cellular met ( cmet ) promote cell survival , proliferation , migration , and invasion via modulation of epithelial - mesenchymal interactions . 
 hgf - cmet signaling is critical for normal processes such as embryogenesis , organogenesis , and postnatal tissue repair after acute injury . 
 hgf - cmet axis activation is also implicated in cellular invasion and metastases through induction of increased proliferation ( mitogenesis ) , migration and mobility ( motogenesis ) , three - dimensional epithelial cell organization ( morphogenesis ) , and angiogenesis . 
 hgf was first discovered in 1984 as a mitogenic protein for rat hepatocytes in vitro . 
 hgf was subsequently found to be indistinguishable from scatter factor , a fibroblast - derived motility factor promoting epithelial cell dispersal   and three - dimensional branching tubulogenesis . 
 hgf is secreted primarily by mesenchymal cells ( or by stellate and endothelial cells in the liver ) as an inactive single - chain precursor ( pro - hgf ) which is bound to heparin proteoglycans within the extracellular matrix . 
 hgf transcription is upregulated by inflammatory modulators such as tumor necrosis factor alpha , il-1 , il-6 , tgf - beta , and vegf [ 11 , 12 ] . 
 circulating pro - hgf undergoes proteolytic conversion via extracellular proteases including hgf activator ( hgfa ) , urokinase - type plasminogen activator , factors xii and xi , matriptase , and hepsin   into an active two - polypeptide chain heterodimeric linked by a disulfide bond . 
 hgfa is a serine protease which is secreted primarily by the liver and circulates as pro - hgfa ; pro - hgfa is activated by thrombin in response to tissue injury and malignant transformation [ 13 , 14 ] . 
 the active form of hgf includes an -chain containing four kringle domains ( k1 to k4 ) and an amino - terminal loop domain ( n ) , and a -chain ( c - terminal ) containing a serine protease homology ( sph ) domain . 
 hgf is a ligand for the met receptor , also known as cellular met or cmet . 
 the met protooncogene was first isolated in 1984 from a human osteosarcoma - derived cell line driven by a chromosomal rearrangement tpr - met , resulting from fusion of translocated promoter region located on chromosome 1q25 and met sequence located on chromosome 7q31 . 
 the tpr - met rearrangement encodes for a prototype of the cmet receptor tyrosine kinase family . 
 the cmet receptor is expressed predominantly on the surface of endothelial and epithelial cells of many organs , including the liver , kidney , prostate , pancreas , kidney , muscle , and bone marrow . like hgf , cmet is synthesized as an inactive single - chain precursor and undergoes proteolytic cleavage into a disulfide linked heterodimer consisting of an extracellular -chain and transmembrane -chain . 
 the extracellular domain includes an amino - terminal semaphorin domain , terminal cysteine - rich psi domain , and four ipt repeat immunoglobulin domains . 
 the cytoplasmic portion includes a juxtamembrane region including two phosphorylation sites , a tyrosine kinase ( tk ) domain and a carboxyl terminal region for substrate docking . under normal conditions , hgf interacts with cmet via a paracrine signaling loop and triggers cmet kinase activation within the cytoplasmic portion ( see figure 1 ) . 
 hgf contains two cmet binding sites including a high affinity constitutively active site on the -chain ( n - terminal and first kringle domain , or n - k1 ) and a low affinity site on the -chain ( c - terminal ) [ 8 , 19 ] . 
 the exact mechanism of cmet receptor activation and the contribution of both binding sites to receptor activation by full length hgf are not yet established , although it is evident that both - and -chain sites have distinct functions . while -chain n - k1 portion activates cmet and induces met dimerization , -chain residues bind cmet once the receptor is already occupied by hgf n - k4 and subsequently induce phosphorylation and downstream signaling . 
 hgf binding induces autophosphorylation of tyrosine residues y1234 and y1235 within the cmet tk domain activation loop . 
 phosphorylation also occurs at two sites within the carboxyl terminal region ( y1349 and y1356 ) , forming a multifunctional high affinity binding docking site that recruits a range of intracellular adaptors containing src homology-2 domains [ 2124 ] . 
 recruited adaptor proteins and kinase substrates include growth factor receptor - bound protein 2 ( grb2 ) , grb2-associated binder ( gab1 ) , phospholipase c- , stat3 , shc , src , shp2 , pi3k , and ship1 [ 23 , 24 ] . 
 these and other adaptor proteins provide scaffolding for a larger apparatus of network proteins , ultimately promoting activation of multiple signaling pathways . of these , grb2 and gab1 are critical effectors that interact directly with the receptor ; grb2 binding to the cmet docking site through y1356 results in downstream signaling via the ras / mapk pathway , while gab1 phosphorylation by met kinase activates the pi3k pathway . 
 other signaling proteins that are activated by cmet include p38 , jnk , and nuclear factor kb . 
 alterations in transcription induce cell cycle progression , antiapoptosis , increased cell motility , angiogenesis , and survival . 
 the hgf - cmet pathway serves as a hub for multiple heterogeneous signaling networks and is also modulated by the activation of other receptor tk families such as the epidermal growth factor receptor ( egfr ) , human epidermal growth factor receptor 2 , insulin - like growth factor 1 receptor , raf kinase , and vegf . 
 the hgf - cmet pathway plays an essential role in mammalian development in terms of morphogenesis , mitogenesis , and motogenesis , and angiogenesis hgf - cmet signaling is likely to be critical in many aspects of adult homeostasis including cardiac and hepatic tissue injury repair [ 26 , 27 ] , skin wound repair , and skin immunity regulation . targeted disruption of the hgf or met genes results in embryonically lethal knockouts with impaired organogenesis of the liver and placenta . 
 preclinical models demonstrate that hgf functions as a hepatotrophic factor enhancing hepatic regeneration and suppressing hepatocyte apoptosis [ 31 , 32 ] ; expression of hgf is increased in response to liver injury , while neutralization of endogenous hgf or met knockout facilitates liver damage and fibrotic changes with delayed repair . under normal physiologic conditions , 
 hgf - induced cmet activation is strictly controlled by paracrine ligand delivery followed by ligand activation at target cell surfaces and ligand - activated receptor internationalization / degradation . despite multiple checkpoints , 
 hgf - cmet axis dysregulation occurs in a variety of solid tumors and hematopoietic derived malignancies and plays a key role in malignant transformation by promoting tumor cell migration , epithelial to mesenchymal transition , invasion , proliferation , and angiogenesis . 
 dysregulated cmet signaling upregulates protease production ( plasminogen dependent and independent ) and increased cell dissociation via extracellular matrix degradation , facilitating tumor invasiveness and metastasis . 
 mechanisms of pathogenic activation include aberrant paracrine or autocrine ligand production , overexpression of hgf and cmet , upregulation of genes encoding proteases for hgf / cmet processing , constitutive kinase activation independent of cmet gene amplification , and cmet gene mutations leading to ligand - independent kinase activity [ 3436 ] . 
 the hgf - cmet axis is implicated in hepatocarcinogenesis through multiple mechanisms , many of which are still being elucidated . 
 overexpression of hgf   and cmet [ 3741 ] is observed in 33% and 2048% of human hcc samples , respectively . 
 the prognostic utility of cmet and hgf overexpression is uncertain ; while some studies show no correlation between cmet overexpression and tumor size or invasive behavior [ 38 , 42 ] or hgf levels and survival , others demonstrate an inverse relationship with survival . 
 specifically , cmet overexpression was found to correlate with poorly differentiated hcc   and increased intrahepatic metastases along with decreased five - year survival . 
 after hepatectomy , cmet overexpression in hcc tissue has been correlated with early tumor recurrence , metastasis , and shorter 5-year survival [ 41 , 45 , 46 ] . 
 a cmet - regulated gene expression signature was found to define a subset of human hcc with poor prognosis and aggressive phenotype and correlated with increased vascular invasion , increased microvessel density , and decreased mean survival time . 
 higher hgf levels negatively correlate with survival per biomarker analysis of the sharp trial   and prior data   and positively correlate with tumor size ; however given that hgf is secreted as an inactive precursor , overexpression alone is unlikely to guarantee pathway dysregulation . 
 hgf has also been investigated as a potential biomarker for hcc development , but may also be a specific marker for liver cirrhosis . 
 while gains in 7q have been reported in hcc , gene amplification has not been reported as a significant source of increased cmet . 
 crosstalk between cmet and egfr [ 55 , 56 ] and cmet and vegf signaling pathways is also implicated in promoting tumor survival . 
 cmet - csrc has been shown to mediate egfr phosphorylation and cell survival in the presence of egfr inhibitors . 
 cmet is both an independent angiogenic factor and interacts with angiogenic survival signals promoted through vegf . by upregulating hypoxia - inducible factor , hypoxia results in increased hgf expression in tumor and surrounding normal interstitial cells and 
 hgf - cmet signaling induces upregulation of tumoral vegf expression and endothelial vegfr2 expression and downregulation of endogenous inhibitors of angiogenesis [ 58 , 59 ] . 
 dual vegf and cmet axis activity demonstrates increased capillary formation in vivo , tubulogenesis in vitro , and tumoral microvessel density . 
 given the predominant role of dysregulated hgf - cmet signaling in hepatocarcinogenesis , pharmacologic cmet inhibition is a promising therapeutic strategy . 
 targets for inhibition of the cmet signal transduction pathway include ligand - receptor interaction , cmet kinase activity , and cmet and adaptor protein interaction . 
 hgf - cmet axis inhibitors can be broadly classified into biologic antagonists , c - met adaptor protein inhibitors , small - molecule downstream pathway inhibitors , and small synthetic met tyrosine kinase inhibitors ( tki ) . of these , tkis are the most common and the only ones to have completed phase 2 testing in hcc as of march 2013 . 
 table 1 shows the selected hgf - cmet inhibitors in active clinical trials for either hcc or advanced solid malignancies ( including hcc ) as of march 2013 . 
 a comprehensive listing of hgf - cmet inhibitors in active clinical trials for all malignancies is maintained by the bottaro nci lab and is available at https://ccrod.cancer.gov/confluence/display/ccrhgf/home . 
 biologic antagonists inhibit cell surface interactions such as ligand - receptor binding or receptor clustering , preventing activation of downstream signaling . 
 these include hgf competitive analogs , met decoy receptor , and anti - hgf - cmet monoclonal antibodies . 
 hgf competitive analogs compete with ligand for receptor binding without causing cmet dimerization and activation and include nk2 [ 60 , 61 ] , nk4 [ 6264 ] , and uncleavable hgf ; preliminary safety and drug development data in humans are pending . 
 nk2 may be the least promising hgf competitive analog due to being a potent mitogen   and promoting metastases . met decoy receptors 
 are soluble forms of the cmet extracellular domain which compete with hgf and inhibit cmet dimerization ; in vitro and in vivo mice models demonstrate suppression of hgf - induced tumor cell migration and metastasis [ 68 , 69 ] . 
 monoclonal antibodies targeting hgf and the extracellular domain of cmet are currently being explored in clinical trials ( see table 1 ) , but data are not yet available for hcc .   given the importance of adaptor proteins in propagating downstream cmet signaling , cmet adaptor inhibitors offer unique potential for cmet specific inhibition . 
 as described above , cmet signaling is initiated through autophosphorylation of cytoplasmic tyrosines that form docking sites for adaptor proteins . 
 grb2 and gab1 are critical effectors that interact directly with the cmet receptor , ultimately recruiting a larger apparatus of network proteins necessary for cmet signaling . 
 gab1 coupling with cmet requires gab1 binding to the sh3 domain of grb2 , and cmet binding to the sh2 domain of grb2 [ 72 , 73 ] . 
 c90 is a selective grb2 antagonist with demonstrable inhibition of gastric cancer cell motility and matrix invasion in vitro and impaired metastatic spread of human prostate cancer cells in vivo ; data in human studies have not been reported to date [ 74 , 75 ] . 
 small - molecule downstream pathway inhibitors directed towards inhibition of stat3 phosphorylation showed preliminary tolerability in a phase i trial of advanced solid tumors , but data are not yet available for hcc . 
 synthetic small met tkis inhibit downstream signal transduction by preventing phosphorylation , either via competitive binding of the intracellular adenosine triphosphate ( atp ) site in cmet 's tk domain , or noncompetitive binding of a cmet region outside of the atp binding site . 
 while some of the tkis in development are cmet specific , others target multiple pathways including vegf , pdgfr , fms - related tyrosine kinase 3 ( flt3 ) , v - kit feline sarcoma viral oncogene homolog protein ( kit ) , and anaplastic lymphoma kinase ( alk ) . preclinical studies and clinical trials show tolerability and efficacy of cmet tkis across a variety of solid malignancies . as of march 2013 , 
 promising results in the phase 2 randomized setting for hcc are available for two cmet inhibitors : tivantinib and cabozantinib . 
 tivantinib ( arq 197 ) is an oral low - molecular - weight tki which is non - atp competitive . 
 safety and tolerability without drug - related worsening of hepatic function were previously reported in a phase ib trial of 20 cirrhotic patients ( child - pugh a and b ) with hcc , with 2 or less prior systemic chemotherapy regimens . 
 rimassa and colleagues reported results at asco 2012 of a phase ii trial assigning 107 patients with unresectable hcc with ecog ps 0 - 1 and child - pugh a in a 2  :  1 randomization to either second - line tivantinib or placebo with crossover allowed . 
 although no difference in median os occurred , the primary endpoint of time to progression ( ttp ) was met and favored tivantinib versus placebo ( 6.6 versus 6.2 months , hr = 0.90 , p = 0.63 ; 6.9 versus 6 weeks , hr = 0.64 , p = 0.04 , resp . ) . 
 patients with high met expression ( defined as 50% or more cells in the tumor specimen with 2 + or 3 + staining intensity ) versus low met expression receiving tivantinib demonstrated a significant improvement in both os and ttp 
 ( 7.2 versus 3.8 months , hr 0.38 p = 0.01 ; 11.7 versus 6.8 weeks , hr 0.43 , p = 0.03 , resp . ) . 
 no detrimental effect was reported in patients with low met expression , and common adverse events were neutropenia , asthenia , poor appetite , and anemia . 
 as of november 2012 , a phase iii trial for tivantinib and hcc patients is in development . 
 cabozantinib ( xl 184 ) is an unselective oral multikinase tki targeting cmet , kit , rearranged during transfection ( ret ) , vegfr1 and 2 and 3 , flt3 , axl receptor tyrosine kinase ( axl ) , and tie family angiopoietin 1 receptor . 
 cabozantinib has shown efficacy in the phase 3 setting for medullary thyroid carcinoma with progression - free survival ( pfs ) improvement and phase 2 setting for advanced solid tumors ( including hcc ) . 
 verslype and colleagues reported preliminary results of a phase 2 randomized discontinuation study of cabozantinib in 41 patients with hcc , child - pugh score a , and one prior line of systemic treatment . 
 all patients initiated cabozantinib for a 12-week lead in time frame ; patients with partial response continued on study drug while patients with progression of disease discontinued . 
 32 patients with stable disease were blindly randomized 1  :  1 to continue cabozantinib or receive placebo . 
 the primary endpoint was overall response rate ( rr ) during the lead in time phase and pfs for patients entering the randomization phase . 
 independent of prior sorafenib use , median os was 15.1 months , median pfs was 4.4 months , and median time on study was 6 months . 
 common grade 3 adverse effects were hand - foot syndrome , diarrhea , and thrombocytopenia . met expression was not evaluated prospectively , and given the broad activity of cabozantinib , it is unclear how much activity is attributable to met inhibition alone . 
 in fact , the inhibition of both vegf and met concurrently may be particularly effective . 
 vegf inhibition leads to increased met signaling , either from resultant hypoxia or direct interactions between vegfr2 and met [ 81 , 82 ] . 
 preliminary evidence of efficacy against hcc was seen in a phase i combination study of tivantinib with sorafenib including a complete response and another prolonged partial response lasting greater than one year . 
 inc280 is a selective met tki in phase i testing for early hcc , currently accruing . 
 foretinib ( gsk 1363089 ) is a small - molecule tki targeting both met and vegf in phase i / ii testing for advanced hcc . 
 based on preclinical trials and phase 2 data for tivantinib and cabozantinib , inhibition of cmet signaling is a promising therapeutic strategy in hcc . 
 although it is unclear which genetic and molecular abnormalities implicated in hgf - cmet dysregulation are predictive of sensitivity to cmet targeted therapy , ongoing trials must address the challenge of identifying patients most likely to achieve maximal benefit and minimal toxicity . 
 two current strategies for patient selection based on tumor biomarkers are quantification of tumor cmet content via immunohistochemical ( ihc ) and immunoassay tissue analysis and assessment of met sequence status . 
 pharmacodynamic serum markers are under evaluation in ongoing trials as a strategy to assess clinical response . 
 tumoral cmet overexpression as measured by commercially available ihc kits appears to correlate with efficacy of cmet inhibitors in hcc and other solid tumors , although more prospective data are necessary for validation . 
 identification of cmet phosphorylation status is a potentially powerful target for targeted antibodies ; two site immunoassays of flash - frozen tissue samples yielding precise measurements of met content and phosphorylation activation are currently under development . 
 another promising stratification strategy is assessment of met sequence status including met mutations , met amplification , and chromosome 7 polysomy . 
 preclinical studies of cmet targeted agents demonstrate variable efficacy based on met mutation location ; for example , pf-2341066/4217903 is a selective inhibitor with increased activity against certain cmet atp binding site mutations in comparison to met kinase domain activation loop mutations . in vitro studies of su11274 , a small - molecule tk competitive atp binding site inhibitor , show selective inhibition of two out of four identified met mutations . 
 amplification of cmet is associated with increased clinical response to foretinib ( xl 880 ) in phase 2 gastric cancer data , and cmet copy number correlates with increased clinical response to tivantinib in addition to erlotinib in advanced nsclc . 
 ancillary pharmacodynamic and pharmacokinetic marker studies in a variety of solid tumors show variable correlation with clinical response . 
 preclinical data with crizotinib in gastric cancer cell lines demonstrate variable biomarker modulation of cmet inhibition with cmet dependent gastric cancer cell lines versus cmet - independent lines [ 88 , 89 ] . 
 clinical data shows changes in plasma concentrations of hgf , vegf , soluble met , soluble vegfr2 , pigf , and epo during treatment with various tkis [ 90 , 91 ] . the predictive utility of these biomarkers in patients with cmet - dependent hcc is unclear ; further analyses and prospective validation are necessary . 
 hgf - cmet is an intriguing target for the development of companion diagnostic tools as an adjunct tool for patient selection and stratification for cmet therapy . in vitro and in vivo animal studies 
 suggest that radiolabelled dyes containing cmet binding peptide successfully targets cmet receptors with higher imaged based tumor uptake . 
 a variant of su11274 with radiomethylation modification is being utilized as a pet visualization agent to quantify cmet receptor in xenograft models . 
 inhibition of cmet is a promising therapeutic strategy in hcc . given the heterogeneous mechanisms underlying cmet dysregulation , there is an urgent and unmet need for the development of predictive biomarkers to identify which subsets of cmet - dependent hcc tumors are most likely to benefit from specific classes of inhibitors . 
 as more agents move into phase 2 and 3 trials for hcc , one important consideration is the emergence of acquired and primary resistance mechanisms from de novo or preexisting mutations . 
 these may be overcome by rational combination therapy directed against multiple pathways , different levels of ligand - receptor - tki interaction , and the presence of cmet addiction . 
 innovative clinical trial designs ( such as the discontinuation cabozantinib design ) with incorporation of enriched patient cohorts , biomarker analyses , pharmacodynamic markers , and companion diagnostics are essential in moving forward .","under normal physiological conditions , the hepatocyte growth factor ( hgf ) and its receptor , the met transmembrane tyrosine kinase ( cmet ) , are involved in embryogenesis , morphogenesis , and wound healing . 
 the hgf - cmet axis promotes cell survival , proliferation , migration , and invasion via modulation of epithelial - mesenchymal interactions . 
 hepatocellular cancer ( hcc ) is the third most common cause of worldwide cancer - related mortality ; advanced disease is associated with a paucity of therapeutic options and a five - year survival rate of only 10% . 
 dysregulation of the hgf - cmet pathway is implicated in hcc carcinogenesis and progression through activation of multiple signaling pathways ; therefore , cmet inhibition is a promising therapeutic strategy for hcc treatment . 
 the authors review hgf - cmet structure and function in normal tissue and in hcc , cmet inhibition in hcc , and future strategies for biomarker identification .",pubmed
"the associations between diabetes and cancer have been the subject of debate for over a century . 
 the length of this debate suggests , perhaps , that any such associations are relatively weak , and it is safe to say that most practicing diabetologists do not view cancer as a specific , diabetes - associated risk in day - to - day practice . that diabetes is not more clearly associated with cancer 
 is surprising , since diabetes may be viewed as a disease of increased intracellular oxidative stress , as well as accelerated  biochemical aging caused by the accumulation of  advanced glycation end - products ( ages ) on proteins and other macromolecules ( reviewed in ) . 
 an additional concern is the role of insulin , which , in various forms , is increasingly used in the management of type 2 diabetes , a condition that is now epidemic worldwide . 
 specifically , the ability of insulin to bind and activate the insulin - like growth factor-1 ( igf-1 ) receptor has raised concerns that it may promote the growth of nascent malignancies and even the establishment of new ones . 
 , there has been a recent surge in interest in the role of diabetes , insulin therapy , and specifically the effects of newer forms of insulin in promoting cancer . 
 recent rigorous epidemiologic studies have confirmed that type 2 diabetes does indeed confer increased risk for certain types of malignancies , in particular breast , colon , and pancreatic   tumors . 
 many patients with type 2 diabetes eventually require insulin , and the long - acting insulin analogue , glargine , is one of the most commonly used agents . 
 several recent studies have demonstrated the potential mitogenic and anti - apoptotic effects of glargine on cultured cancer cells , and have implicated activation of the igf-1 pathway . 
 however , the clinical implications remain controversial , particularly when studies pertaining to untransformed cells did not support any mitogenic potential . in addition , according to some authors , glargine compared with human insulin does not seem to display increased mitogenic effects , despite its higher affinity for the igf-1 receptor ; rather , any type of insulin has the potential to promote growth in cancer cells , especially if administered at sufficiently high , supra - physiologic doses . 
 the question now is whether glargine is associated with increased cancer risk compared with other commonly used forms of insulin . 
 this question has come to the fore with the publication of several studies in diabetologia [ 12 - 15 ] , which will be reviewed briefly here . 
 the epidemiologic study that initiated the recent debate was performed in germany   and suggested a specific cancer risk associated with glargine . based upon the mixed opinions of six reviewers , the editors of diabetologia , the journal to which it was submitted , commissioned three additional studies [ 13 - 15 ] , aiming to confirm or refute its findings . in the initial study , hemkens et al 
 .   followed 127,031 german patients who were being treated with human insulin and human insulin analogues ( aspart , lispro , or glargine ) over a mean of 1.6 years between january 1998 and june 2005 . 
 those on two insulin analogues or a combination of human insulin and an analogue were excluded from the analysis . 
 patients with known or suspected malignant disease within 3 years prior to study start were also excluded . 
 the primary outcome was occurrence of a malignant neoplasm , and the secondary outcome was all - cause mortality . the gross malignancy incidence rates for human insulin , aspart , lispro , and glargine were 2.50 , 2.16 , 2.13 , and 2.14 per 100 patient - years , respectively , in the unadjusted analysis . 
 however , when the data were adjusted for daily insulin dose , glargine was found to be associated with a greater risk of malignancy , and the risk was incremental with higher glargine doses when compared with human insulin { adjusted hazard ratio [ hr ] of 1.09 for a daily dose of 10 iu [ 95% confidence interval ( ci ) 1.00 - 1.19 ] , 1.19 for a daily dose of 30 iu [ 95% ci 1.10 - 1.30 ] , and 1.31 for a daily dose of 50 iu [ 95% ci 1.20 - 1.42]}. aspart and lispro were not associated with an increased risk for cancer compared with human insulin . 
 in addition , regardless of the type of insulin , higher doses and longer duration of exposure increased the risk of malignancy . 
 finally , the mean insulin dose until occurrence of a neoplasm ( cumulative dose divided by time until an event ) was significantly lower for glargine ( 25.9 iu / day ) than for human insulin , aspart , and lispro ( 43.8 , 38.9 , and 36.2 iu / day , respectively ) . the major weaknesses of the study included the fact that patients were not allocated to treatment groups in a prospective manner and that insulin dose was calculated during follow - up from mean values , and not determined at baseline . 
 in addition , the exclusion of patients on combination insulin therapy may not reflect common practice , in which patients are typically on basal / bolus regimens requiring the use of different types of insulin . 
 finally , the duration of diabetes , as well as smoking status , body mass index ( bmi ) , and other potential confounding factors were not taken into account . 
 the three additional , commissioned studies were published in the same issue of diabetologia . in sweden , jonasson and 
 colleagues   evaluated 114,842 patients with diabetes on insulin and noted that women on glargine alone , but not glargine combined with other types of insulin , had a higher risk of breast cancer than women on other types of insulin ( hr adjusted for age , smoking , bmi , and other confounding factors was 1.97 , 95% ci 1.29 - 3.00 ) . 
 however , this increased risk was not associated with an increased mortality rate ; instead , all - cause mortality was actually decreased in women on glargine alone versus women on other types of insulin ( relative risk [ rr ] 0.83 , 95% ci 0.71 - 0.96 ) . 
 the associations with other malignancies such as prostate and gastrointestinal tumors did not reach statistical significance . 
 no analysis taking into account insulin dose was possible in this study , and therefore the incremental risk conferred by higher doses as observed by hemkens et al . 
 the short duration of exposure and observation as well as the lack of data on how much insulin the patients were taking limit the strength of the conclusions . in a study in scotland , no increased risk of total or site - specific cancer associated with overall glargine use was found in a cohort of 12,852 patients followed for 4 years ; in particular , no increase in breast cancer risk ( hr 1.49 , 95% ci 0.79 - 2.83 ) was found . 
 however , exclusive use of glargine did seem to confer higher risk ( adjusted hr 3.39 , 95% ci 1.46 - 7.85 ) , but the number of cases was small ( six events ) . 
 in addition , glargine - only users tended to be older , were more likely to be women , and to have a higher bmi , reflecting a baseline imbalance . 
 finally , the restricted use of glargine in scotland may have contributed to a skewing of the results , since there were a limited number of patients taking this form of insulin . in a uk 
 studied 62,809 patients with diabetes on metformin , sulfonylurea monotherapy , a combination of both , or insulin therapy . 
 the latter were further divided into glargine - treated versus other human insulin - treated ( long - acting , biphasic , and biphasic analogue ) . the results indicated that any type of insulin therapy increased the risk of occurrence of solid tumors and that concomitant metformin therapy attenuated this effect . 
 indeed , the adjusted hrs relative to metformin monotherapy were 1.08 ( 95% ci 0.96 - 1.21 ) for the metformin / sulfonylurea combination , 1.36 ( 95% ci 1.19 - 1.54 ) for sulfonylurea alone , and 1.42 ( 95% ci 1.27 - 1.60 ) for insulin - based therapies . 
 concomitant metformin therapy decreased the risk of malignancy associated with insulin by 46% ( hr 0.54 , 95% ci 0.43 - 0.66 ) . when compared with glargine 
 , other human insulins displayed comparable rates , suggesting no added risk attributable to glargine ( adjusted hr 1.24 , 95% ci 0.90 - 1.17 for basal human insulin ; hr 0.88 , 95% ci 0.66 - 1.19 for biphasic human insulin ; and hr 1.02 , 95% ci 0.76 - 1.37 for analogue biphasic insulin ) . 
 malignancies that had the strongest association with insulin therapy compared with metformin were colorectal ( hr 1.69 , 95% ci 1.23 - 2.33 ) and pancreatic ( hr 4.63 , 95% ci 2.64 - 8.10 ) tumors . 
 breast and prostate cancer risks did not seem to be affected by treatment with insulin or insulin secretagogues , and a prior history of solid tumor strongly increased the overall risk ( hr 3.86 , 95% ci 3.46 - 4.31 ) . in a letter published in a subsequent issue of diabetologia , rosenstock et al . 
 , reported a study of glargine versus neutral protamine hagedorn ( nph ) insulin in type 2 diabetes patients randomly assigned to one insulin or the other for over 4 years . although malignancy risk was not a primary endpoint for the study , no increased cancer risk with glargine compared to nph was found ( rr 0.90 , 95% ci 0.64 - 1.26 ) . home and lagarenne , using the sanofi - aventis 
 pharmacovigilance database for all randomized company - sponsored clinical trials , published a systematic review of 31 trials comparing glargine to other agents ( nph for most studies ) . 
 no evidence for an increased risk of malignancy attributable to glargine was found ( rr 0.90 , 95% ci 0.60 - 1.36 ) . 
 all but one of these studies lasted less than 12 months ( on average , 6 months ) , so their major limitation was the short duration of exposure and follow - up . 
 indeed , the majority of reported cases of malignancy were derived from the study of rosenstock and colleagues , discussed above , in which follow - up exceeded 4 years . 
 finally , a meta - analysis comparing cancer risk associated with another new , long - acting insulin , insulin detemir , versus nph and glargine was performed . 
 it used data from randomized controlled trials sponsored by novo nordisk ( bagsvaerd , denmark ) and found comparable malignancy risks associated with detemir and glargine in the five trials that compared both drugs . 
 however , the number of events was very small ( n = 8 ; event rate per 100 exposure - years = 0.87 for detemir , and n = 8 ; event rate per 100 exposure - years = 1.27 for glargine ) . 
 the issues are highly emotive : cancer and diabetes eventually affect a large proportion of the world 's population . 
 in addition , there are enormous financial implications surrounding the controversy , and unfortunately , complex issues tend to be oversimplified and are often sensationalized . 
 either a diagnosis of cancer or the institution of insulin therapy for diabetes provokes great anxiety in many people . 
 criticism of the publication of the diabetologia papers came in the correspondence section of the lancet , where pocock and smeeth   raised concerns regarding the quality of the statistical analyses and contended that these analytical flaws , especially in the german paper , had raised unsubstantiated anxieties . 
 they criticized the swedish study for its finding of an association of insulin glargine with breast cancer , arguing that the latter was not a 
 pre - defined site for analysis but rather a secondary finding ultimately not  confirmed by the scottish study   ( the latter did find an increased risk of breast cancer associated with the exclusive use of glargine , but only affecting very few , older patients ) . 
 further criticism came in an editorial by garg , hirsch , and skyler   in diabetes technology and therapeutics , questioning the motivations behind the german study and raising concerns similar to those brought up by pocock and smeeth . 
 this paper also emphasized the lack of evidence of an increased overall cancer risk associated with glargine based on the swedish , scottish , and uk studies , concluding that  glargine use should not be discontinued based on unsubstantiated allegations. the letter of pocock and smeeth elicited a prompt response from the editor of diabetologia . 
 the editor , while acknowledging the imperfections of the studies ( which he had already discussed in detail in an accompanying editorial ) , argued that the epidemiologic data can not and should not be dismissed , especially when considered in the context of animal and cell culture work , but rather should generate further research to define risk . 
 indeed , in his initial editorial on the subject , he had earlier stated that 
 the evidence presented in this set of papers is sufficient to establish that there is a case to answer , but is entirely insufficient to bring a verdict. at least on the latter conclusion , all parties agreed . 
 these thought - provoking studies , although inconclusive , do raise concerns about the long - term effects of diabetes itself , insulin treatment , and specifically , glargine , on cancer development . 
 it must be remembered that the greatly increased mortality among patients with diabetes has little to do with cancer , but instead is related to vascular complications . 
 the overall benefit - to - risk ratio of good glycemic control is likely to support extensive use of insulin . 
 all are agreed that the current evidence does not warrant any change in clinical practice , specifically with regard to the use of glargine . 
 further research will need to address the relationship between diabetes , insulin , insulin analogues and secretagogues , and cancer . 
 it will also explore potentially protective effects of metformin and perhaps the most tantalizing question : why is diabetes not more strongly associated with cancer ? 
 ","diabetes , in particular type 2 , is associated with an increased incidence of cancer . 
 although the mortality attributable to cancer in type 2 diabetes is overshadowed by that due to cardiovascular disease , emerging data from epidemiologic studies suggest that insulin therapy may confer added risk for cancer , perhaps mediated by signaling through the igf-1 ( insulin - like growth factor-1 ) receptor . 
 co - administered metformin seems to mitigate the risk associated with insulin . 
 a recent series of publications in diabetologia addresses the possibility that glargine , the most widely used long - acting insulin analogue , may confer a greater risk than other insulin preparations , particularly for breast cancer . 
 this has led to a heated controversy . despite this 
 , there is a consensus that the currently available data are not conclusive and should not be the basis for any change in practice . 
 further studies and more thorough surveillance of cancer in diabetes are needed to address this important issue .",pubmed
"adamantinoma of the tibia is a rare slow - growing primary tumor of the bone . 
 there has been much controversy on the origins of this tumor with one major focus being the relationship between osteofibrous / fibrous dysplasia and adamantinoma . 
 there have been approximately 300 cases of tibial adamantinoma reported in the published english language literature . of these cases , 1520% 
 the most common site of metastasis is to the lungs . as metastases to the regional lymph nodes 
 are rare , there is limited information regarding the best practice plan for their management in the literature . 
 we report 2 cases of tibial adamantinoma , which were problematic from a diagnostic point of view . 
 case 2 is of a tibial adamantinoma that presented with groin metastases which was initially diagnosed as a cutaneous adnexal carcinoma . 
 a review of inguinal nodal metastatic adamantinoma is outlined in addition to the clinic - pathological data on these cases . 
 in 2007 , a 29-year - old female presented with a swelling in her left inguinal area . 
 investigations including a computed tomography ( ct ) scan showed an extremely large heterogeneous , hyperdense , lobular lymph node centered in the left inguinal region measuring 6.54.6 cm . 
 histopathological examination revealed the presence of a malignant neoplasm that was composed of sheets of basaloid cells with round vesicular nuclei and minimal cytoplasm . 
 this lesion was interpreted as a cutaneous eccrine carcinoma ( figure 1a  c ) . due to the unusual diagnosis , 
 an external consultation and a request for additional clinical history were obtained . on detailed review , 
 the pathological report was reinterpreted as metastatic adamantinoma in conjunction with a confirmed past clinical history of surgical excision of a left tibial adamantinoma three years previously in 2004 . 
 the specimen consisted of a firm rubbery mass of fibro - fatty tissue that measured 18.511.37.2 cm with an ellipse of skin measuring 820.3 cm . 
 the cut surface revealed a multi - lobulated light brown well - circumscribed mass that measured 10.475.5 cm . on microscopic examination , 
 the neoplastic cells were composed of predominantly basaloid and tubular nests of malignant cells with some peripheral palisading ( figure 1d  f ) . 
 the histomorphological appearance of these cells was identical to the previous histology in this case ( figure 1a  c ) confirming the presence of inguinal lymph nodal metastases from adamantinoma of the tibia . 
 biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) . 
 lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node (  ) ( d ) . 
 the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) . 
 biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) . 
 lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node (  ) ( d ) . 
 the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) . in april 2009 , 
 a new node along the left external iliac in the retroperitoneum was detected and biopsied under radiological guidance . 
 histopathological examination showed similar findings confirming the presence of metastatic adamantinoma ( figure 2a and b ) . 
 ct scans of the chest showed the presence of two nodules in the right lung , which remained stable on follow up over the following year . 
 follow up in early 2011 shows stable residual disease with no additional findings of note . 
 photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm . 
 the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 . 
 photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm . 
 the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 . in 1999 , a 58-year - old female presented with a painful swelling on the mid shaft of her left tibia . 
 histopathological examination showed the presence of islands of woven bone in a background of bland spindled fibrous tissue , which was interpreted as fibrous dysplasia ( figure 3a and b ) . 
 nine years later , in 2008 , she was seen for a painful swelling in the same region , which had been increasing in size since her previous operation . 
 a wide excision of the lesion with a tibial allograft and intramedullary nail fixation was performed in october 2009 . 
 there was a predominant fibrous dysplasia like pattern consisting of cellular bland , spindled fibroblastic proliferation with scattered islands of woven bone . embedded within the spindle cells were occasional clusters of plump epitheliod appearing cells that had a corded arrangement in focal regions ( figure 3c ) . 
 immunohistochemistry confirmed the epithelial nature of these cells with strong positive staining to pankeratin antibodies ( figure 3d ) . 
 such areas were not identified even on retrospective detailed examination of the previous biopsy from 1999 . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) . 
 medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma . 
 ( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) . 
 medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma . 
 ( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) . 
 the patient underwent revision surgery for non - union of the left tibia in 2010 . 
 in 2007 , a 29-year - old female presented with a swelling in her left inguinal area . 
 investigations including a computed tomography ( ct ) scan showed an extremely large heterogeneous , hyperdense , lobular lymph node centered in the left inguinal region measuring 6.54.6 cm . 
 histopathological examination revealed the presence of a malignant neoplasm that was composed of sheets of basaloid cells with round vesicular nuclei and minimal cytoplasm . 
 this lesion was interpreted as a cutaneous eccrine carcinoma ( figure 1a  c ) . due to the unusual diagnosis , 
 an external consultation and a request for additional clinical history were obtained . on detailed review , 
 the pathological report was reinterpreted as metastatic adamantinoma in conjunction with a confirmed past clinical history of surgical excision of a left tibial adamantinoma three years previously in 2004 . 
 the specimen consisted of a firm rubbery mass of fibro - fatty tissue that measured 18.511.37.2 cm with an ellipse of skin measuring 820.3 cm . 
 the cut surface revealed a multi - lobulated light brown well - circumscribed mass that measured 10.475.5 cm . on microscopic examination , 
 the neoplastic cells were composed of predominantly basaloid and tubular nests of malignant cells with some peripheral palisading ( figure 1d  f ) . 
 the histomorphological appearance of these cells was identical to the previous histology in this case ( figure 1a  c ) confirming the presence of inguinal lymph nodal metastases from adamantinoma of the tibia . 
 biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) . 
 lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node (  ) ( d ) . 
 the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) . 
 biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) . 
 lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node (  ) ( d ) . 
 the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) . in april 2009 , 
 a new node along the left external iliac in the retroperitoneum was detected and biopsied under radiological guidance . 
 histopathological examination showed similar findings confirming the presence of metastatic adamantinoma ( figure 2a and b ) . 
 ct scans of the chest showed the presence of two nodules in the right lung , which remained stable on follow up over the following year . 
 follow up in early 2011 shows stable residual disease with no additional findings of note . 
 photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm . 
 the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 . 
 photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm . 
 the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 . 
 in 1999 , a 58-year - old female presented with a painful swelling on the mid shaft of her left tibia . 
 histopathological examination showed the presence of islands of woven bone in a background of bland spindled fibrous tissue , which was interpreted as fibrous dysplasia ( figure 3a and b ) . 
 nine years later , in 2008 , she was seen for a painful swelling in the same region , which had been increasing in size since her previous operation . 
 a wide excision of the lesion with a tibial allograft and intramedullary nail fixation was performed in october 2009 . 
 there was a predominant fibrous dysplasia like pattern consisting of cellular bland , spindled fibroblastic proliferation with scattered islands of woven bone . embedded within the spindle cells were occasional clusters of plump epitheliod appearing cells that had a corded arrangement in focal regions ( figure 3c ) . 
 immunohistochemistry confirmed the epithelial nature of these cells with strong positive staining to pankeratin antibodies ( figure 3d ) . 
 such areas were not identified even on retrospective detailed examination of the previous biopsy from 1999 . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) . 
 medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma . 
 ( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) . 
 photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) . 
 medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma . 
 ( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) . 
 the patient underwent revision surgery for non - union of the left tibia in 2010 . 
 a literature search limited to the english language using the search terms adamantinoma of tibia and tibial adamantinoma and metastases and inguinal lymph node metastases was conducted . 
 research engines used included pubmed , medline , scopus , embase , and google / google scholar . 
 table 1 provides a comprehensive chronological review of 10 cases with inguinal lymph node metastases with details of reference number , year reported , author , sex , age at diagnosis , bone involved , location in bone , character and duration of symptoms + / history of trauma , latent period , treatment , and outcome . 
 , six cases with lymph node metastases as described in keeney 's study are not included in this table due to the lack of supporting clinical information . 
 similarly , cases with inguinal lymph node metastases as described in moon 's review have been excluded from this table as the original reports are not in english . 
 table 1inguinal lymph node metastases from adamantinoma of the tibia as reported in the english literature ( pub med , medline , scopus , embase , google / google scholar ) since 1930-search terms included adamantinoma of the tibia , and tibial adamantinoma and metastases , and inguinal lymph node metastases.reference numberyear reportedauthorsexage at diagnosisbone involvedlocation in bonecharacter and duration of symptoms + /history of traumalatent periodtreatmentoutcome#101938dunnem32left tibiaupper and middle thirdsswelling , 4yr ; pain later months severe contusionnine monthshigh voltage roentgen , ak amputation 9 months laterdied , 8 years inguinal lymph nodes*#111942dockerty , meyerdingf24left tibiamiddlerecurrent pain , 8 yr . , swelling , 4yr . 
 no history of traumanonelocal excision , ak amputation 15 months laterdied , 1 year , inguinal lymph node metastases#121952mangalik , lal mehrotram40femur , tibia , fibuladistal proximal middleduration of symptoms for 12 months . 
 previous injury to leg1 yearak amputation , irradiation of metastatic nodes#131962knappf14left tibiamiddlemass for 4 years no history of traumaak amputationtotal survival 16 years . 
 pulmonary and inguinal lymph node metastasis#141976winter wgm30left tibiadistalnoneresection , disarticulation of lk , inguinal node dissection , pleurectomydied with inguinal nodes , pulmonary , brain - right occipital metsm37tibiamiddlenoneresection and bone grafting very late knee disarticulationdied , 8 years after initial surgery inguinal lymph nodes and pulmonary metastasis#151990de keyserf13tibiapathological fractureintramedullary nailing several curettages , en bloc resection bk amputation , ak amputationdied 4 years after ak amputation had pulmonary and lymph node metastasis#61994hazelbagm26left tibiadistalnoneamputation metastasis to groin lymph node , pelvisdied of diseasem15left tibiadistalnonechemotherapy , amputationdied of disease . 
 lung / lymph metastases#162008freym20tibianadnadtotal tibia allograft knee disarticulation polychemotherapypulmonary and inguinal lymph node metastases amputation2011tharmabala et al.f24left tibiaswelling left tibia swelling in left groin4 yearssurgical excision in left tibia 2004 . 
 left retroperitoneal mass excision 2009left inguinal nodal metastasis , stable pulmonary nodules with routine follow up in 2011*this is reported in reference # 17 by moon in 1965 . 
 it was first described by maier in 1900 as a primary bone tumor with epithelial characteristics . later in 1913 , fischer named the tumor primary adamantinoma of the tibia because of its resemblance to adamantinoma ( also known as ameloblastoma ) of the jaw and the pituitary gland . 
 although , adamantinoma of the tibia histologically resembles adamantinomas of the jaw and pituitary , there has been no evidence to suggest that these tumors are of similar origin . 
 adamantinoma has also been associated with osteofibrous / fibrous dysplasia and in this regard is considered by some authors to represent a spectrum of disease with evidence of progression from osteofibrous / fibrous dysplasia to adamantinoma in some cases . due to the rarity of this lesion and the varied histological phenotypes , adamantinoma of the tibia 
 can be a diagnostic dilemma leading to misdiagnosis , delay , and suboptimal treatment , as seen in our cases . 
 it can present at any age but mostly occurs in the second to fourth decade . 
 they are most commonly ( 8590% ) localized in the tibial diaphysis rarely , it has been described in other bones of the appendicular skeleton including the fibula , ulna , femur , humerus , radius , and short bones of the feet . 
 , , adamantinoma of the tibia can be divided into two main groups based on their histology and biological behavior . 
 classical adamantinoma refers to the prototype cases with a predominance of epithelial tumor cells and are associated with a destructive growth pattern on radiology . 
 patients with classical adamantinoma are usually of an older age group with a reported range of 1565 years . 
 this variant of classical adamantinoma has a stronger predisposition to local recurrences and a higher potential for distant metastases . 
 in contrast , differentiated adamantinoma of the tibia has a predominance of a osteofibrous / fibrous dysplasia like pattern with scant epithelial tumor elements and is commonly seen in an intracortical location . 
 many authors suggest that osteofibrous/ fibrous dysplasia , a benign lesion occurring commonly in the tibia of children under 10 years of age , is related to adamantinomas and may even be a precursor lesion for adamantinomas . 
 , in such scenarios , tibial adamantinomas have often been misdiagnosed as fibrous dysplasia , as seen in our case . 
 the association of fibrous dysplasia with adamantinoma was first described by cohen et al . in 1962 who proposed that the fibrous pattern seen in such cases was different from that seen in classical fibrous dysplasia . 
 this variant of differentiated adamantinoma is considered by some authors as a secondary reparative process that is associated with a more benign behavior and better prognosis . 
 distant metastases have been reported in adamantinoma of the long bones in about 1520% of cases this may be falsely low due to the slow growing nature of the tumor as long term follow up is required to evaluate its true metastatic potential . 
 regional or other distant metastases as seen in the lung are found more frequently in patients who have a history of repeated local recurrences linked to inadequate primary excision of this lesion . 
 however , in our case ( case 1 ) inguinal nodal metastases occurred with no evidence of local recurrence . 
 risk factors for metastases include male sex , persistent pain , symptoms of less than five years duration , and inadequate initial treatment by biopsy curettage / excision , or resection . 
 the only histological feature associated with an increased recurrence rate was lack of squamous differentiation . in view of the relationship between adamantinoma and osteofibrous dysplasia , czerniak et al . proposed two biological courses that a tibial adamantinoma may follow . 
 one is by the differentiated adamantinomas , which are predominantly intracortical and behave as a reparative process with a low potential to metastasize . 
 the second , such as that seen in our case ( case # 2 ) of classical adamantinoma , is one of progressive tumor growth with a higher potential to metastasize . 
 early metastases within the first two years of initial diagnosis is commonly seen to occur to the regional lymph nodes , whereas delayed metastases is almost always pulmonary or osseous . 
 given that lymph node metastasis usually occurs within the first two years , this is suggestive that these were present at the time of initial diagnosis . in this context , we recommend ultrasound imaging of regional lymph nodes as an essential component in the follow - up examinations of these cases . 
 the current practice recommends surgical extirpation as the primary mode of treatment for localized regional lymph node metastases with radiotherapy and chemotherapy as suggested alternatives . 
 the exact role for sentinel node biopsy and its cost / benefit ratio is unknown due to limited clinical experience in these rare lesions . in conclusion , 
 tibial adamantinoma is a rare primary bone tumor that can cause diagnostic dilemmas especially with clinical presentations of the uncommon pattern of regional nodal metastases . 
 increased clinical awareness of such rare neoplasms with recognition of uncommon patterns of metastases is essential for accurate diagnosis and optimal patient management . 
 additionally , due to the slow - growing nature of tibial adamantinomas we recommend continuous long - term follow up for these patients .","adamantinoma is a rare primary bone tumor that commonly arises in the jaw and has also been described in the appendicular skeleton such as the tibia . 
 we report 2 cases of tibial adamantinomas that were originally misdiagnosed ; one as fibrous dysplasia of the tibia and the other as a cutaneous eccrine carcinoma in a groin mass , which was metastatic adamantinoma to the inguinal lymph nodes . 
 such metastatic adamantinoma to the groin lymph nodes is extremely rare . 
 the clinical and pathological data with a review of the available literature on inguinal lymph node metastases from primary tibial adamantinoma are reported . 
 increased clinical awareness and accurate recognition of such uncommon patterns of inguinal nodal metastases are imperative for appropriate planning of therapeutic strategies and risk management in these patients .",pubmed
"antireflection ( ar ) technology plays an important role in the fabrication of high - efficiency solar cells by increasing light coupling into the active region of devices . 
 generally , a relatively low - light reflectivity can be obtained for crystalline silicon solar cells by combining surface texturization with sixny - thin - film deposition . however , in order to further reduce the light reflectivity for the improvement of solar cell efficiency , a variety of nanostructures with size comparable to the wavelength of m : most of the utilized solar spectrum have been proposed , such as photonic crystals , porous structures , plasm : monics [ 5 - 8 ] , etc . 
 the subwavelength structures can be nanowires , nanocones , frustum nanorods , biomimetic nanostructures [ 13 - 16 ] , dielectric nanoparticles   etc . 
 all these nanostructures should be based on the wide bandgap materials which , themselves , can not absorb sunlight . 
 the zno material has a wide bandgap , which has been popularly used in the fields of photocatalysis   and optoelectronic nanodevices . for photovoltaic application , the zno nanostructures as antireflective layers could be fabricated by various approaches like pulsed laser deposition , chemical vapor deposition , vapor  liquid - solid growth , hydrothermal method , and electrochemical deposition technique . 
 am : mong them , a seeding and growth two - step process in zinc salt and amide mixed solutions seems quite acceptable for photovoltaics due to its low cost and good potential for scale - up . 
 experiments have shown that zno nanorod arrays can exhibit better antireflective properties than other oxide compounds . 
 a weighted global reflectance of 6.6% has been achieved in the broadband range from 400 to 1,200  nm . 
 however , m : most work on zno nanorod arrays as antireflective layers is performed on the naked silicon wafer by now . the application of zno nanostructures in the practical silicon solar cells is seldom reported . in this work , 
 we have grown zno nanowhiskers ( nws ) with different lengths by the seeding and growth two - step process , and meanwhile have integrated the zno nw technique into the fabrication of czochralski ( cz ) silicon solar cells based on the current standard celling technology . the density and the length of zno nws 
 have been optimized for the improvement of solar cell performances by controlling the time of growth . 
 the result shows that the efficiency of cz silicon solar cell can be improved by a value of 3% relatively using the zno nws as the ar layer in practice . 
 the starting samples are p - type cz silicon solar cells fabricated by a standard process including surface texturization , phosphorus diffusion , sixny film deposition , screen printing , and metallization . 
 the seed layer of zno was first deposited on the front surface of solar cells by a magnetron sputtering system . 
 the deposition temperature is 150c , and the seed layer thickness is about 10  nm . after protecting the parts without the zno seed film with waterproof tapes , 
 the samples were immersed in the solution of zinc nitrate hexahydrate ( zn(no3)26h2o ) and ( ch2)6  n4 mixture for 30 to 60  min for the growth of zno nw . 
 the zno nw growth temperature is 90c . for characterization , a scanning electron microscope ( sem ; hitachi s-4800 , hitachi , ltd . 
 , chiyoda , tokyo , japan ) was used to observe the zno nws on the solar cells . 
 the reflection spectra of the samples were measured by a uv  vis spectrophotometer ( hitachi u-4100 ) in the wavelength range of 300 to 1,100  nm . 
 the external quantum efficiencies ( eqes ) of the final solar cells were obtained from the spectral responses . 
 the current  voltage characteristics of the cells under the dark and illumination circumstances were measured . for illumination , 
 one sun ( 100  mw / cm ) with am 1.5 global spectrum was used . 
 figure  1 shows the sem micrographs of a silicon solar cell with the zno nws after a two - step growth process . 
 it can be seen that the zno nws are uniformly fabricated on the surface of silicon solar cell . generally , the fabrication of zno nws based on aqueous solution consists of nucleation   and growth . 
 the main chemical reactions through the whole process can be described as follows : ( 1)znno326h2ozn2++  2no3+6h2o ( 2)ch26n4 + 6h2o6hcho+4nh3   sem images of top ( a ) and cross - sectional ( b ) views of zno nw - coated solar cell surface . 
 the decomposition of hexamethylenetetramine produces amm : monia that can supply the oh needed for the zno nw formation reaction and acts as a ph buffer to regulate the ph value of the solution . 
 the ph value of the solution should be 6 or 7 for the whole reaction process . during the process 
 , the zno nanocrystals existing in the seed layer serve as the nucleation centers where the growth of zno nws takes place . due to its growth habit , 
 c - axis is the m : most preferred growth orientation , which leads to the formation of zno nws . 
 figure  2 shows the sem micrographs of zno nws on the solar cells after growing at different times . 
 figure  2a , d shows the plan view and its cross - sections of zno nws after growing 30  min , respectively . 
 one can see that the tapered zno nws with the length of approximately 100  nm are obtained on the pyramid - like textured surface of solar cell via this method . with the growth time increasing , the density of zno nws increases ( see figure  2b , c , e , f ) . 
 meanwhile , the length of zno nws becomes larger , e.g. , approximately 200  nm after 45  min and approximately 300  nm after 60  min . 
 sem images of zno nws grown under different times and their cross - section view . 
 ( a ) and ( d ) 30  min , ( b ) and ( e ) 45  min , and ( c ) and ( f ) 60  min . 
 figure  3 shows the reflectance spectra of the solar cells without or with the zno nw coating . it can be seen that , compared with the solar cell without zno nw coating ( reference ) , all the solar cells with zno nw coating exhibit lower reflectance than in the whole wavelength range . 
 the reflectivity of zno nw - coated solar cells decreases with an increase of zno nw growth time . 
 the solar cell with zno nws grown for 45  min has the lowest reflectivity . 
 however , when the zno nw growth time reaches to 60  min , the reflectivity of coated solar cell becomes higher in the wavelength range of 500 to 1,100  nm . 
 since the density and length of zno nws increase with an increase of growing time , it is suspected that the zno nws grown for 60  min could have formed something like 
 this finally results in a higher reflection of zno nw grown for 60  min . 
 nevertheless , these results clarify that the zno nw - coated solar cells have better antireflection effect than the reference without zno nw coating . 
 the decrease of reflectivity in the long wavelength range mainly results from the effect of zno nw trapping light by the multiple scattering of the incident light . 
 meanwhile , their gradually and continuously changing refractive index profile from the air to the substrate is also beneficial for its antireflection property . 
 the drastic decrease of reflectance in the low - wavelength range should be mainly attributed to the absorption of light by zno nws , themselves , which hardly makes any contribution to the improvement of the solar cell performance . after a prolonged growing time 
 , e.g. , 60  min , the light absorption of long zno nws becomes even stronger , and therefore , their contribution to the solar cell efficiency is further smaller . 
 the reflectance ( r ) spectra for the solar cell with zno nw grown under different times . 
 figure  4 shows the eqes of the solar cells with zno nws grown for different times . 
 it can be seen that the solar cells with zno nws grown for 30 and 45  min generally have higher eqes than the referenced one in the whole spectral region . 
 it signifies that zno nws on cz silicon solar cells can effectively improve the response of light spectrum . for the solar cell with 
 zno nws grown for 60  min , the eqes in the short - wavelength range are smaller than that of the referenced due to the stronger light absorption of long zno nws . 
 table  1 lists the detailed parameters of the solar cells coated with zno nws grown for different times . 
 it can be seen that even though the open - circuit voltages for all the solar cells keep the same , the short - circuit current density of the solar cells coated by zno nws are higher . 
 this finally results in that the efficiency of the solar cells coated by zno nws grown for 30 and 45  min are improved by a value of 0.1% and 3.0% , respectively , in comparison with the referenced cell . 
 figure  5 examples the current density as a function of bias voltage for the solar cells with zno nws grown for 45  min . 
 it reveals that zno nw - coated solar cells have higher short - circuit current density than the referenced one , while the open - circuit voltages keep constant . 
 the improvement of short - circuit current density is obviously a result of zno nws suppressing the light reflectivity . in the case of zno nws 
 grown for 60  min , the fill factor of the cell has a low fill factor and , therefore , a low efficiency . 
 this deterioration of the cell performance should be related to the long - time immersion of the solar cell in the 90c aqueous solution during the zno nw growth process . 
 the external quantum efficiencies ( eqes ) of the solar cells with zno nws grown for different times . 
 performance parameters of c - si solar cells with and without zno nws ff fill factor , jsc short - circuit current density , voc , open - circuit voltage 
 . the current density - voltage curves of the solar cells with 45-min grown zno nws . under a standard am 1.5 illumination . 
 in summary , around 100 to 300  nm zno nws are grown on the surface of czochralski si solar cells based on a low cost aqueous solution method . 
 zno nws grown for different times , such as 30 , 45 , and 60  min , can reduce the reflectivity of the solar cells effectively ( r  <  3% ) in a large spectral region and improve the external quantum efficiency in the corresponding range . 
 furtherm : more , zno nws with 45-min growth time can improve the efficiency of the solar cells by 3% , corresponding to their increment of short - circuit current density and fill factor . 
 finally , long - time immersion of solar cells in aqueous solution during the zno nw growth process can deteriorate the solar cell performance drastically . 
  
  
 deren yang and xuegong yu are both professors and doctorate degree holders in the state key laboratory of silicon materials , department of materials science & engineering , zhejiang university . 
 dong wang , dong lei , and genhu li are masters degree holder candidates of the same university . 
 this work is supported by the national natural science foundation of china ( grant nos . 
 60906002 and 50832006 ) , national key technology r&d program ( 2011bae03b13 ) , key laboratory of optoelectronic materials chemistry and physics , cas ( 2008dp173016 ) , and innovation team project of zhejiang province ( 2009r50005 ) .","an efficient antireflection coating is critical for the improvement of silicon solar cell performance via increased light coupling . here 
 , we have grown well - aligned zno nanowhisker ( nw ) arrays on czochralski silicon solar cells by a seeding - growth two - step process . it is found that the zno nws have a great effect on the macroscopic antireflection effect and , therefore , improves the solar cell performance . 
 the zno nw array - coated solar cells display a broadband reflection suppression from 500 to 1,100  nm , and the minimum reflectance smaller than 3% can easily be achieved . by optimizing the time of zno nw growth 
 , it has been confirmed that an increase of 3% relatively in the solar cell efficiency can be obtained . 
 these results are quite interesting for the application of zno nanostructure in the fabrication of high - efficiency silicon solar cells .",pubmed
"little is understood about this condition 's mechanism , exacerbating factor , and definite treatment . 
 hyperhidrosis generally occurs in the axillae , the palms , plantar surface of the feet , and the face . 
 it is estimated to affect 0.6 to 1.0% of the population , generally young adults . 
 the principal characteristics of this condition include intense discomfort experienced by the patients , affecting their social , affective , and professional lives . 
 primary hyperhidrosis generally manifests in childhood and early adolescence , does not occur during sleep , and can be exacerbated by emotional stress . 
 excessive sweating of the palms and axillae can cause utmost embarrassment in professional and social interaction . 
 the fact that such sweating is not socially accepted , and the discomfort , odor , and stained clothes caused by constant wetness significantly decrease quality of life ( qol ) . 
 although various treatment options are available , including topical and systemic therapies , iontophoresis , regional nerve block , and botulinum toxin injection , have their limitations . 
 video - assisted thoracoscopic sympathetic surgery has been accepted globally as a form of treatment for primary palmar hyperhidrosis . in the literature , different methods , including resection ( sympathectomy ) , transection ( sympathicotomy ) , and clipping 
 much effort to reduce compensatory hyperhidrosis and determine its contributory factors has been attempted during surgical procedures . 
 it has been used to reduce severe compensatory hyperhidrosis related to the procedural level of the sympathetic chain . 
 procedures that involve r3 or / and r4 sympathetic ganglions are widely accepted in many treatment centers , and the results have been favorable . however , some patients still present with postoperative compensatory sweating or overly dry hands . 
 the aim of this study was to compare two simplified methods for the treatment of palmar and axillary hyperhidrosis , in which the sympathetic chain was transected in merely one segment at the level of either the third or fourth rib , respectively referred to as r3 or r4 sympathicotomy . 
 emphasis was placed on comparing the impact of sympathicotomy at r3 and r4 , and to evaluate the efficacy and adverse effects of palmar and axillary hyperhidrosis by surgery . 
 between january of 1999 and april of 2009 , 133 patients with primary hyperhidrosis underwent video - assisted thoracic sympathicotomy . there were 62 males and 71 females , with a mean age of 22.56.5 years . 
 the most common clinical manifestation of hyperhidrosis was palmer and axillary sweating ( in 39.9% ) , followed by palmer sweating alone ( in 33.8% ) , and axillary sweating alone ( in 26.3% ) . 
 the severity of palmar sweating , sweating in other parts of the body , other concomitant symptoms and past medical history were recorded . 
 chest radiography , electrocardiogram ( ecg ) and routine blood examination were performed before surgery . 
 severe primary palmar hyperhidrosis that significantly interfered with daily life , including work , was the common primary indication for surgery . 
 secondary hyperhidrosis caused by hyperthyroidism or neurotic anxiety , and patients with bradycardia ( heart rate < 60/min ) were excluded from surgery . 
 patients were randomized into two therapeutic groups ( table 1 ) . patients were placed under single lumen intubation general anesthesia in the semi - fowler 's position ( dorsal decubitus position with back lifted 45 degrees from horizontal , and abduction of both arms ) . 
 a 2 mm trocar was introduced into the pleural cavity through the fourth intercostals space of the mid - axillary line . 
 after thoracoscope insertion , an initial panoramic view of the upper pleural cavity and mediastinal structures was obtained . 
 another operating channel ( 2 mm ) was made through the fourth intercostal space of the anterior axillary line . 
 after identification of the thoracic sympathetic ganglion , the t3 or t4 sympathetic chains were then destroyed by electric cautery . the anesthesiologist re - inflated the lung . 
 following lung re - expansion by positive inflation with a 6 fr feeding catheter , the trocar was then removed . 
 the patients were asked simple questions related to symptom resolution , surgical outcome satisfaction , and occurrence / intensity of reflex sweating . 
 compensatory sweating in the other regions of the body was graded as "" none "" when compensatory sweating was not noticeable and did not interfere with daily activities , "" mild "" when it was tolerable but sometimes interfered with daily activities , "" moderate "" when it was barely tolerable and frequently interfered with daily activities , and "" severe "" when it was intolerable and always interfered with daily activities . 
 the degree of satisfaction in each patient was recorded as "" satisfied "" or "" dissatisfied "" . 
 self - reported satisfaction , recurrence , and improvement of sweating after intervention constituted the three recorded aspects of the patients ' subjective experience with sweat - triggering situations . 
 recurrence was defined as severe discomfort experienced by patients similar to that experienced before surgery , despite improvement in sweating . statistical analysis of comparisons between the 2 groups was conducted using the student 's unpaired t test and chi - square test with spss 13.0 ( spss , inc , chicago , il ) . 
 all operations were successfully performed using video - assisted thoracoscopy without associated severe morbidity and mortality . no life - threatening event or death occurred and 
 other complications , such as horner syndrome , hemothorax , intercostal neuralgia , pulmonary edema and atelectasis were not observed . 
 mild subcutaneous emphysema and residual pneumothorax on follow - up chest radiograph resolved spontaneously , and no further therapy was required . 
 there was no significant difference between the 2 groups in terms of age , sex , and positive family history ( table 1 ) . 
 in addition , there was no significant difference between the 2 groups in the rate of satisfaction and recurrence . 
 the incidence of "" moderate "" to "" severe "" compensatory sweating was higher in the r3 ( 71.8% ) group than r4 ( 33% , p=0.02)(table 2 ) . 
 no patient requested reoperation in either group , and no patient expressed regret after surgery . 
 the dominating indication for sympathetic surgery is hyperhidrosis of the upper limb . when conservative and medical treatment fails , endoscopic thoracic surgery is performed . 
 the first sympathectomy for the treatment of hyperhidrosis was performed in 1920 , in which anterior cervical access was used . in 1942 , 
 great advance in surgical technique was made , which rendered it unnecessary to sacrifice the stellate ganglion in order to denervate the upper limb . 
 as such , one of the most feared complications in thoracic sympathectomy - claude bernard - horner syndrome - was eliminated . in this study , patients were informed about the possible side effects of sympathicotomy , including compensatory sweating , prior to the procedure . 
 chou et al . asserted that reflex response in the sweating center of the hypothalamus could cause the underlying mechanism of compensatory sweating . 
 according to previous studies , various factors , such as the location of hyperhidrosis , sympathicotomy level , and surgical procedures including clamp , cut and resect can induce compensatory sweating . 
 also , the severity of compensatory sweating is influenced by patient condition , such as obesity , primary complaint severity and t2 sympathetic ganglion resection . 
 therefore , more selective sympathicotomy was proposed to minimize side effects , by lowering the level to preserve sympathetic tone in place of upper t2 sympathicotomy or multilevel sympathicotomy . 
 selective sympathicotomy is an effective surgical method to eliminate compensatory sweating in the treatment of hyperhidrosis . 
 although compensatory sweating remains the main secondary effect , numerous studies have reported that sympathicotomy is a safe and effective procedure for the management of hyperhidrosis and reduction of compensatory sweating . 
 the frequency and severity of compensatory sweating were reduced , with less interruption of sympathetic trunk in group t4 compared to group t3 ( table 2 ) . 
 this explains the differences at the other levels in sympathicotomy at palmar sweating and compensatory sweating are still controversial topic . 
 lin et al . suggested that postoperative results including compensatory sweating is influenced by changes in the sympathetic tone and disturbance of the reflex arc in the hypothalamus secondary to the procedures of the upper thoracic sympathetic system . 
 furthermore , anatomical variation , a mistake at the level of interruption , and incomplete resection of the sympathetic trunk may also cause compensatory sweating . the high personal expectations of patients could be the reason of dissatisfaction despite the development of minor complications . 
 difference in surgical technique , such as sympathicotomy level and surgical procedure ( clamp , cut and resect ) , and classification of the extent of compensatory sweating could impact on the complication rate . 
 many authors felt that the more sympathetic segments excised , particularly including t2 , the greater the incidence of severe compensatory symptoms . 
 dewey et al .   limited the extent of resection for hyperhidrosis to a single level , and found reduction in the incidence of severe compensatory symptoms . 
 however , until now , no prospective study has been performed to compare r3 and r4 single level sympathicotomy for the treatment of palmar and axillary hyperhidrosis . in this study , patients did not experience discomfort in their daily activities . 
 these results suggest that , compared to r3 sympathicotomy , r4 sympathicotomy decreases the extent of side effects but does not compromise efficacy . as such , it should be the method of choice for the treatment of palmar and axillary hyperhidrosis . 
 r3 and r4 have similar success rates in the treatment of palmar and axillary hyperhidrosis .","backgroundvideo - assisted thoracic sympathicotomy plays an important for the treatment of essential hyperhidrosis . 
 patients are usually satisfied with the surgical outcome at the early post - operative period , but suffer recurrence and compensatory sweating in the late post - operative period . 
 there are many sympathicotomy methods to minimize recurrence and compensatory sweating . 
 we compared the outcome of sympathicotomy methods above the third rib ( r3 ) and the fourth rib ( r4 ) with regards to symptoms , satisfaction , recurrence , and compensatory palmar and axillary hyperhydrosis.materials and methodsfrom january 1999 to april 2009 , 39 cases of thoracoscopic sympathicotomy at the third rib ( r3 ) , and 94 cases of thoracoscopic sympathicotomy at the fourth rib ( r4 ) for palmar and axillary hyperhidrosis were compared for early and late post - operative satisfaction , compensatory sweating and recurrence.resultsthere was no sex or age difference between groups . 
 early satisfaction was 94.9% and 98.9% in the r3 group and r4 group , respectively . 
 there was no difference in early satisfaction ( 94.9% in r3 and 98.9% in r4 ) , late satisfaction ( 84.6% in r3 and 89.4% in r4 ) , or recurrence ( 17.9% in r3 and 17.0% in r4 ) between groups . 
 there was significant difference in compensatory sweating ( 71.8% in r3 and 33% in r4 , p=0.002).conclusionr4 sympathicotomy demonstrated superior efficacy in the treatment of compensatory sweating compared to r3 in palmar and/or axillary hyperhidrosis .",pubmed
"parkinson disease ( pd ) , which is the most common neurodegenerative parkinsonian syndrome , is characterized by the degeneration of both dopaminergic and nondopaminergic neurons with neuronal intracytoplasmic inclusions known as lewy bodies 1 . 
 although the clinical diagnosis of pd is often straightforward and robust in the cases with the typical cardinal symptoms and characteristic signs , the correct diagnosis can be challenging , especially in clinically mild or uncertain cases . furthermore , it remains difficult to accurately differentiate pd from other neurodegenerative parkinsonian syndromes ( non - pd ) , such as multiple system atrophy ( msa ) , progressive supranuclear palsy ( psp ) , and corticobasal degeneration . 
 an accurate and early diagnosis is the key to correct management and prognostication . in recent years 
 , molecular imaging techniques using pet or single - photon emission computed tomography ( spect ) have offered a variety of tests that can improve the diagnostic accuracy of pd and non - pd . 
 dopamine transporter ( dat ) ligands 24 and the sympathetic innervation of the heart 1,5 have become very popular and are used widely in clinical practice . 
 for example , imaging of dat binding with [ i]-2-carbomethoxy-3-(4-iodophenyl)-n-(3-fluoropropyl)-nortropane ( fp - cit ) successfully visualizes the presynaptic dopaminergic degeneration of the nigrostriatal tract 4,6 . 
 dat spect abnormalities can help to support a diagnosis of degenerative parkinsonian syndromes by excluding patients with essential tremor 7 , and psychogenic 8 and vascular parkinsonism 9 who have a normal nigrostriatal function . however , dat imaging as a stand - alone tool can not differentiate between the various types of degenerative parkinsonian syndromes with sufficient accuracy 10,11 . 
 similarly , several studies have shown the utility of meta-[i]-iodobenzylguanidine ( mibg ) myocardial scintigraphy in assessing the sympathetic cardiac nerve terminals in pd patients with a decreased cardiac mibg uptake in comparison with other parkinsonian syndrome patients in whom the mibg uptake is normal 12 . because these molecular imaging tools are limited in terms of their test accuracy ( ta ) , the use of either dat spect or mibg scintigraphy alone is insufficient for distinguishing pd from non - pd 13 . in this retrospective study , we compared the results of spect studies of striatal dat receptors and myocardial mibg scintigraphy with the clinical diagnoses in 34 patients with suspected parkinsonian syndromes to evaluate the diagnostic accuracy and usefulness of combined spect imaging in the same patient population . 
 thirty - four patients ( male / female : 19/15 , mean age : 62.113.7 years , age range : 3181 years ) , who underwent both [ i]-mibg scintigraphy and dat spect within a 4-month period for a differential diagnosis between pd and other parkinsonian syndromes were consecutively enrolled in this retrospective study , which was carried out from april 2014 until june 2016 . because many patients underwent two tests during a period ranging from 3 to 4 months at our hospital , we established a 4-month interval between the tests . 
 the study was approved by the institutional ethics committee and all procedures were in accordance with the ethical standards on human investigation and with the principles of the declaration of helsinki . 
 all of the patients underwent molecular in - vivo brain and cardiac diagnostic examinations , which consisted of presynaptic striatal dat scintigraphy with fp - cit and the assessment of myocardial sympathetic innervation with mibg . 
 the final diagnosis was made by the neurologists of tokushima university hospital , department of neurology , who carefully assessed the medical history and symptoms , the possible response to dopaminergic treatment , and the diagnostic mri findings . 
 furthermore , five patients were diagnosed with probable msa - p according to the established criteria 15,16 and two patients fulfilled the national institute of neurologic disorders and stroke criteria for probable psp 17 . 
 the brain spect scans were acquired according to the european association of nuclear medicine procedure guidelines for brain neurotransmission spect using [ i]-labeled dat ligands ( fp - cit , datscan ; nihon medi - physics co. ltd , tokyo , japan ) 18 . 
 after an injection of 167 mbq of datscan , the projection data were obtained in a 128128 matrix on a ecam , toshiba ( toshiba medical systems corp . , 
 tochigi , japan ) ( 15 april 201414 april 2015 ) , or symbia t16 , siemens ( siemens healthcare , erlangen , germany ) ( 15 april 201530 july 2016 ) mounted with low - energy to medium - energy general - purpose collimators . 
 the data were reconstructed using the filtered back projection method without attenuation and scatter correction from 15 april 2014 to 14 april 2015 . 
 the data were reconstructed by the ordered subset expectation maximization method ( iteration 12 , subset 6 ) using a syngo miva10c with the symbia net software program ( siemens ag , munich , germany ) and corrected for attenuation by computed tomography and the triple energy window scatter correction method from 15 april 2015 to 30 july 2016 . the specific binding ratio ( sbr ) was calculated semiquantitatively using the dat view software program ( nihon medi - physics , tokyo , japan ) on the basis of bolt s method as described in detail elsewhere 19 . for the quantitative analysis , irregular regions of interest ( rois ) were drawn on single - slice views in areas corresponding to the left and right striata on either side . 
 although dat spect in pd typically shows an asymmetric loss of dat binding ( most prominent in the putamen ) , the tracer uptake is more symmetric in msa 20 and psp 21 . in one report of pathology - confirmed pd and msa cases with a similar disease duration 22,23 , visual inspection and a semiquantitative analysis with 
 dat spect detected a bilateral reduction in striatal dat binding in all patients , but the trend toward greater asymmetric binding was greater in msa than in pd . 
 for this study , we adopted the average value of the right and left sbrs as the sbr . 
 after the patient had rested for 15  min in the supine position , 111  mbq of [ i]-mibg ( myomibg ; fuji ri pharma , tokyo , japan ) was injected intravenously . 
 early and delayed spect were performed at 15  min and 3  h after the injection , respectively . 
 planar imaging for 5  min in the anterior projection was performed automatically during spect . 
 planar scanning and spect were performed using a dual - head gamma camera equipped with a low - energy to medium - energy general purpose parallel - hole collimator ( toshiba ecam ) . 
 the heart to mediastinum ( h / m ) ratio was calculated by dividing the count density of the left ventricular roi by that of the mediastinal roi , according to the standard method , which has been described previously 23 , 24 . 
 the h / m ratios calculated from the roi counts obtained by delayed spect were used for the comparison study because delayed scans show the neuronal uptake of mibg more explicitly 24 . 
 the values were expressed as meansd and were analyzed using student s t - test and -test . 
 p values of less than 0.05 were considered to indicate a statistically significant difference between pd and non - pd groups . 
 the sensitivity and specificity of the respective diagnostic index ( h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for differentiation between pd and non - pd were assessed using a receiver - operating characteristic ( roc ) analysis . for the combined use of dat spect and mibg myocardial scintigraphy , the cutoff delayed - phase h / m and sbr values were 2.745 and 3.24 , respectively . 
 the statistical analyses were carried out using the spss software program ( ibm spss statistics , version 23 ; ibm corp . , armonk , new york , usa ) . 
 the normality of the data distribution was assessed using the shapiro  wilk test . the mann 
 whitney u - test was used for the comparison of non - normally distributed data . 
 differences with a p value of less than 0.05 were considered to be biologically significant . 
 the analysis of the descriptive statistics and basic comparisons were carried out using the spss software program . 
 the sensitivity and specificity of the respective diagnostic index ( the h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for the differentiation between pd and non - pd were assessed in a roc analysis . 
 we also calculated the specificity , sensitivity , positive predictive value , negative predictive value , and ta of the combination of the two methods using the cutoff value for their combination . 
 the brain spect scans were acquired according to the european association of nuclear medicine procedure guidelines for brain neurotransmission spect using [ i]-labeled dat ligands ( fp - cit , datscan ; nihon medi - physics co. ltd , tokyo , japan ) 18 . 
 after an injection of 167 mbq of datscan , the projection data were obtained in a 128128 matrix on a ecam , toshiba ( toshiba medical systems corp . , tochigi , japan ) ( 15 april 201414 april 2015 ) , or symbia t16 , siemens ( siemens healthcare , erlangen , germany ) ( 15 april 201530 july 2016 ) mounted with low - energy to medium - energy general - purpose collimators . 
 the data were reconstructed using the filtered back projection method without attenuation and scatter correction from 15 april 2014 to 14 april 2015 . 
 the data were reconstructed by the ordered subset expectation maximization method ( iteration 12 , subset 6 ) using a syngo miva10c with the symbia net software program ( siemens ag , munich , germany ) and corrected for attenuation by computed tomography and the triple energy window scatter correction method from 15 april 2015 to 30 july 2016 . the specific binding ratio ( sbr ) was calculated semiquantitatively using the dat view software program ( nihon medi - physics , tokyo , japan ) on the basis of bolt s method as described in detail elsewhere 19 . for the quantitative analysis , 
 irregular regions of interest ( rois ) were drawn on single - slice views in areas corresponding to the left and right striata on either side . 
 although dat spect in pd typically shows an asymmetric loss of dat binding ( most prominent in the putamen ) , the tracer uptake is more symmetric in msa 20 and psp 21 . in one report of pathology - confirmed pd and msa cases with a similar disease duration 22,23 , visual inspection and a semiquantitative analysis with 
 dat spect detected a bilateral reduction in striatal dat binding in all patients , but the trend toward greater asymmetric binding was greater in msa than in pd . 
 for this study , we adopted the average value of the right and left sbrs as the sbr . 
 after the patient had rested for 15  min in the supine position , 111  mbq of [ i]-mibg ( myomibg ; fuji ri pharma , tokyo , japan ) was injected intravenously . 
 early and delayed spect were performed at 15  min and 3  h after the injection , respectively . 
 planar imaging for 5  min in the anterior projection was performed automatically during spect . 
 planar scanning and spect were performed using a dual - head gamma camera equipped with a low - energy to medium - energy general purpose parallel - hole collimator ( toshiba ecam ) . 
 the heart to mediastinum ( h / m ) ratio was calculated by dividing the count density of the left ventricular roi by that of the mediastinal roi , according to the standard method , which has been described previously 23 , 24 . 
 the h / m ratios calculated from the roi counts obtained by delayed spect were used for the comparison study because delayed scans show the neuronal uptake of mibg more explicitly 24 . 
 the values were expressed as meansd and were analyzed using student s t - test and -test . 
 p values of less than 0.05 were considered to indicate a statistically significant difference between pd and non - pd groups . 
 the sensitivity and specificity of the respective diagnostic index ( h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for differentiation between pd and non - pd were assessed using a receiver - operating characteristic ( roc ) analysis . for the combined use of dat spect and mibg myocardial scintigraphy , the cutoff delayed - phase h / m and sbr values were 2.745 and 3.24 , respectively . 
 the statistical analyses were carried out using the spss software program ( ibm spss statistics , version 23 ; ibm corp . , armonk , new york , usa ) . 
 the mann  whitney u - test was used for the comparison of non - normally distributed data . 
 differences with a p value of less than 0.05 were considered to be biologically significant . 
 the analysis of the descriptive statistics and basic comparisons were carried out using the spss software program . 
 the sensitivity and specificity of the respective diagnostic index ( the h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for the differentiation between pd and non - pd were assessed in a roc analysis . 
 we also calculated the specificity , sensitivity , positive predictive value , negative predictive value , and ta of the combination of the two methods using the cutoff value for their combination . 
 although there was no significant difference in the age of the patients in the two groups , a statistically significant difference was observed in the male - to - female ratio . 
 the demographic and clinical features of the pd and non - pd patients the mean h / m ratio of the mibg uptake in the delayed phase ( 2.190.55 vs. 2.440.80 , cutoff=2.745 , p=0.025 ) was significantly lower in the pd patients than in the non - pd patients ; however , the mean sbr ( 2.460.87 vs. 2.941.40 , cutoff=3.24 , p=0.08 ) did not differ to a statistically significant extent ( table 1 and fig . 
 roc analyses were used to set the cutoff sbr value ( in dat spect ) and the h / m ratio ( in delayed scanning ) in mibg scintigraphy for differentiating pd from non - pd ( fig . 
 2 ) . in the comparison with the separate use of the sbr and the delayed h / m ratio , the combined use of the two semiquantitative analyses using cutoff values of less than 3.24 and less than 2.745 , respectively , allowed for more accurate differentiation between pd and non - pd . 
 the sensitivity and specificity of sbr were 86.7 and 52.6% , respectively ( table 2 ) . 
 the sensitivity and specificity of the delayed h / m ratio on mibg scintigraphy were 93 and 47% , respectively . 
 the accuracy of these analyses in differentiating between pd and non - pd was the same using each of the molecular methods ( dat spect , 67.6% ; mibg scintigraphy , 67.6% ) . in comparison with the sbr and delayed h / m ratio , the combined use of two semiquantitative analyses enabled pd and non - pd to be differentiated more accurately ( cutoff values : sbr<3.24 ; delayed h / m ratio<2.745 ) . 
 these indices distinguished pd from non - pd with a sensitivity of 86.7% , a specificity of 73.7% , an accuracy of 79.4% , a positive predictive value of 72.2% , and a negative predictive value of 87.5% ( table 2 ) . 
 box plots of the striatal fp - cit and cardiac delayed mibg accumulation in the pd and non - pd patients . 
 the box plots show the striatal fp - cit and cardiac delayed mibg accumulation in the pd and non - pd patients . 
 whiskers represent the minimum and maximum values . there was a statistically significant difference in the delayed h / m ratios of the pd and non - pd groups . 
 the sbr values of the two groups did not differ to a statistically significant extent . 
 - cit , n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)-nortropane ; h / m , heart to mediastinum ratio ; mibg , meta-[i]-iodobenzylguanidine ; non - pd , degenerative parkinsonian syndrome other than pd ; pd , parkinson s disease ; sbr , specific binding ratio . the receiver - operating characteristic curves of the delayed h / m ratio and sbr . delayed h / m ratio : auc=0.698 ; cutoff value=2.745 ; sensitivity=0.93 ; specificity=0.47 . 
 auc , area under the curve ; h / m , heart to mediastinum ratio ; sbr , specific binding ratio . 
 the statistical parameters that were evaluated when investigating the combined use of fp - cit spect and mibg scintigraphy in differentiating pd from non - pd 
 this retrospective study investigated the extent to which the combined use of mibg scintigraphy and dat spect may improve diagnostic accuracy in differentiation between pd and non - pd . 
 the analysis showed that the individual molecular approaches had a mild ta in differentiating between pd and non - pd ( mibg scintigraphy , 62% ; fp - cit spect , 69% ) . in agreement with previous studies 25,26 
 , we confirmed that the combined use of dat spect and mibg scintigraphy was more useful for differentiating between pd from non - pd in comparison with the use of either method alone . 
 dat spect is therefore very popular in the clinical setting and enables highly accurate differentiation between degenerative parkinsonian syndromes and movement disorders that are not associated with a dopamine deficit 79 . 
 according to plotkin , the resulting accuracy , sensitivity , and specificity of fp - cit spect in the detection of pd and non - pd were 94 , 93 , and 100% , respectively 28 . 
 the loss of dopaminergic neurons in the substantia nigra and the reduction of striatal dopamine projections are histopathological characteristics that are common to the degenerative parkinsonian syndromes and the low accumulation of striatal dat shows a moderate ta in differentiating pd from non - pd . 
 the sensitivity ( 86.7% ) and specificity ( 52.6% ) that were observed in the present study were consistent with previous studies 10,20,29,30 . in our study , the sensitivity and specificity in differentiating pd from non - pd using the h / m ratio of the mibg uptake were 93 and 47% , respectively . 
 the usefulness of mibg myocardial scintigraphy in the diagnosis of dlb was suggested recently in a meta - analysis . the sensitivity and specificity of mibg scintigraphy in the differential diagnosis between pd and other parkinsonian syndromes ranged from 71 to 100% and from 50 to 100% , respectively , with pooled estimates of 88% ( 95% confidence interval : 8690% ) and 85% ( 95% confidence interval : 8188% ) 5 . 
 the specificity observed in the present study was lower than expected . in terms of the diagnostic performance , mibg scintigraphy is usually a sensitive , but not specific , test for pd 5 . 
 there might be several reasons for this . as the myocardial [ i]-mibg uptake in patients with non - pd ( especially in patients with msa and psp ) is often found to be slightly reduced in comparison with healthy controls 31 , 
 [ i]-mibg scintigraphy may yield some false - positive results in patients with msa and psp . 
 the myocardial sympathetic nervous system of msa patients typically shows mild degeneration 32 ; thus , false - positive results may account for this scintigraphic finding . 
 in addition to lbd - related postganglionic sympathetic degeneration , false - positive results on [ i]-mibg scintigraphy may also occur because of age - related degeneration because the myocardial [ i]-mibg uptake has been reported to decrease significantly with age 30,33 . 
 the combination of dopaminergic and sympathetic scintigraphic imaging tests for differentiating between pd and non - pd has been explored by other authors 25,26,28,30,34 . 
 some studies showed the complementary role of [ i]-mibg scintigraphy and dat spect in the differential diagnosis between pd and non - pd 25,34 . 
 a recent study showed that [ i]-fp - cit spect presents high sensitivity in the diagnosis of lewy body disease ; thus , [ i]-mibg scintigraphy may play a complementary role in the differential diagnosis between pd and other parkinsonism 30 . in the present study 
 , we therefore explored the extent to which the combination of these two tracers would improve the ta . 
 our data show that by combining fp - cit spect and mibg scintigraphy , the ta can be increased to 79.4% . 
 first , this study was carried out in a single university hospital ; thus , the study population was small . 
 second , the clinical diagnosis was used as the gold standard in our study because of the absence of histopathological confirmation ; thus , the clinical diagnosis may have been incorrect in some cases . 
 third , before a molecular examination is performed , it is necessary to establish an appropriate withdrawal period in which to cease the administration of interfering drugs  by considering their biological half - lives . 
 dat spect is therefore very popular in the clinical setting and enables highly accurate differentiation between degenerative parkinsonian syndromes and movement disorders that are not associated with a dopamine deficit 79 . 
 according to plotkin , the resulting accuracy , sensitivity , and specificity of fp - cit spect in the detection of pd and non - pd were 94 , 93 , and 100% , respectively 28 . 
 however , the loss of dopaminergic neurons in the substantia nigra and the reduction of striatal dopamine projections are histopathological characteristics that are common to the degenerative parkinsonian syndromes and the low accumulation of striatal dat shows a moderate ta in differentiating pd from non - pd . 
 the sensitivity ( 86.7% ) and specificity ( 52.6% ) that were observed in the present study were consistent with previous studies 10,20,29,30 . 
 in our study , the sensitivity and specificity in differentiating pd from non - pd using the h / m ratio of the mibg uptake were 93 and 47% , respectively . 
 the usefulness of mibg myocardial scintigraphy in the diagnosis of dlb was suggested recently in a meta - analysis . the sensitivity and specificity of mibg scintigraphy in the differential diagnosis between pd and other parkinsonian syndromes ranged from 71 to 100% and from 50 to 100% , respectively , with pooled estimates of 88% ( 95% confidence interval : 8690% ) and 85% ( 95% confidence interval : 8188% ) 5 . 
 the specificity observed in the present study was lower than expected . in terms of the diagnostic performance , mibg scintigraphy is usually a sensitive , but not specific , test for pd 5 . 
 there might be several reasons for this . as the myocardial [ i]-mibg uptake in patients with non - pd ( especially in patients with msa and psp ) is often found to be slightly reduced in comparison with healthy controls 31 , 
 [ i]-mibg scintigraphy may yield some false - positive results in patients with msa and psp . 
 the myocardial sympathetic nervous system of msa patients typically shows mild degeneration 32 ; thus , false - positive results may account for this scintigraphic finding . 
 in addition to lbd - related postganglionic sympathetic degeneration , false - positive results on [ i]-mibg scintigraphy may also occur because of age - related degeneration because the myocardial [ i]-mibg uptake has been reported to decrease significantly with age 30,33 . 
 the combination of dopaminergic and sympathetic scintigraphic imaging tests for differentiating between pd and non - pd has been explored by other authors 25,26,28,30,34 . 
 some studies showed the complementary role of [ i]-mibg scintigraphy and dat spect in the differential diagnosis between pd and non - pd 25,34 . a recent study showed that [ i]-fp - cit spect presents high sensitivity in the diagnosis of lewy body disease ; thus , [ i]-mibg scintigraphy may play a complementary role in the differential diagnosis between pd and other parkinsonism 30 . in the present study 
 , we therefore explored the extent to which the combination of these two tracers would improve the ta . 
 our data show that by combining fp - cit spect and mibg scintigraphy , the ta can be increased to 79.4% . 
 first , this study was carried out in a single university hospital ; thus , the study population was small . 
 second , the clinical diagnosis was used as the gold standard in our study because of the absence of histopathological confirmation ; thus , the clinical diagnosis may have been incorrect in some cases . 
 third , before a molecular examination is performed , it is necessary to establish an appropriate withdrawal period in which to cease the administration of interfering drugs  by considering their biological half - lives . 
 the combination of dat spect and mibg myocardial scintigraphy may improve the sensitivity of the diagnosis of pd . 
 in particular , this approach is based on commercially available radiotracers and can therefore be used in clinical practice without short - lived pet tracers . 
 the sample size of the present study was small and the study was carried out in a single center . 
 the results should therefore be confirmed in a multicenter study with a large population of pathologically diagnosed patients . 
 ","objectivemolecular imaging of nigrostriatal dopamine transporters ( dat ) and sympathetic cardiac innervation with single - photon emission computed tomography ( spect ) are useful tools for differentiating idiopathic parkinson disease ( pd ) from other degenerative parkinsonian syndromes ( non - pd ) . nevertheless , these modalities are often insufficient for achieving a definite diagnosis . 
 the aims of this study were to evaluate the diagnostic accuracy of the combination of these tools.materials and methodsthe spect radiotracers [ 123i]-n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)-nortropane ( fp - cit ) and meta-[123i]-iodobenzylguanidine ( mibg ) were used to research presynaptic dopaminergic projections ( dat spect ) and myocardial adrenergic innervation ( mibg scintigraphy ) , respectively . 
 pd patients ( n=15 ; age : 61.513.6 years ) and non - pd patients ( n=19 ; age : 62.614.2 years ) who underwent both tests were enrolled in this study . 
 receiver - operating characteristic analyses were used to set the cutoff values of the specific binding ratio in dat spect and the heart to mediastinum ratio in delayed scan in mibg scintigraphy for differentiating pd from non - pd . 
 we calculated the sensitivity , specificity , and test accuracy of the individual methods and also the combination of these two modalities.resultswhen dat spect and mibg scintigraphy were used individually , they showed mild accuracy in differentiating pd from non - pd ( dat , 67.6% ; mibg , 67.6% ) . 
 the combination of the two approaches using cutoff values of less than 3.24 for the specific binding ratio and less than 2.745 for the delayed heart to mediastinum ratio enabled more accurate differentiation between pd and non - pd . 
 the accuracy of these indices in distinguishing pd from non - pd was 79.4%.conclusionthese results suggested that the combination of dat spect and mibg scintigraphy may improve the diagnostic accuracy in differentiating pd from non - pd .",pubmed
"the ticks that belong to the genus amblyomma are large , ornate , longirostrate blood - feeding ectoparasites of a wide variety of vertebrates . 
 amblyomma testudinarium is one of the species that has been frequently reported to bite humans in asian countries . 
 usually , only a few ticks attached to and fed on a human , but 3 reported cases were infested with more than 100 larval ticks . in malaysia , a patient repeatedly infested with a. testudinarium was reported . 
 it is well known that a. testudinarium is distributed in korea . however , human infestation of a. testudinarium tick is rare , with 2 previously reported cases in elderly korean persons . 
 we report here an additional case of perianal infestation with a. testudinarium and provide histopathologic observations of its attachment to the skin . 
 a 73-year - old korean man with a round nodule in the left perianal area presented to our clinic in september 2010 . 
 the solitary nodule had been found about 2 weeks before admission and was not accompanied by hemorrhage and pain . 
 he recalled a trip to a water reservoir located at yeonggwang - gun ( between 35 to 36 north latitude ) , a rural county in jeollanam - do province , south korea 1 month before the presentation . at that time 
 , he defecated in the sitting posture on the grass near the water reservoir and felt the pain of sting around anus . 
 gross appearance of the tick bite site was characterized by a mild swelling and erythema . 
 the patient agreed to surgical treatment of the skin lesion to remove the perianal tick . 
 1a ) , placed in 10% buffered formalin and submitted for evaluation . during histologic processing , the tick was accidentally separated from the biopsied skin tissue . 
 it was 25 mm in length and 18 mm in width and engorged with blood meal ( fig . 
 the pedipalps had a long article ii with approximately 2.5 times as long as the article iii . 
 the denticles of the inner file were much smaller than those of the other files . 
 the ornamented scutum was relatively small , and its punctuations were dark brown or brownish - black spots on a pale yellow background ( fig . 
 the eyes were located on the lateral edges of the scutum but not projecting ( fig . 
 2c ) and located on the ventrolateral surface posterolaterally to the coxa iv ( fig . 
 the genital aperture was located at the level between the coxa i and coxa ii ( fig . 
 the anus was located at the level posterior to the spiracular plates and embraced by y - shaped anal groove posteriorly ( fig . 
 the coxa i had 2 subequal spurs , and the external spur was slightly longer than the internal spur ( fig . 
 the coxae ii and iii had a single salient ridge - like spur ( fig . 
 the coxa iv had a single spur which was longer than those of the coxae ii and iii ( fig . 
 microscopical examinations revealed an intense skin reaction at the site where the tick had attached . 
 due to deep penetration , the pathological changes were observed through all skin layers from the epidermis to the subcutaneous fat layer . in a low power field , 
 the lesion comprised of central amorphous eosinophilic material and surrounding inflammatory cell infiltrations ( fig . 
 4 ) . at the site of the tick bite , a small epidermal defect and invaginated squamous epithelium were seen . 
 the epidermal defect was about 1.8 mm in width and adjacent epidermis revealed parakeratosis , hyperkeratosis , and mild spongiosis . 
 scattered inflammatory cells , mainly neutrophils , were seen in the epidermis . in the dermis , a characteristic feeding lesion was formed immediately beneath the attachment site . 
 it was composed of 2 different portions ; superficial perirostral portion and deep cavitary portion ( fig . 
 the perirostral portion was 2.1 mm in depth and much wider at the distal part than at its entrance . 
 the cement , however , was found slightly at the dermal entrance of the tick mouthparts but not on the surface of the epidermis . 
 it was lamellate and had projections into the adjacent tissue in some areas ( fig . 
 it was relatively small and filled with purulent exudates containing inflammatory cells and erythrocytes ( fig . 
 a dense and mixed cellular infiltrate was also noted around the feeding lesion and extended into neighboring dermis and subcutaneous tissues . 
 however , there were very few inflammatory cells immediately adjacent to the site which abutted the eosinophilic cement ( fig . 5 ) . 
 there was no evidence of infection or residual disease up to 1 month after surgical removal 
 of the tick . 
 among the hard ticks , only 3 genera , ixodes , haemaphysalis , and amblyomma have been recorded from humans in the republic of korea . 
 this means that it does not belong to the genus ixodes which is the most common cause of human tick - bites in korea . 
 the pedipalps of the present specimen are slender and longer than the basis capituli , and their second segment is not salient laterally . 
 the eyes and fastoons are present , the small scutum is ornate , and the spiracular plate is comma - shaped 
 the denticles of the inner file are much smaller than those of the other files . 
 the spur of the coxa iv is longer than those of coxae ii and iii . 
 the tarsi iii and iv have 2 distinct subapical ventral spurs . from the above observations , this tick is easily distinguished from amblyomma nitidum and amblyomma geoemydae which are distributed in the far east asia . 
 therefore , our tick has been identified as a female adult of a. testudinarium based on morphological characteristics . 
 the warm temperature and subtropical vegetation fauna are known to be a proper environmental condition for a. testudinarium . 
 thus , this tick is more common in the tropic areas , including south east asia and indian peninsula . in japan , 
 the closest geographical neighbor to korea , a. testudinarium is one of the most common ticks infesting humans and especially important in southwestern areas . 
 in korea , it is also distributed in jeju island and the southern coastal area of the mainland . 
 the first human case was reported in suncheon city ( 35 north latitude ) , a southern coastal area of jeollanam - do . 
 the second case occurred in tongyeong city ( below 35 north latitude ) , southern coastal area of gyeongsangnam - do . in the present case , 
 the patient was infested in yeonggwang - gun , a western coastal area of jeollanam - do . 
 therefore , a. testudinarium extends the geographic range of distribution from the southern coastal area more northwards to the southwestern costal area ( below 36 north latitude ) of the korean peninsula . 
 after a site for feeding has been chosen , ticks embed their anterior part calling capitulum into the skin . the capitulum is composed of 4 different components ; the palps , chelicerae , hypostome , and basis capituli . 
 ticks attached on the skin lacerate the epidermal layers using the chelicerae which are paired appendages for cutting , ripping , and tearing skin . 
 after epidermal laceration , ticks insert midline mouthparts ( the chelicerae and hypostome ) into the opening at a depth that varies by species . in the present case , 
 the depth of penetration is similar to the length of the hypostome which has been deeply inserted into the dermis . 
 however , the width of epidermal laceration is not similar to that of mouthparts but similar to that of the basis capituli . 
 this means that the basis capituli is embedded shallowly in the host epidermis during the feeding period . 
 the histopathology of skin responses to infection with the present female a. testudinarium shows that the dermis is infiltrated with various inflammatory cells associated with both acute and chronic reactions , i.e. , lymphocytes , neutrophils , histiocytes , and eosinophils . 
 these inflammatory findings are similar to those reported by other researchers on amblyomma species experimentally and naturally infested in various vertebrate hosts . around the lesion abutting the cement , we observed an interesting area having relatively few inflammatory cells . 
 this cell - free area is called the paucicellular zone . the biologic factors causing a zone of minimal cellular inflammation 
 are not exactly known but are likely due to the anti - inflammatory effects of the tick saliva . according to the study on the effects of tick saliva and salivary gland extracts , this paucicellular zone might reflect tick - induced suppression of cellular migration into the injury site as a result of saliva constituents suppressing the movement of cells into the feeding lesion . 
 the salivary glands of hard ticks secret eosinophilic cement material that help attachment of the tick to the host for the feeding period . 
 it is well known that there is considerable difference in the distribution of the cement within the host tissue . 
 generally , ixodids with long mouthparts ( longirostrate ticks ) penetrate the skin deeply to the dermis and have a perirostal secretion of the cement . 
 ixodids with short mouthparts ( brevirostrate ticks ) have a copious superficial secretion of the cement which spreads over the skin . 
 amblyomma and hyalomma are longirostrate ticks with relative long mouthparts . in the present amblyomma case , 
 eosinophlic cement was found mostly in the perirostral portion but little on the skin surface .","a perianal tick and the surrounding skin were surgically excised from a 73-year - old man residing in a southwestern costal area of the korean peninsula . 
 microscopically a deep penetrating lesion was formed beneath the attachment site . 
 dense and mixed inflammatory cell infiltrations occurred in the dermis and subcutaneous tissues around the feeding lesion . 
 amorphous eosinophilic cement was abundant in the center of the lesion . 
 the tick had y - shaped anal groove , long mouthparts , ornate scutum , comma - shaped spiracular plate , distinct eyes , and fastoons . 
 it was morphologically identified as a fully engorged female amblyomma testudinarium . 
 this is the third human case of amblyomma tick infection in korea .",pubmed
"hypertrophic cardiomyopathy ( hcm ) is characterized by left and/or right ventricular hypertrophy , with predominant involvement of the interventricular septum in the absence of other causes of hypertrophy , such as hypertension , valvular heart disease , or metabolic disease [ 17 ] . 
 familial hcm is an autosomal dominant disorder caused by mutations in genes that encode sarcomere proteins . 
 it has been reported that at least 15 genes are implicated in 5570 % of hcm , and the major genes causing hcm include cardiac -myosin heavy chain ( myh7 ) , -tropomyosin , cardiac troponin t ( tnnt2 ) , cardiac myosin - binding protein c ( mybpc3 ) , cardiac troponin i ( tnni3 ) , cardiac myosin regulatory light chains ( myl2 ) , and cardiac myosin essential light chains ( myl3 ) [ 1 , 4 , 5 , 811 ] . 
 cardiac troponin t is a thin - filament protein that takes part in muscle contraction . 
 the troponin complex on the actin filament regulates the force and velocity of muscle contraction . 
 troponin c functions as a calcium receptor while troponin i inhibits adenosine triphosphatase ( atpase ) activity when bound to actin . 
 troponin t fixes the troponin group to tropomyosin . during relaxation , the troponin group is bound to actin and tropomyosin , blocking the interaction of myosin and actin . 
 mutations of tnnt2 were thought to account for approximately 15 % of familial hcm , with most missense mutations located in exons 816 , and were associated with a particularly severe form of the disease characterized by a poor overall prognosis with a high incidence of sudden death despite only mild left ventricular hypertrophy ( lvh ) [ 14 ] . 
 most tnnt2 mutations of familial hcm alter the contractile properties of cardiac muscle , especially the ca sensitivity of force development and atpase activity in vitro and in vivo [ 1317 ] . 
 previous reports have suggested that there is a more consistent relationship between certain genetic mutations and clinical outcome , allowing for the classification of  benign  and  malignant  mutations [ 1830 ] . 
 for example , a favorable prognosis has been reported in patients with a phe110ile mutation of the tnnt2 mutation ( in 16 individuals of 6 japanese families ) [ 19 , 20 ] . from these data , genetic analysis and determination of genotype 
 however , these findings were based on limited experience ; to date , only 30 different mutations have been identified in tnnt2 [ 911 , 18 ] . 
 in addition to this genetic diversity , the phenotypic expression of these mutations varies considerably , ranging from asymptomatic individuals with a normal life expectancy to those with sudden cardiac death or the need for an early heart transplant . 
 clinical parameters such as the degree of lvh , the presence or absence of a left ventricular outflow tract gradient , and electrophysiology testing have not been predictive markers of poor prognosis [ 15 ] . 
 more recently , it was reported that late gadolinium enhancement with cardiac magnetic resonance can be a predictive marker of the ventricular arrhythmia and poor prognosis in hcm . in recent years 
 , mutation - specific risk stratification was considered to be not possible , but genetic test - based risk stratification seemed to be clinically informative . 
 the tnnt2 mutation has the third - ranked incidence of familial hcm , and the genotype  phenotype correlation of this gene still remains insufficient in japanese familial hcm . 
 we genetically evaluated 173 patients ( 101 men and 72 women ; 079 years old , median age 20 years ) who were clinically diagnosed with familial hcm . 
 written informed consent was obtained from all study subjects in accordance with the declaration of helsinki . 
 if patients were younger than 16 years , informed consent was given by their guardians . 
 we assessed each patient by taking their history and performing a physical examination , and reviewed their medical records . 
 all assessments were done with the approval of the ethics committees of tokyo women s medical university . 
 the diagnosis of hcm was determined through clinical evaluation , chest radiography , electrocardiography , echocardiography , and cardiac catheterization based on current international consensus criteria . 
 diseases of the heart , hypertension , valvular heart disease , or metabolic disease attributable to hcm were excluded from this study by pediatric cardiologists . 
 genomic dna was prepared from peripheral blood lymphocytes or lymphoblast cell lines transformed by the epstein 
 tnnt2 coding regions and exon  intron boundaries , including regions approximately 30100 bp upstream and downstream from the exons , were amplified from genomic dna using primers , as described in previous reports [ 34 , 35 ] . 
 genomic dna ( 50 ng ) was amplified through the use of primers designed from flanking intron sequences ( table 1).table 1primers used for amplification of fragments from tnnt2 
 exonforward ( 53)reverse ( 53)2gagctcttctgaggaaggcactacccagaatccgagggac3 and 4cagggcagcgtggactccccccagggctcccaggattt5ccattctctgctctgggttcgtgcacatgggaaagctgttct6tagggcttatctgtggggaaggccttccctggaaagagcactg7gaaatggaaatccacagggatactgcaccccgttccatca8 and 9ctctaggaaggatcagggcccctcacaaaagggatggagga10gcgatgtcaccttctccctacaccgcacccggccaata11ggtttccaatcctttcccctaagctgcagtggacacctcattc12gcctttgtcttcctgccttctccagccagcccaatctcttcact13acagggagggggcaatctggcccccagagcagatgcgggcagtg14actgggtgctgccgtctggtcaagggggctgttggggaatagg15cactcagcccccttctccaagcttctccgccccacatttc16ggcaccccagtcctacccgtccccctcaacagcactttt primers used for amplification of fragments from tnnt2 
  amplified products were purified using a multiscreen polymerase chain reaction ( pcr ) plate ( millipore , billerica , ma , usa ) and directly sequenced using the abi - prism bigdye - terminator cycle sequencing reaction kit and abi 3130xl genetic analyzer ( applied biosystems , foster city , ca , usa ) . 
 when a mutation was detected , we confirmed that it was not presenting 363 japanese normal chromosomes by direct sequencing . 
 the pcr conditions of these genes were modified and fragments were analyzed by electrophoresis , as described previously [ 36 , 37 ] . 
 proband patients bearing tnnt2 mutations were additionally examined for reported hcm mutations , the coding regions of myh7 , mybpc3 , tnni3 , -tropomyosin , mly2 , mly3 , and all exons , and a promoter region of the -galactosidase a gene . 
 primer sequences and detailed pcr conditions for these additional analyses are available upon request . reference sequences and single - nucleotide polymorphism information were obtained from the national center for biotechnology information . 
 we genetically evaluated 173 patients ( 101 men and 72 women ; 079 years old , median age 20 years ) who were clinically diagnosed with familial hcm . 
 written informed consent was obtained from all study subjects in accordance with the declaration of helsinki . 
 if patients were younger than 16 years , informed consent was given by their guardians . 
 we assessed each patient by taking their history and performing a physical examination , and reviewed their medical records . 
 all assessments were done with the approval of the ethics committees of tokyo women s medical university . 
 the diagnosis of hcm was determined through clinical evaluation , chest radiography , electrocardiography , echocardiography , and cardiac catheterization based on current international consensus criteria . 
 diseases of the heart , hypertension , valvular heart disease , or metabolic disease attributable to hcm were excluded from this study by pediatric cardiologists . 
 genomic dna was prepared from peripheral blood lymphocytes or lymphoblast cell lines transformed by the epstein 
 tnnt2 coding regions and exon  intron boundaries , including regions approximately 30100 bp upstream and downstream from the exons , were amplified from genomic dna using primers , as described in previous reports [ 34 , 35 ] . 
 genomic dna ( 50 ng ) was amplified through the use of primers designed from flanking intron sequences ( table 1).table 1primers used for amplification of fragments from tnnt2 
 exonforward ( 53)reverse ( 53)2gagctcttctgaggaaggcactacccagaatccgagggac3 and 4cagggcagcgtggactccccccagggctcccaggattt5ccattctctgctctgggttcgtgcacatgggaaagctgttct6tagggcttatctgtggggaaggccttccctggaaagagcactg7gaaatggaaatccacagggatactgcaccccgttccatca8 and 9ctctaggaaggatcagggcccctcacaaaagggatggagga10gcgatgtcaccttctccctacaccgcacccggccaata11ggtttccaatcctttcccctaagctgcagtggacacctcattc12gcctttgtcttcctgccttctccagccagcccaatctcttcact13acagggagggggcaatctggcccccagagcagatgcgggcagtg14actgggtgctgccgtctggtcaagggggctgttggggaatagg15cactcagcccccttctccaagcttctccgccccacatttc16ggcaccccagtcctacccgtccccctcaacagcactttt primers used for amplification of fragments from tnnt2 
  amplified products were purified using a multiscreen polymerase chain reaction ( pcr ) plate ( millipore , billerica , ma , usa ) and directly sequenced using the abi - prism bigdye - terminator cycle sequencing reaction kit and abi 3130xl genetic analyzer ( applied biosystems , foster city , ca , usa ) . 
 when a mutation was detected , we confirmed that it was not presenting 363 japanese normal chromosomes by direct sequencing . 
 the pcr conditions of these genes were modified and fragments were analyzed by electrophoresis , as described previously [ 36 , 37 ] . 
 proband patients bearing tnnt2 mutations were additionally examined for reported hcm mutations , the coding regions of myh7 , mybpc3 , tnni3 , -tropomyosin , mly2 , mly3 , and all exons , and a promoter region of the -galactosidase a gene . 
 reference sequences and single - nucleotide polymorphism information were obtained from the national center for biotechnology information . 
 to investigate mutations of tnnt2 , we performed dna analysis in 173 unrelated japanese patients with familial hcm . 
 we identified three reported mutations and a new mutation of tnnt2 in 11 individuals from four families ( a , b , c , d ) ( table 2).table 2clinical features of patients with tnnt2 mutation in families a , b , c , and dhcm individualsa iii-2a iii-1a ii-6a ii-6 ( iii-2/mother)b iii-7b iii-2b iv-3c iv-3c iii-3c ii-3d iii-2exon9 and 109 and 109109991010108nucleotide substitutiont328a and c388tt328a and c388tt328ac388tc274c274c274c388c388c388t236camino substitutionp110i and r130cp110i and r130cp110ir130cr92wr92wr92wr92wr92wr92wi79tsexfmmffmmffffage ( years)121455463541242158025outcome ( years)12 , vf survivor14 , died suddenly24 , vf survivorclinical diagnosishcmhcmcavb , pminormaldhcm ( hcm)abnormal ecghcmhcmhcmhcmhcmelectrocardiogramsr + crbbbsr + crbbb , abnqpm rhythm ( cavb)wnlr - wave progressiondetails unknownrvh , deep qlvh , st depdetails unknowndetails unknownst dep , inv t2d echocardiogramlvh , ashlvh , ashlvhwnllvh , apical hypertrophydetails unknownlvhlvh , ashlvhdetails unknownlvh , ashcardiac catheterizationlv relaxation abnormalitieslv relaxation abnormalitieslv relaxation abnormalitiesndndndndndndndnd 
 hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , dhcm dilated phase of hcm , lvh left ventricular hypertrophy , vf ventricular fibrillation , cavb complete atrioventricular block , sr sinus rhythm , crbbb complete right bundle branch block , abnq abnormal q wave , lvh left ventricular hypertrophy , rvh right ventricular hypertrophy , st dep st depression , inv t inversion t , pmi pacemaker implantation , ecg electrocardiogram , ash asymmetric septal hypertrophy , aph apical hypertrophy , nd not determined , wnl within normal limits clinical features of patients with tnnt2 mutation in families a , b , c , and d 
 hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , dhcm dilated phase of hcm , lvh left ventricular hypertrophy , vf ventricular fibrillation , cavb complete atrioventricular block , sr sinus rhythm , crbbb complete right bundle branch block , abnq abnormal q wave , lvh left ventricular hypertrophy , rvh right ventricular hypertrophy , st dep st depression , inv t inversion t , pmi pacemaker implantation , ecg electrocardiogram , ash asymmetric septal hypertrophy , aph apical hypertrophy , nd not determined , wnl within normal limits in family a , all five members who carried or were suspected of having hcm also had arrhythmia . 
 four of them had blocks , including a complete atrioventricular block ( cavb ) and complete right bundle branch block ( crbbb ) ( fig . 1 ; ii-1 , 6 , iii-1 , 2 ) , and one had bradycardia . 
 two of them died suddenly ( fig . 1 ; i-2 , iii-1 ) , and one was a ventricular fibrillation ( vf ) survivor ( fig . 
 1 ; iii-2 ) . the proband ( fig . 1 ; iii-2 ) , aged 12 years , with sinus rhythm ( sr ) and a crbbb , had an episode of vf , and her elder brother ( fig . 1 ; iii-1 ) , 
 the proband and her brother and father ( fig . 1 ; iii-2 , iii-1 , i-2 ) showed mild lvh ( maximum wall thickness < 20 mm ) . 
 1pedigree and phenotype of family a. pedigree with three generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , cavb complete atrioventricular block , pmi pacemaker implantation , crbbb complete right bundle - branch block , vf ventricular fibrillation pedigree and phenotype of family a. pedigree with three generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , cavb complete atrioventricular block , pmi pacemaker implantation , crbbb complete right bundle - branch block , vf ventricular fibrillation dna analysis showed that the proband and her elder brother had the double reported mutations , arg130cys and phe110ile , of tnnt2 ( fig . 
 her father , who had arrhythmia and mild hcm , had the phe110ile mutation , and her mother ( ii-6 ) , who had no symptoms or abnormal findings , had the arg130cys mutation . in family b 
 , 3 of 10 members who carried or were suspected of having hcm were thought to have obstructive hypertrophic cardiomyopathy . 
 2 ; iii-7 ) showed apical hypertrophy and apical aneurysm at a comparatively young age . 
 her aunt ( fig . 2 ; ii-4 ) , who had a heart murmur , was thought to have hypertrophic obstructive cardiomyopathy ( hocm ) , and died when she was 19 years old . 
 2 ; iii-9 ) did not show obstructive hcm , but died suddenly when she was 21 years old . her grandmother ( fig . 
 iii-7 ) , her son ( fig . 2 ; iv-3 ) , and her cousin ( fig . 2 ; iii-2 ) , who carried hcm , had an arg92trp mutation of tnnt2.fig . 2pedigree and phenotype of family b. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , ecg electrocardiogram pedigree and phenotype of family b. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , ecg electrocardiogram in family c , the proband ( fig . 3 ; iv-3 ) , her mother ( fig . 3 
 ; iii-3 ) , and her grandmother ( fig . 3 ; ii-3 ) showed mild cardiac hypertrophy ( maximum wall thickness < 20 mm ) and asymmetric septal hypertrophy , and her myocardial hypertrophy had been gradually increasing . 
 her mother was suspected of having a shifting dilated phase by recent cardiac magnetic resonance imaging ( mri ) . 
 3 ; iv-3 ) had symptomatic west syndrome following perinatal brain damage , and was treated with corticotropin . 
 3 ; iv-3 ) and her mother ( fig . 3 ; iii-3 ) had the arg92trp mutation of tnnt2.fig . 
 3pedigree and phenotype of family c. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , wpw syndrome wolff - parkinson - white syndrome pedigree and phenotype of family c. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , wpw syndrome wolff - parkinson - white syndrome in family d , two of three members who carried or were suspected of having hcm died suddenly ( fig . 4 ; 
 4 ; iii-2 ) . the proband ( fig . 4 ; iii-2 ) had relatively strong cardiac hypertrophy ( maximum wall thickness > 20 mm ) , and cardiac standstill from ventricular fibrillation at the age of 24 years . 
 her cousin ( fig . 4 ; iii-3 ) died suddenly at a young age ( 13 years ) , and her granduncle also died at the age of 40 . 
 this transition was observed in codon 79 , and converted an isoleucine residue to a threonine residue ( fig . 
 ile79thr occurred in conserved residues found in tnnt2 orthologues of human , mouse , rat , cat , and ox ( fig . 
 iii-2 ) also had a 5-bp ( cttct ) deletion / deletion polymorphism of intron 3 of tnnt2 ( fig . 
 4pedigree and phenotype of family d. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 c the insertion / deletion ( i / d ) polymorphism pedigree and phenotype of family d. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , vf ventricular fibrillation 
 a the new ile79thr mutation of tnnt2 ( exon 8) . 
 this domain is well preserved in mammals 
 a the deletion / deletion ( d / d ) polymorphism . 
 c the insertion / deletion ( i / d ) polymorphism to clarify the clinical importance of this polymorphism , we performed genetic analysis in 47 hcm patients from 173 unrelated japanese patients with familial hcm . in these 47 patients , 
 24 had the deletion / deletion polymorphism ( 51 % , 14 men and 10 women ; 072 years old , median age 30 years old ) . 
 five of the 24 ( 21 % ) patients who had the deletion / deletion polymorphism were diagnosed with apical hcm or hocm . in 6 of the 10 ( 60 % ) with available data of echocardiography among these 24 patients , 
 on the other hand , in the 47 hcm patients , the remaining 23 did not have the deletion / deletion polymorphism , but had the deletion / insertion or insertion / insertion polymorphism ( 49 % , 15 men and 8 women ; 076 years old , median age 18 years ) . 
 two of the 23 ( 9 % ) patients were diagnosed with apical hcm , and there were no hocm patients . 
 two of the seven ( 29 % ) with available data of echocardiography among these 23 patients showed the maximum wall thickness of more than 30 mm . 
 to investigate mutations of tnnt2 , we performed dna analysis in 173 unrelated japanese patients with familial hcm . 
 we identified three reported mutations and a new mutation of tnnt2 in 11 individuals from four families ( a , b , c , d ) ( table 2).table 2clinical features of patients with tnnt2 mutation in families a , b , c , and dhcm individualsa iii-2a iii-1a ii-6a ii-6 ( iii-2/mother)b iii-7b iii-2b iv-3c iv-3c iii-3c ii-3d iii-2exon9 and 109 and 109109991010108nucleotide substitutiont328a and c388tt328a and c388tt328ac388tc274c274c274c388c388c388t236camino substitutionp110i and r130cp110i and r130cp110ir130cr92wr92wr92wr92wr92wr92wi79tsexfmmffmmffffage ( years)121455463541242158025outcome ( years)12 , vf survivor14 , died suddenly24 , vf survivorclinical diagnosishcmhcmcavb , pminormaldhcm ( hcm)abnormal ecghcmhcmhcmhcmhcmelectrocardiogramsr + crbbbsr + crbbb , abnqpm rhythm ( cavb)wnlr - wave progressiondetails unknownrvh , deep qlvh , st depdetails unknowndetails unknownst dep , inv t2d echocardiogramlvh , ashlvh , ashlvhwnllvh , apical hypertrophydetails unknownlvhlvh , ashlvhdetails unknownlvh , ashcardiac catheterizationlv relaxation abnormalitieslv relaxation abnormalitieslv relaxation abnormalitiesndndndndndndndnd 
 hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , dhcm dilated phase of hcm , lvh left ventricular hypertrophy , vf ventricular fibrillation , cavb complete atrioventricular block , sr sinus rhythm , crbbb complete right bundle branch block , abnq abnormal q wave , lvh left ventricular hypertrophy , rvh right ventricular hypertrophy , st dep st depression , inv t inversion t , pmi pacemaker implantation , ecg electrocardiogram , ash asymmetric septal hypertrophy , aph apical hypertrophy , nd not determined , wnl within normal limits clinical features of patients with tnnt2 mutation in families a , b , c , and d 
 hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , dhcm dilated phase of hcm , lvh left ventricular hypertrophy , vf ventricular fibrillation , cavb complete atrioventricular block , sr sinus rhythm , crbbb complete right bundle branch block , abnq abnormal q wave , lvh left ventricular hypertrophy , rvh right ventricular hypertrophy , st dep st depression , inv t inversion t , pmi pacemaker implantation , ecg electrocardiogram , ash asymmetric septal hypertrophy , aph apical hypertrophy , nd not determined , wnl within normal limits in family a , all five members who carried or were suspected of having hcm also had arrhythmia . 
 four of them had blocks , including a complete atrioventricular block ( cavb ) and complete right bundle branch block ( crbbb ) ( fig . 1 ; ii-1 , 6 , iii-1 , 2 ) , and one had bradycardia . 
 two of them died suddenly ( fig . 1 ; i-2 , iii-1 ) , and one was a ventricular fibrillation ( vf ) survivor ( fig . 
 1 ; iii-2 ) . the proband ( fig . 1 ; iii-2 ) , aged 12 years , with sinus rhythm ( sr ) and a crbbb , had an episode of vf , and her elder brother ( fig . 1 ; iii-1 ) , 
 the proband and her brother and father ( fig . 1 ; iii-2 , iii-1 , i-2 ) showed mild lvh ( maximum wall thickness < 20 mm ) . 
 1pedigree and phenotype of family a. pedigree with three generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , cavb complete atrioventricular block , pmi pacemaker implantation , crbbb complete right bundle - branch block , vf ventricular fibrillation pedigree and phenotype of family a. pedigree with three generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , cavb complete atrioventricular block , pmi pacemaker implantation , crbbb complete right bundle - branch block , vf ventricular fibrillation dna analysis showed that the proband and her elder brother had the double reported mutations , arg130cys and phe110ile , of tnnt2 ( fig . 
 her father , who had arrhythmia and mild hcm , had the phe110ile mutation , and her mother ( ii-6 ) , who had no symptoms or abnormal findings , had the arg130cys mutation . in family b 
 , 3 of 10 members who carried or were suspected of having hcm were thought to have obstructive hypertrophic cardiomyopathy . 
 2 ; iii-7 ) showed apical hypertrophy and apical aneurysm at a comparatively young age . 
 her aunt ( fig . 2 ; ii-4 ) , who had a heart murmur , was thought to have hypertrophic obstructive cardiomyopathy ( hocm ) , and died when she was 19 years old . 
 2 ; iii-9 ) did not show obstructive hcm , but died suddenly when she was 21 years old . her grandmother ( fig . 
 iii-7 ) , her son ( fig . 2 ; iv-3 ) , and her cousin ( fig . 2 ; iii-2 ) , who carried hcm , had an arg92trp mutation of tnnt2.fig . 2pedigree and phenotype of family b. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , ecg electrocardiogram pedigree and phenotype of family b. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , ecg electrocardiogram in family c , the proband ( fig . 3 ; iv-3 ) , her mother ( fig . 3 
 ; iii-3 ) , and her grandmother ( fig . 3 ; ii-3 ) showed mild cardiac hypertrophy ( maximum wall thickness < 20 mm ) and asymmetric septal hypertrophy , and her myocardial hypertrophy had been gradually increasing . 
 her mother was suspected of having a shifting dilated phase by recent cardiac magnetic resonance imaging ( mri ) . 
 3 ; iv-3 ) had symptomatic west syndrome following perinatal brain damage , and was treated with corticotropin . 
 3 ; iv-3 ) and her mother ( fig . 3 ; iii-3 ) had the arg92trp mutation of tnnt2.fig . 
 3pedigree and phenotype of family c. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , wpw syndrome wolff - parkinson - white syndrome pedigree and phenotype of family c. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , wpw syndrome wolff - parkinson - white syndrome in family d , two of three members who carried or were suspected of having hcm died suddenly ( fig . 4 ; 
 4 ; iii-2 ) . the proband ( fig . 4 ; iii-2 ) had relatively strong cardiac hypertrophy ( maximum wall thickness > 20 mm ) , and cardiac standstill from ventricular fibrillation at the age of 24 years . 
 her cousin ( fig . 4 ; iii-3 ) died suddenly at a young age ( 13 years ) , and her granduncle also died at the age of 40 . 
 this transition was observed in codon 79 , and converted an isoleucine residue to a threonine residue ( fig . 
 ile79thr occurred in conserved residues found in tnnt2 orthologues of human , mouse , rat , cat , and ox ( fig . 
 iii-2 ) also had a 5-bp ( cttct ) deletion / deletion polymorphism of intron 3 of tnnt2 ( fig . 
 4pedigree and phenotype of family d. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 c the insertion / deletion ( i / d ) polymorphism pedigree and phenotype of family d. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . 
 hcm hypertrophic cardiomyopathy , vf ventricular fibrillation 
 a the new ile79thr mutation of tnnt2 ( exon 8) . 
 this domain is well preserved in mammals 
 a the deletion / deletion ( d / d ) polymorphism . 
 c the insertion / deletion ( i / d ) polymorphism to clarify the clinical importance of this polymorphism , we performed genetic analysis in 47 hcm patients from 173 unrelated japanese patients with familial hcm . in these 47 patients , 
 24 had the deletion / deletion polymorphism ( 51 % , 14 men and 10 women ; 072 years old , median age 30 years old ) . 
 five of the 24 ( 21 % ) patients who had the deletion / deletion polymorphism were diagnosed with apical hcm or hocm . in 6 of the 10 ( 60 % ) with available data of echocardiography among these 24 patients , 
 on the other hand , in the 47 hcm patients , the remaining 23 did not have the deletion / deletion polymorphism , but had the deletion / insertion or insertion / insertion polymorphism ( 49 % , 15 men and 8 women ; 076 years old , median age 18 years ) . 
 two of the 23 ( 9 % ) patients were diagnosed with apical hcm , and there were no hocm patients . 
 two of the seven ( 29 % ) with available data of echocardiography among these 23 patients showed the maximum wall thickness of more than 30 mm . 
 previous studies have reported familial hcm cases where a consistent relationship between certain genetic mutations and clinical outcome was observed , allowing for the classification of  benign  and  malignant  mutations [ 1830 ] . however , in recent years it was considered that the specific gene mutation can not be the sole factor that dictates clinical phenotype . 
 mutation , mutation type was not seen to be clinically useful in predicting prognosis in hcm given this very low incidence rate [ 32 , 3840 ] . 
 in addition , it was reported that mutation of a sarcomeric protein gene can cause rcm , hcm , and dcm within the same family , and patients with a benign mutation experienced a very serious clinical course [ 41 , 42 ] . in the present study , 
 the phenotype of each family was often different from reported cases , even if they had the same genetic mutation . 
 in addition , families with the same genetic mutation showed a similar trend in the phenotype , but it was not exactly the same . 
 however , sudden death in youth was observed in all of these families . on the other hand 
 mutation , which did not show the phenotype of hcm or a family history of sudden death . 
 for example , in family a , two tnnt2 mutations were found , the arg130 cys and phe110ile mutations . 
 the arg130 cys mutation has been reported by a chinese research group , whose clinical features had an early onset , syncope , sudden death in youth , heart failure , and arrhythmia as atrial fibrillation . 
 the phe110ile mutation has been reported to show a favorable prognosis , and have comparatively slight hypertrophy or apical hypertrophy ( in 16 individuals of 6 japanese families ) [ 19 , 20 ] . 
 a , whereas the proband and her elder brother , who carried these two mutations , showed similar reported phenotypes of the arg130 cys mutation , her mother and mother s family members , who had the arg130cys mutation , showed no abnormal clinical features such as cardiac hypertrophy , sudden death , or abnormal electrocardiograms . 
 furthermore , her father and father s family members , who had the phe110ile mutation , showed atrioventricular blocks , and these phenotypes have not been reported . in family b , 
 the mutation showed comparatively slight cardiac hypertrophy or a high incidence of sudden death in males ( 19 individuals of two mixed racial families ) [ 2224 ] . 
 however , the proband and her family , who carried hcm , showed relatively severe cardiac hypertrophy , with a high incidence of sudden death in females . in family c , an arg92trp mutation was also found . 
 the proband and her family , who carried hcm , showed mild cardiac hypertrophy and asymmetric septal hypertrophy . 
 the proband s myocardial hypertrophy had been gradually increasing , and her mother had a suspected shift to the dilated phase . 
 she survived following an episode of vf , and her cousin died suddenly at the age of 13 years . 
 previously , a mutation of the 79 residue , the ile79asn mutation , had been reported [ 24 , 25 ] , and this mutation also showed a poor prognosis . in our mutated case , even if the amino acid mutation ( ile79thr ) was different from the reported case ( ile79asn ) , the patient showed a malignant prognosis . in family d , a 5-base - pair ( cttct ) deletion / deletion ( d / d ) polymorphism in intron 3 of the tnnt2 was also found . 
 it has been reported that this polymorphism had caused skipping of exon 4 of tnnt2 , and that the deletion allele could be associated with a predisposition for prominent lvh . although it was in a very limited range , from our genetic study on this polymorphism we gained the impression that a patient carrying the tnnt2 deletion / deletion polymorphism had a stronger tendency toward hypertrophy . to conduct further analysis , further examination including new cases is required . 
 we considered that , at least in japanese familial hcm , only the type of genetic mutation of tnnt2 did not seem useful in distinguishing the prognosis . 
 however , if mutations were found , there was a risk of sudden death in youth . 
 therefore , regardless of the type of genetic mutation , it would be more important to observe the patient in detail from birth in each lineage . 
 the development of a  case - based  method would be useful to treat and help each individual , and more attention needs to be paid toward searching for the modifier and environmental factors including diet , lifestyle , exercise , and the modification gene and polymorphism .","hypertrophic cardiomyopathy ( hcm ) is an autosomal dominant disorder resulting from mutations in genes for at least 15 various sarcomere - related proteins including cardiac -myosin heavy chain , cardiac myosin - binding protein c , and cardiac troponin t. the troponin t gene ( tnnt2 ) mutation has the third incidence of familial hcm , and the genotype  phenotype correlation of this gene still remains insufficient in japanese familial hcm . 
 therefore , in the present study , we focused on screening the tnnt2 mutation in 173 unrelated japanese patients with familial hcm , and found three reported mutations and a new mutation of tnnt2 in 11 individuals from four families . in these families , two individuals from one family had double mutations , arg130cys and phe110ile , six individuals from two other families had an arg92trp mutation , and one individual of another family had a new mutation , ile79thr , of tnnt2 . 
 the phenotype of each family was often different from reported cases , even if they had the same genetic mutation . 
 in addition , families with the same genetic mutation showed a similar trend in the phenotype , but it was not exactly the same . 
 however , sudden death in youth was observed in all of these families . 
 although the type of genetic mutation is not useful for predicting prognosis in hcm , the possibility of sudden cardiac death remains . 
 therefore , the prognosis of individuals bearing the tnnt2 mutation with familial hcm should be more carefully observed from birth .",pubmed
"schistosomatidae ( platyhelminthes : digenea ) includes several digenetic endoparasites with complex life cycles , whose developmental stages alternate between intermediate ( freshwater gastropods ) and definitive hosts ( birds , reptiles , fishes , and mammals ) . 
 another important feature is the increased longevity ( over 5 years ) of the schistosoma species in the human host . 
 schistosoma , the avian and mammalian blood flukes , is the best studied genus of schistosomatidae . 
 several species are described , six of which infect humans causing schistosomiasis : s. haematobium , s. intercalatum , s. japonicum , s. malayensis , s. mansoni , and s. mekongi . 
 other species are known to infect a broad range of mammals such as hippopotamus , rodents , carnivores , and ruminant animals like buffalo , cattle , goat , lechwe , and sheep . 
 for example , s. mattheei , more commonly found in cattle , is believed to form hybrids between s. mattheei and s. haematobium thus increasing its snail and definitive host range . 
 schistosomiasis , a chronic and debilitating disease , is considered by the world health organization as one of the most serious public health issues and the second most prevalent tropical disease with high morbidity in the world . 
 schistosomiasis is endemic in 77 countries   and its transmission is dependent on the existence and distribution of intermediate hosts . 
 it is estimated that 237 million people require treatment worldwide and that 600 to 779 million people live in endemic areas , under infection risk . yet , exacerbating this scenario , this disease is responsible for the loss of 1.7 to 4.5 millions of years of life in the world , measured by disability - adjusted life years ( daly ) , one of the highest indexes among all the neglected tropical diseases . 
 control of schistosomiasis represents a great challenge and it is based on drug treatment ( praziquantel ) , snail control , improved sanitation , and health education . 
 the development of powerful and scalable methods to analyse nucleic acids and proteins has changed the way biological data is surveyed . 
 the application of such technologies , together with the development of powerful computational tools and methods , have expanded our perspective of schistosome biology and allowed a better understanding of processes such as host - parasite interaction [ 810 ] . 
 this paper aims to discuss some advances in schistosome research with emphasis on genomics and transcriptomics . 
 first , we summarize the current status of sequencing projects of nuclear and mitochondrial genomes . 
 then , we present key findings in transcriptomic analyses . finally , we point out the main challenges of the current research and suggest some future directions in schistosoma genomic and transcriptomic studies . 
 in order to strengthen traditional methods and provide new information to improve success towards schistosomiasis control , the scientific community joined efforts to assess the schistosoma genomic information , starting in 1994 , as an initiative of who / tdr ( unicef - undp - world bank - who special programme for research and training in tropical diseases ) [ 11 , 12 ] . at that time , 
 only a few hundred expressed sequence tags ( ests ) from s. mansoni were available . 
 the who / tdr support opened the possibility to generate data that could be translated into new tools for schistosomiasis diagnostics and treatment , representing the  kickoff  of schistosoma nuclear genome studies . 
 the karyotype of known schistosoma species comprises seven pairs of autosomes and one pair of sex chromosomes ( female = zw , male = zz ) , ranging in size from 18 to 73 megabases ( mb ) [ 14 , 15 ] . 
 the s. japonicum ( anhui isolate ) and s. mansoni ( puerto rico isolate ) genomes were decoded and simultaneously published as an initiative of the wellcome trust sanger center institute in collaboration with the institute for genomic research ( tigr ) and the schistosoma japonicum genome sequencing and functional analysis consortium [ 16 , 17 ] . recently 
 , the s. haematobium ( egyptian isolate ) genome was sequenced opening new possibilities in comparative genomics of schistosomes . 
 s. haematobium , s. japonicum , and s. mansoni carry a nuclear genome of 385 , 397 , and 363 mb composed by approximately , 13,073 , 13,469 , and 10,852 protein - coding genes , respectively [ 1619 ] . 
 it is worth to mention that the number of predicted protein - coding genes does not reflect the genome structure per se , but the actual state of analyses of the different draft genome data . 
 the genetic linkage map of s. mansoni was obtained and allowed the refinement of the genome sequencing data providing a means for gene discovery and gene function analysis [ 20 , 21 ] . 
 more importantly , it has allowed the identification of genetic loci that determine important traits such as host specificity , virulence , and drug resistance . 
 in fact , although the quality and annotation of the s. mansoni genome sequence data have been greatly improved ( see below ) ; the corresponding data of s. haematobium and s. japonicum are still considered drafts [ 1619 ] . 
 their genome sequences are distributed in a large number of contigs ; therefore extensive work is required . 
 constant data refinement is necessary to provide a reliable comprehension of the genome and gene models and to provide a curated annotation . 
 such strategies include new assembly of existing capillary reads supplemented with an additional ~90,000 fosmid and bac end sequences , deep sequencing of clonal dna ( nmri strain , puerto rican origin ) using 108-based paired reads on illumina genome analyzer iix platform , and rna - seq data of different parasite life stages . 
 progress on the knowledge of s. mansoni genome features is still expanding , especially with the application of the next - generation sequencing platforms and single nucleotide polymorphism typing methods . 
 for instance , 14 new microsatellite markers were recently identified by de novo assembly of massive sequencing reads ; these new features can be employed for pedigree studies . yet , newer sequencing technologies will allow for the development of population genomics studies of field , laboratory , and clinical isolates . as a result of the availability of the schistosoma genome sequence data , comparative analyses across different species became possible bringing together different areas of research [ 21 , 23 , 24 ] . 
 comparative genomics of three schistosoma species revealed several conserved features such as the overall gc content , sequence identity , presence of repetitive elements , and synteny . 
 this genome - wide analysis corroborates previous genetic studies and supports the evolutionary hypothesis of s. haematobium and s. mansoni as the most related species , followed by s. japonicum , to the exclusion of other trematoda such as clonorchis , fasciola , and schmidtea . 
 mitochondrial genes have been used as molecular markers for species and strain identification , which is key to a variety of studies such as phylogenetics , population genetics , biogeography , and molecular ecology [ 2529 ] . besides helping to elucidate the evolutionary relationships among taxa 
 , mitochondrial genes have been successfully applied into epidemiological studies , monitoring , and control of microbes , parasites , and vectors of medical and socioeconomic importance . 
 the analysis of the whole mitochondrial genome of diverse taxa has changed the perspective of such studies . 
 the mitochondrial genome of six schistosoma species was sequenced ( table 1 ) including s. haematobium ( nc_008074 ) , s. japonicum ( nc_002544 ) , s. mansoni ( nc_002545 ) , s. malayensis , s. mekongi ( nc_002529 ) , and s. spindale ( nc_008067 ) [ 27 , 3032 ] . 
 these genomes range in size from 13,503 to 16,901  bp and encode 36 genes comprising two ribosomal genes ( large and small subunit rrna genes ) , and 22 transfer rna ( trna ) genes , as well as 12 protein - encoding genes ( atp6 , cob , cox13 , nad16 , and nad4l ) . 
 the atp8 gene , commonly found in other phyla , is absent from platyhelminthes , whose mitochondrial genomes have been analysed . moreover 
 , each schistosoma mitochondrial genome contains a long noncoding region that is divided into two parts by one or more trna genes , which is found in other platyhelminthes and vary in length according to each taxon . 
 comparative mitogenomics have revealed a highly conserved gene order among distinct platyhelminthes with the exception of some schistosoma species [ 25 , 3033 ] . 
 the mitochondrial genome of the asian schistosomes , s. japonicum and s. mekongi , displays the same gene order as other digenea and cestoda [ 25 , 32 ] . in contrast , the gene order in the mitochondrial genome of s. haematobium , s. mansoni , and s. spindale is strikingly different from other taxa [ 25 , 30 ] . 
 the unique gene order rearrangements identified in these species constitute valuable information to the reconstruction of the phylogenetic relationships of schistosoma and other platyhelminthes ( section 3.1 ) . 
 the use of genomic data is valuable to the understanding of the evolutionary history of schistosoma especially due to the absence of fossil records . 
 molecular markers have been used to test hypotheses in a variety of schistosoma phylogenetic , phylogeographic and epidemiological studies [ 4 , 25 , 3436 ] . 
 traditionally , these markers include the nuclear ribosomal rna genes ( 18s , 5.8s , and 28s ) and the internally transcribed spacer ( its ) region besides a number of mitochondrial genes ( cob , cox13 , nad16 , etc . ) . 
 based on mitochondrial data from different studies , a standard phylogeny of 23 schistosoma species was proposed . 
 these clades include ( 1 ) the s. japonicum complex ( s. japonicum , s. malayensis , s. mekongi , s. ovuncatum , and s. sinensium ) found in central and south eastern asia ; ( 2 ) the s. hippopotami clade ( s. edwardiense and s. hippopotami ) distributed in africa ; ( 3 ) the proto - s . 
 mansoni clade ( s. incognitum and s. turkestanicum ) in central asia , near east , and eastern europe ; 
 ( 4 ) the s. mansoni clade ( s. mansoni and s. rodhaini ) found in africa and south america ; ( 5 ) the s. haematobium clade ( s. bovis , s. curassoni , s. guineensis , s. haematobium , s. intercalatum , s. kisumuensis , s. leiperi , s. margrebowiei , and s. mattheei ) occurring in different parts of africa ; ( 6 ) the s. indicum clade ( s. indicum , s. nasale , and s. spindale ) found in india and south asia . 
 there are divergent opinions concerning the origin of the genus schistosoma and its intermediate snail host [ 25 , 3739 ] . 
 the  out of asia  hypothesis is the most generally accepted , indicating a migration followed by dispersion of these parasites from asia to africa . in this regard , studies of molecular phylogeny suggested that the genus schistosoma had its origin in asia and ( at least two descendants ) colonized the african continent independently , where they easily radiated , becoming exclusive parasites of planorbid snails . returning to asia , the parasites diversified into groups of species , characterized by the position of the spike in the eggs . 
 the molecular phylogeny of seven schistosoma species suggested that schistosomes were endoparasites of rodents and ruminants and were transmitted to the first hominids in africa as a migration to savanna areas occurred . 
 however , in order to better define the schistosoma origin , there is a need to sample a broader range of species . 
 most of the widely accepted schistosoma phylogenies are primarily based on sequence alignments of mitochondrial genes such as cox13 , and nad16 , [ 25 , 30 ] . 
 indeed , these gene rearrangements , considered to be rare evolutionary events , have been applied to an increasing number of studies ( cf . ) . 
 as mentioned before , major changes in the mitochondrial gene order were identified in some schistosoma species ( section 2.2 ) , more specifically in the african clades ( s. mansoni and s. haematobium ) and the s. indicum group ( s. spindale ) . 
 this provides further evidence for the asian ancestry of schistosomes and corroborates the concept of the reinvasion of asia by members of the s. indicum group from africa [ 27 , 30 ] . besides reconstructing the phylogenetic relationships of diverse taxa 
 , the evolutionary framework has been applied to improve functional annotation of genes and gene products , as well as to study gene / protein family evolution . 
 phylogenomics has allowed the identification and characterization of all eukaryotic protein kinases encoded in the s. mansoni genome and improved the functional annotation of over 40% of them , highlighting the molecular diversity of these enzymes [ 42 , 43 ] . 
 a phylogenetic analysis was also employed in the classification of s. mansoni histone proteins , which play a key role in epigenetic modifications that might reflect the parasite complex lifestyle . by applying an evolutionary framework to analyse a large sequence dataset from a broad range of organisms , functional annotation of the s. mansoni histone proteins 
 moreover , phylogenomics has unraveled the distinct evolutionary histories of three expanded endopeptidase families in s. mansoni . 
 this analysis included members of the metallopeptidase m8 , serine peptidase s1 , and aspartic peptidase a1 families , which were originated from successive events of gene duplication followed by divergence in the parasite lineage after its diversification from other metazoans . 
 other protein families such as nuclear receptors and antioxidants have been characterized in s. mansoni by applying evolutionary and functional approaches [ 46 , 47 ] . 
 the identification of smnr4a , a member of the nuclear receptor subfamily 4 , and its relatedness to the human homologs were corroborated by phylogenetic analysis . 
 thioredoxin and glutathione systems , involved in a variety of cellular processes including antioxidant defense , differ in parasitic and free - living platyhelminthes . by applying different phylogenetic methods and experimental testing 
 , it has been shown that the canonical enzymes of such systems were lost in the parasitic lineages . 
 the availability of sequencing data from mitochondrial genomes from closely related taxa to schistosoma has provided a basis for studies ranging from phylogenetic systematics to molecular ecology and so forth [ 27 , 29 , 48 , 49 ] . 
 importantly , it allowed the assessment of molecular markers prior to broad scale sampling by comparing data from individual genes versus completely sequenced mitochondrial genomes . 
 data from african and asian schistosomes were compared to an s. mansoni intraspecific dataset showing a positive correlation between polymorphism and species divergence . a positive correlation rather than random distribution 
 furthermore , partial sequences of cox1 have been shown not to be the ideal marker for either species identification or population studies of schistosoma species . instead , 
 authors have suggested the use of cox3 and nad5 sequences for both phylogenetic and population studies of such species . 
 dna barcoding was performed for samples of s. haematobium using cox1 and nad1 as molecular markers . 
 the results supported the identification of group 1 and 2 haplotypes based on phylogenetic analysis . 
 moreover , no change in genetic diversity was detected across samples collected over different time points . 
 this approach allowed the development of new assays based on group - specific pcr primers and snapshot probes to assist genetic screening of schistosome isolates . 
 another study employing mitochondrial sequences was performed in schistosoma cercariae harvested from biomphalaria choanomphala as part of parasitological and malacological surveys for s. mansoni across lake victoria . 
 dna barcoding of cercarial samples revealed the presence of hybrid species between s. mansoni and s. rodhaini , which is typically found in small mammals . 
 dna barcoding studies are underway for an increasing number of schistosoma species ( table 1 ) , whose data is made available through the barcode of life database ( bold ) system ( http://www.boldsystems.org/ ) . 
 this initiative includes contributors from the biodiversity institute of ontario , canada , and the university of new mexico , usa , among others . 
 other schistosomatidae genera are subject to dna barcoding analysis such as allobilharzia , austrobilharzia , bilharziella , bivitellobilharzia , dendritobilharzia , gigantobilharzia , griphobilharzia , heterobilharzia , macrobilharzia , orientobilharzia , ornithobilharzia , schistosomatium , and trichobilharzia . 
 advances in dna barcoding of a broader range of schistosomatidae will create a framework for species identification from environmental and clinical samples as well as specimens deposited in biological collections . 
 moreover , dna barcoding analysis will help reconstructing the evolutionary relationships across different platyhelminthes and ultimately improve our understanding of parasite biology and evolution . 
 transcriptomic analyses are valuable not only for studies of differential regulation in gene expression , but also for the identification of splicing variants . 
 these approaches have proved to be essential in the identification of gene - coding regions during the process of genome annotation . in this section 
 , we aim to review the achievements in schistosoma transcriptomic studies that range from the whole organism and tissue analyses to those performed at the cellular level . 
 transcriptomics in physiological conditions and in response to a variety of treatments are investigated with respect to the interaction with the host as well as other environments . 
 this section summarizes the accomplishments of gene function studies and the diverse techniques that are being employed . 
 a fine tuning in gene expression of parasites must occur to enable them to survive in such a complex and extremely diverse milieu , adapting to diverse environments as evading the immune system and , at the same time , exploiting different hosts . 
 transcriptomic studies came along allowing a temporal and quantitative analyses of gene expression , which are crucial to completely exploit different datasets and assess parasite biology at the molecular level . aiming to elucidate the differential regulation on the schistosoma transcriptomics , 
 a large number of projects employed diverse strategies , such as the ones using expressed sequence tags ( ests ) , open reading frame expressed sequence tags ( orestes ) , and serial analysis of gene expression ( sage ) [ 52 , 53 ] . 
 the increasing number of projects resulted in the production of a vast amount of sequence data and their availability in public databases . 
 it is noteworthy that only s. japonicum , s. mansoni and , not until very recently , s. haematobium had some of their transcriptome explored ( table 1 ) . 
 the large number of transcriptome projects accelerated the studies in this area by providing means to the use of complementary approaches , especially microarray analysis . 
 together , these approaches have been used to answer a wide range of questions . in the early years 
 , microarray datasets conceived the profile of gene expression between genders of schistosoma species such as s. japonicum and s. mansoni [ 5456 ] . 
 this greatly extended the number of known sex - associated genes and also provided new insights into changes in gene expression promoted by parasite pairing . 
 in addition , gender biased alternative splicing patterns were observed , which are perhaps involved in the generation and maintenance of the sexual dimorphism . 
 later , studies profiled the s. mansoni gene expression in different life stages broadening our understanding of factors controlling schistosome development [ 8 , 53 , 58 ] . while comparing different parasite life stages , 
 information acquired from microarray datasets highlighted putative antischistosome candidate molecules to be used as vaccine targets including dyp - type peroxidases , fucosyltransferases , g - protein coupled receptors , leishmanolysins , tetraspanins , and the netrin / netrin receptor complex . some of these candidates ( fucosyltransferases , leishmanolysins , and tetraspanins ) are members of protein families , which are expanded in the s. mansoni predicted proteome in comparison with other metazoans as inferred by phylogenomic analysis ( http://phylomedb.org/ ) . 
 leishmanolysins , which are members of the metallopeptidase m8 family , were analysed in detail corroborating the potential of these enzymes as future therapeutic targets . 
 another study identified upregulated genes during the first five days of schistosomula development in vitro that are involved in blood feeding , tegument and cytoskeletal development , cell adhesion , and stress responses . 
 the highly upregulated genes included a tegument tetraspanin sm - tsp-3 , a protein kinase , a novel serine protease and serine protease inhibitor , and intestinal proteases belonging to distinct mechanistic classes . 
 other groups have analysed immunity towards schistosoma using protein microarrays as a vaccine discovery tool . 
 they selected a subset of proteins from genomic , transcriptomic , and proteomic data to perform experimental validation aiming at identifying novel antischistosome vaccine candidates [ 60 , 61 ] . 
 recently , an elegant approach to investigate specific tissues such as gastrodermis and both genders reproductive tissues was performed by applying laser microdissection microscopy combined to microarray . 
 these studies brought the perspective of expediting tissue - specific profiling [ 62 , 63 ] . 
 additionally , expression profiles of s. japonicum were explored in studies comparing two chinese isolates ( anhui and zhejiang ) , as well as the anhui chinese and philippines isolates [ 55 , 64 ] . 
 later , an interspecies study compared the s. japonicum and s. mansoni transcriptomes . surprisingly , despite their large phenotypic differences , there was a reduced number of differentially expressed genes based on the expression profile , which might reflect a limitation of the technique ( preferential hybridization related to species polymorphisms ) . bypassing these pitfalls , 
 the next generation sequencing platforms allow exploiting expression data ( rna - seq ) of any organism without previous knowledge of the genome or transcriptome and , further , will reveal new features of the parasite transcriptional landscape . 
 the most recent achievement in this area was the refinement of the genomics and transcriptomics data using a combination of sanger capillary sequencing , next generation dna sequencing , genetic markers , and rna - seq data from several s. mansoni life stages . a previous study , explored the transcriptome of s. mansoni adult males obtaining 11 novel microexon genes ( megs ) , as well as mapping 989 and 1196 contigs to intergenic and intronic genomic regions , respectively . 
 this data could represent new protein - coding genes or noncoding rnas ( ncrnas ) . 
 together , these approaches have significantly advanced our understanding of the dynamics parasite transcriptomes [ 9 , 65 ] . 
 however , there is a need to polish and translate sequence data and gene expression profiles into functional information by computational predictions and experimental characterization . 
 the vast amount of available data opened a myriad of possibilities towards the elucidation of gene functions . 
 transcriptomics is also a key approach to study posttranscriptional regulatory mechanisms such as alternative and transsplicing [ 57 , 6668 ] . 
 alternative splicing of transcripts of secreted proteins , like megs and polymorphic mucin isoforms , has been largely studied in schistosomes [ 51 , 66 ] . 
 this posttranscriptional processing has been proposed as a distinct genetic system to generate protein variation that would allow parasite adaptation to the immune response of different hosts . 
 additionally , alternative splicing has been shown to generate protein variation involved in transcriptional control and splicing . in the case of smca150 , a spliceosome interacting transcriptional cofactor 
 , it could impact many other transcripts leading to several effects in the parasite , especially in sexual dimorphism . 
 spliced leader ( sl ) transsplicing has been shown to be an important posttranscriptional regulation mechanism for s. mansoni . 
 at least 11% of all transcripts has been described as undergoing transsplicing in this organism . 
 sl - rnas are small ncrnas , products of small intronless genes , transcribed by dna polymerase ii and tandemly repeated in the genome . differently from nematodes , 
 s. mansoni carries a single sl sequence , which is composed by 36 nucleotides with an 3-terminal aug codon . 
 although the role of transsplicing is poorly understood , there is an evidence suggesting that sl transsplicing would function providing an initiator methionine for translation initiation of some recipient mrnas in platyhelminthes . 
 this mechanism is also involved in the resolution of polycistronic operons in monocistronic transcripts in s. mansoni [ 19 , 70 ] . despite the great potential revealed by the increasing number of genome and transcriptome studies in schistosomes , there are still several schistosoma species that remain neglected . 
 in contrast to the vast amount of data available in public databases , limited data has been decoded in functional studies and/or in the development of successful tools to fight schistosomes . 
 some of them include the convolution of schistosomes life cycle , the complex interaction with their hosts , time and labor - demand functional studies , the difficulties to nurture the complete life cycle in vitro , and the absence of immortalized cell lines , which has delayed the development of transgenesis tools and models . 
 lately , the posttranscriptional suppression of genes through rna interference ( rnai ) techniques has been a promise to fetch progress on new interventions for schistosomiasis and other helminthiases . in 2003 
 simultaneously gene knockdown of glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) and a glucose transporter ( sgtp1 ) in the larval stage ( sporocysts )   and cathepsin b in adult worms   of s. mansoni were reported . 
 currently , this powerful technique is widely used in schistosomes and has been employed in two studies of functional screening . 
 the first study encompassed the exposure of sporocysts by soaking parasites to dsrna targeting 33 genes , from calcium binding proteins , transcription factors , to receptors , and antioxidant enzymes . in this work , despite the efficacy of this tool to deliver suppression of 11 different genes , it showed that , in s. mansoni , there is a gene - to - gene variability and exposure to dsrna could trigger gene specific upregulation . 
 later , in a different study using electroporation to expose schistosomula to dsrna of 11 target genes ( expressed in distinct tissues ) , the efficiency of rnai in a target specific fashion was observed . 
 other issues with the use of rnai have been widely discussed , not only in schistosomes , but also in other helminth parasites , showing that care should be taken when conducting and evaluating such experiments . despite the difficulties in exploring gene function by rnai in schistosomes , 
 many proteases ( cathepsins and aminopeptidases ) [ 7779 ] , kinases , multidrug resistance transporters , and type v collagen , among other proteins have been studied in diverse life stages of s. japonicum and s. mansoni . 
 recently , this tool has been described targeting tetraspanin 2 in diverse developmental stages of s. haematobium indicating the rnai feasibility in this species . 
 tetraspanins belong to a family of integral membrane proteins present in the outer surface membranes of the parasite tegument . 
 studies have shown that tetraspanins play important roles in the tegument development , maturation , or stability . 
 importantly , the knockdown of peroxiredoxin   and thioredoxin glutathione reductase , antioxidant enzymes , were amongst the first reports of lethality after gene silencing in the parasite , demonstrating the potential of rnai on drug target discovery . 
 moreover , enzymes such as glutathione - s - transferases ( gst26 and gst28 ) , peroxiredoxins ( prx1 and prx2 ) , and superoxide dismutase have been shown to play important roles in the protection of s. mansoni sporocysts in the host - parasite interplay . 
 knockdown of those targets increased parasite susceptibility to oxidative stress and to intermediate host immune cells as described elsewhere . recently 
 researchers are developing new tools for the introduction and expression of homologous or heterologous gene constructs via lentiviruses , replication - defective retroviruses , transposons , retrotransposons , and dna plasmid vectors . 
 sophisticated systems applied to assess gene function in the parasite are being achieved [ 8891 ] . to date , 
 eggs and miracidia are indicated as the preferred life stages for successful transformation approaches in order to enter the germline . in summary 
 , results from such independent studies have shown that schistosome transgenesis works in all the developmental stages tested and that some success has been achieved in integrating genes into the germline [ 8892 ] . 
 altogether , the increasing information on schistosome genomes and transcriptomes is now raising the possibilities to better explore molecular techniques in the field of parasitology , genomics , and computational biology allowing the development of approaches to unravel mechanisms of parasite infection , host - parasite interaction , and so forth [ 21 , 23 , 24 ] . in order to accelerate both parasite research and development of effective tools for schistosomiasis diagnostics and treatment , 
 user - friendly databases have been created and made publicly available to the scientific community [ 9 , 9395 ] . here , we highlight some of these major resources . 
 schistodb integrates genome and proteome sequence data along with functional annotation of genes and gene products of s. haematobium , s. japonicum , and s. mansoni . 
 this resource also covers results from large - scale analysis including ests , metabolic pathways , and candidate drug targets . 
 helmcop ( helminth control and prevention ) integrates functional , structural , and comparative genomics data of helminths from different taxonomic groups and distinct hosts ( plant , animal , and human ) . 
 helmcop offers a comprehensive suite of structural and functional annotations to support comparative analyses and host - parasite interaction studies . 
 genedb is an annotation database that brings together data from a broad range of pathogens and closely related organisms . 
 genedb offers database - driven annotation tools and pipelines and includes manually curated information maintained by computational and biological scientists . 
 collectively , these computational resources support biological and biomedical research and open new frontiers in evolutionary and functional genomics , transcriptomics , and proteomics of schistosoma species and other parasites . besides biodiversity assessment at the molecular level 
 , these resources provide a framework for the identification and prioritization of vaccine and drug targets against human diseases . as the scientific community progresses in data integration and interpretation 
 , we will meet the goals of current and future initiatives to improve human health [ 9 , 12 , 23 , 24 ] . 
 the -omics studies came along with the aim to fill the gaps of long - established approaches , enabling the use of a broad range of experimental and computational methods and contributing to dissect the biological basis of host - parasite interaction , diagnostics improvement , and the discovery of new drug and vaccine targets . 
 nuclear and mitochondrial genomes of some schistosoma species were completely sequenced opening new frontiers for comparative analyses , which will improve our understanding of parasite biology and evolution . 
 other genomes to be analysed include schistosoma species from each of the six clades currently recognized , and other representatives of the schistosomatidae . 
 improving accuracy / quality of genome assembly and annotation will be crucial for such analyses . despite the efforts in the characterization of genomes and transcriptomes of distinct schistosoma species , many questions regarding the biology and evolution of this important taxon remain unanswered . 
 these difficulties might be due to the size of schistosome genomes ( ~10-times larger compared to protozoan parasites ) , the complexity of its life cycle , the presence of rare features in posttranscriptional regulation ( e.g. , transsplicing ) , and little integration of the current information available , among other reasons . 
 there is an urgent need for data integration to help interpreting the massive amount of data generated over the years in this and other areas or research . 
 we envision that , in the future , more transcriptomic and proteomic data will be used to improve the understanding of the origin and evolution of schistosomatidae . 
 the analysis of structural data generated by experimental or computational approaches will also shed light in the biodiversity of schistosomes at the molecular level . in conclusion , 
 the availability of schistosome genome and transcriptome data has provided an unprecedented resource for many research areas . 
 limitations do exist and should be addressed to allow advances in understanding schistosome biology and evolution and ultimately support vaccine and drug development against schistosomiasis .","schistosomes are digenean blood flukes of aves and mammals comprising 23 species . 
 some species are causative agents of human schistosomiasis , the second major neglected disease affecting over 230 million people worldwide . 
 modern technologies including the sequencing and characterization of nucleic acids and proteins have allowed large - scale analyses of parasites and hosts , opening new frontiers in biological research with potential biomedical and biotechnological applications . 
 nuclear genomes of the three most socioeconomically important species ( s. haematobium , s. japonicum , and s. mansoni ) have been sequenced and are under intense investigation . 
 mitochondrial genomes of six schistosoma species have also been completely sequenced and analysed from an evolutionary perspective . 
 furthermore , dna barcoding of mitochondrial sequences is used for biodiversity assessment of schistosomes . despite the efforts in the characterization of schistosoma genomes and transcriptomes , many questions regarding the biology and evolution of this important taxon 
 remain unanswered . 
 this paper aims to discuss some advances in the schistosome research with emphasis on genomics and transcriptomics . 
 it also aims to discuss the main challenges of the current research and to point out some future directions in schistosome studies .",pubmed
"for patients with synchronous colorectal liver metastases ( sclm ) , hepatic resection is considered the best treatment , with reported 5-year survival rates between 23% and 37%.14 resections of both the primary colorectal lesion and the hepatic metastases are needed for patients with sclm when they are resectable . 
 however , the optimal surgical strategy for resectable sclm still remains controversial . from the perspectives of less operation with less mental stress and simplifying perioperative treatment , simultaneous resection of the primary colorectal and liver tumors 
 is a favorable strategy for patients with sclm.58 however , several papers reported that the morbidity rate after simultaneous resection of primary and liver tumors was high because of greater surgical stress and a longer operation time than for single - organ surgery 
 . staged resection with initial operation for the primary lesion followed by resection of hepatic tumors is regarded as an alternative strategy to avoid excessive surgical stress for patients with sclm , though the efficacy of this strategy and the patients who could benefit from this strategy are unknown.46,9,10 thus , this study was conducted to determine which patients could benefit from staged resections instead of simultaneous resection by identifying predictive factors for postoperative morbidity and anastomotic leakage after simultaneous resection of sclm . 
 the medical records of all consecutive patients who underwent liver resections for colorectal liver metastases from january 1992 to january 2004 at our institution were analyzed retrospectively , with institutional review board approval . 
 all sclm patients received simultaneous resection of primary colorectal and hepatic tumors irrespective of the patient 's or the tumor 's characteristics . 
 all 86 patients underwent contrast enhanced computed tomography ( ct ) of the chest , abdomen , and pelvis , as well as hepatic mri , preoperatively . 
 as a control , the morbidity of 167 patients who underwent hepatectomy for metachronous liver metastasis from colorectal cancer from january 1992 to january 2004 and that of 1,728 patients who underwent only resection for colorectal cancer with colorectal reconstruction during the same period were also reviewed . of the 1,728 colorectal cancer patients , 
 incidences of the following postoperative complications were analyzed : anastomotic leak , rectovaginal fistula , intraperitoneal or pelvic abscess , wound infection , wound dehiscence , ileus , enteroparesis , postoperative delirium , urinary tract infection , dysuria , empyema thoracis , pleural effusion , atelectasis , cholecystitis , perihepatic or subphrenic abscess , bile leak , liver failure , and others . 
 anastomotic leakage was defined as follows : peritonitis and a dehiscence in the anastomosis , discharge of pus from the anus , vaginal fistula , or feces from the abdominal drain . 
 leakage was confirmed by ct scan , contrast enema , re - operation , or digital rectal examination . 
 all complications were graded according to the classification proposed by clavien et al.11 postoperative mortality was defined to include any death during postoperative hospitalization or within 30  days . 
 correlations between postoperative morbidity and the following patient , tumor , and surgical factors were analyzed : age , sex , body mass index ( bmi ) , preoperative comorbidity , site of primary tumor , intestinal obstruction by tumor , size of primary tumor , differentiation of tumor , distribution of hepatic tumors , number of hepatic tumors , hepatic tumor size , operative methods , operation time , intraoperative blood loss , and blood transfusion . 
 survival and follow - up were calculated from the time of the operation to the date of death or last available follow - up . 
 multivariate analyses to evaluate the independent predictive factors for postoperative complications or anastomotic leakage were done by multiple logistic regression analysis . 
 the medical records of all consecutive patients who underwent liver resections for colorectal liver metastases from january 1992 to january 2004 at our institution were analyzed retrospectively , with institutional review board approval . 
 all sclm patients received simultaneous resection of primary colorectal and hepatic tumors irrespective of the patient 's or the tumor 's characteristics . 
 all 86 patients underwent contrast enhanced computed tomography ( ct ) of the chest , abdomen , and pelvis , as well as hepatic mri , preoperatively . 
 as a control , the morbidity of 167 patients who underwent hepatectomy for metachronous liver metastasis from colorectal cancer from january 1992 to january 2004 and that of 1,728 patients who underwent only resection for colorectal cancer with colorectal reconstruction during the same period were also reviewed . of the 1,728 colorectal cancer patients , 
 incidences of the following postoperative complications were analyzed : anastomotic leak , rectovaginal fistula , intraperitoneal or pelvic abscess , wound infection , wound dehiscence , ileus , enteroparesis , postoperative delirium , urinary tract infection , dysuria , empyema thoracis , pleural effusion , atelectasis , cholecystitis , perihepatic or subphrenic abscess , bile leak , liver failure , and others . 
 anastomotic leakage was defined as follows : peritonitis and a dehiscence in the anastomosis , discharge of pus from the anus , vaginal fistula , or feces from the abdominal drain . 
 leakage was confirmed by ct scan , contrast enema , re - operation , or digital rectal examination . 
 all complications were graded according to the classification proposed by clavien et al.11 postoperative mortality was defined to include any death during postoperative hospitalization or within 30  days . 
 correlations between postoperative morbidity and the following patient , tumor , and surgical factors were analyzed : age , sex , body mass index ( bmi ) , preoperative comorbidity , site of primary tumor , intestinal obstruction by tumor , size of primary tumor , differentiation of tumor , distribution of hepatic tumors , number of hepatic tumors , hepatic tumor size , operative methods , operation time , intraoperative blood loss , and blood transfusion . 
 survival and follow - up were calculated from the time of the operation to the date of death or last available follow - up . 
 multivariate analyses to evaluate the independent predictive factors for postoperative complications or anastomotic leakage were done by multiple logistic regression analysis . 
 from 1992 to 2004 , 86 patients were treated with simultaneous resection of primary and hepatic tumors for sclm . 
 there were 37 female and 49 male patients , with a median age of 59  years ( range , 40 to 85  years ) . 
 the primary tumor was staged as t3 in 54 ( 63% ) and t4 in 32 ( 37% ) according to the tnm classification . 
 the mean diameter of the primary tumor was 55  mm ( range , 26140  mm ) . 
 liver metastases were solitary in 29 patients and multiple in 57 patients . in 47 patients ( 55% ) , the hepatic tumor showed a unilobar distribution , while a bilobar tumor distribution was observed in 39 ( 45% ) . 
 the mean diameter of the hepatic tumor was about 43  mm ( range , 5200  mm ) . 
 the mean resected liver volume was 380  g ( range , 101,660  g ) . 
 the operation for primary colorectal cancer was right ( hemi ) colectomy in 17 patients , transverse colectomy in 1 , left ( hemi ) colectomy in 4 , sigmoidectomy in 24 , high anterior resection in 7 , low anterior resection in 20 , very low anterior resection in 6 , inter - sphincteric resection in 2 , hartmann 's operation in 1 , and abdomino - perineal resection in 4 ( table 4 ) . 
 a diverting stoma to prevent anastomotic leakage was made in 22 ( 26% ) patients at the surgeon 's discretion , and lateral lymph node dissection was performed in 20 ( 23% ) . in terms of liver tumor resection , lobectomy was performed in 11 patients , segmentectomy in 22 , bisegmentectomy in 1 , trisegmentectomy in 2 , subsegmentectomy in 3 , and partial resection in 47 . 
 adjuvant therapy was given to only 17 patients ( 19.8% ) because adjuvant chemotherapy for colorectal cancer in stage iii or more was performed since january 2003 . 
 no patients died within 30  days of the operation , but 55 ( 64% ) patients developed complications ( table  1 ) . 
 eighteen patients ( 21% ) experienced leakage , of whom 6 needed urgent re - operation with ileostomy and drainage of an intra - abdominal collection caused by leakage . 
 postoperative bleeding , wound dehiscence , and ileus were the reasons for the three other re - operation cases . 
 the most frequent complication was wound infection.table 1postoperative complications after simultaneous resection for sclm according to clavien gradecomplicationsno . 
 of patientsgr igr iigr iiiagr iiibgr ivacolon and rectum  anastomotic leakage18 ( 21%)126  intrapelvic abscess6 ( 7%)141  intraperitoneal abscess5 ( 6%)1031  rectovaginal fistula4 ( 5%)13liver  bile leakage7 ( 8%)61  hepatic abscess7 ( 8%)511  liver failure3 ( 3%)111  postoperative bleeding1 ( 1%)1other organs  wound infection25 ( 29%)232  pleural effusion12 ( 14%)111  wound dehiscence6 ( 7%)321  enteroparesis5 ( 6%)5  postoperative delirium4 ( 5%)13  dysuria4 ( 5%)4  urinary tract infection3 ( 3%)3  pneumonia2 ( 2%)2  others7 ( 8%)142 postoperative complications after simultaneous resection for sclm according to clavien grade the morbidity rate of the 167 patients who underwent hepatectomy for metachronous colorectal liver metastasis during the same period was 19.8% , and that of 1,728 patients who underwent only resection for colorectal cancer was 32.1% . 
 postoperative complications were significantly correlated with presence of diverting stoma ( p  <  0.01 ) , duration of operation greater than 8  h ( p  <  0.01 ) , amount of intraoperative blood loss ( p  <  0.01 ) , and intraoperative blood transfusion ( p  <  0.01 ) . 
 only a greater amount of blood loss had a predictive value for increased occurrence of postoperative complications . 
 then , the correlations between anastomotic leakage and clinicopathological factors were examined to identify risk factors for anastomotic leakage after simultaneous resection for sclm . 
 patients who underwent abdomino - perineal resection ( n  =  4 ) or hartmann 's operation ( n  =  1 ) were excluded from the analysis . anastomotic leakage was significantly correlated with lateral lymph node dissection ( p  <  0.01 ) , primary site of rectum ( p  =  0.01 ) , duration of operation greater than 8  h ( p  <  0.01 ) , and amount of intraoperative blood loss ( p  =  0.02 ) . 
 neither serum levels of tp and alb , steroid usage , nor neoadjuvant therapy showed correlation with occurrence of anastomotic leakage ( data not shown ) . 
 multivariate analyses revealed operation time greater than 8  h ( p  <  0.01 ) as the only independent predictive factor for anastomotic leakage after simultaneous resection of sclm ( table  2 ) . 
 extent of hepatectomy , timing of anastomosis and hepatectomy , and usage of pringle maneuver did not correlate with occurrence of complication or anastomotic leakage.table 2correlation between anastomotic leakage and clinicopathological factors in patients who underwent simultaneous resection for sclmleakage (  ) ( n  =  63)leakage ( + ) ( n  =  18)univariate analysis p valuemultivariate analysis p value , rr ( 95%ci)patient characteristics  median age ( range ) ( years)59 ( 4085)59 ( 4173)0.81  male / female33/3012/60.42  bmi ( mean    sd)21.9    2.922.5    2.20.44  preoperative comorbidity  absent44120.78  present196primary colorectal tumor  sitecolon4260.01n.s.rectum2112  stenosisabsent5600.34present718  tumor size , mm52.058.00.25  pt stagept34190.25pt4229  pn stagepn01720.22pn+4616  histologywell , mod60150.12poor33liver metastasis  distributionunilobar3890.43bilobar259  number of tumors ( range)2.3 ( 18)2.6 ( 18)0.57  tumor size , mm47330.06operative factors  lateral lymph node dissection  absent5510<0.01n.s .  present88  diverting stoma  absent48110.24  present157  liver resection  partial hx , segmentectomy51160.72 
 lobectomy122  timing of anastomosis  colectomy    anastomosis    hx2040.20  colectomy    hx    anastomosis75  hx    colectomy    anastomosis369  pringle maneuver  absent1010.44  present5317  operation time  < 8  h538<0.01<0.01 , 6.63 ( 2.0920.9 )  8  h1010  blood loss , g ( range)1,345 ( 1626,000)2,487 ( 4306,560)0.02n.s . 
 transfusion  absent3990.37  present249  blood transfusion , ml3431,2120.05rr relative risk , ci confidence interval , hx hepatectomy , n.s . 
 non - significant ( p  >  0.05 ) correlation between anastomotic leakage and clinicopathological factors in patients who underwent simultaneous resection for sclm rr relative risk , ci confidence interval , hx hepatectomy , n.s . non - significant ( p  >  0.05 ) table  3 showed the rates of complication  iiia and anastomotic leakage according to operative procedures of the primary and hepatic resections which were performed in the same patient . 
 complication  iiia and anastomotic leakage were more frequently observed in patients with rectal resection ; however , extent of hepatectomy did not seem to affect occurrence of complication  iiia or anastomotic leakage.table 3rates of complication  gr iiia and anastomotic leakage according to the site of primary colorectal resection and extent of hepatectomyprimary colorectal resectionhepatectomycomplication  gr iiiaanastomotic leakagecolectomy < lobectomy4/40 ( 10%)5/39 ( 13%)lobectomy0/7 ( 0%)1/7 ( 14%)rectal resection < 
 lobectomy11/32 ( 34%)11/28 ( 39%)lobectomy2/7 ( 29%)1/7 ( 14%)one patient who underwent hartmann 's operation was excluded from the analysisfour patients who underwent abdomino - perineal resection were excluded from the analysis rates of complication  gr iiia and anastomotic leakage according to the site of primary colorectal resection and extent of hepatectomy one patient who underwent hartmann 's operation was excluded from the analysis four patients who underwent abdomino - perineal resection were excluded from the analysis hospitalization was significantly longer in the 55 patients with postoperative morbidity ( 32.2  days ) than in the 31 patients without postoperative morbidity ( 17.6  days ) ( p  <  0.01 ) . in addition , hospitalization was significantly longer in the 18 patients with anastomotic leakage ( 43.5  days ) than in the 63 patients without anastomotic leakage ( 22.2  days ) ( p  <  0.01 ) . 
 the overall survival rate after simultaneous resection for sclm of the 86 patients was 61% at 3  years and 45% at 5  years , with mst of 47  months . 
 from 1992 to 2004 , 86 patients were treated with simultaneous resection of primary and hepatic tumors for sclm . 
 there were 37 female and 49 male patients , with a median age of 59  years ( range , 40 to 85  years ) . 
 the primary tumor was staged as t3 in 54 ( 63% ) and t4 in 32 ( 37% ) according to the tnm classification . 
 the mean diameter of the primary tumor was 55  mm ( range , 26140  mm ) . 
 liver metastases were solitary in 29 patients and multiple in 57 patients . in 47 patients ( 55% ) , the hepatic tumor showed a unilobar distribution , while a bilobar tumor distribution was observed in 39 ( 45% ) . 
 the mean diameter of the hepatic tumor was about 43  mm ( range , 5200  mm ) . 
 the mean resected liver volume was 380  g ( range , 101,660  g ) . 
 the operation for primary colorectal cancer was right ( hemi ) colectomy in 17 patients , transverse colectomy in 1 , left ( hemi ) colectomy in 4 , sigmoidectomy in 24 , high anterior resection in 7 , low anterior resection in 20 , very low anterior resection in 6 , inter - sphincteric resection in 2 , hartmann 's operation in 1 , and abdomino - perineal resection in 4 ( table 4 ) . 
 a diverting stoma to prevent anastomotic leakage was made in 22 ( 26% ) patients at the surgeon 's discretion , and lateral lymph node dissection was performed in 20 ( 23% ) . in terms of liver tumor resection , lobectomy was performed in 11 patients , segmentectomy in 22 , bisegmentectomy in 1 , trisegmentectomy in 2 , subsegmentectomy in 3 , and partial resection in 47 . 
 adjuvant therapy was given to only 17 patients ( 19.8% ) because adjuvant chemotherapy for colorectal cancer in stage iii or more was performed since january 2003 . 
 no patients died within 30  days of the operation , but 55 ( 64% ) patients developed complications ( table  1 ) . 
 eighteen patients ( 21% ) experienced leakage , of whom 6 needed urgent re - operation with ileostomy and drainage of an intra - abdominal collection caused by leakage . 
 postoperative bleeding , wound dehiscence , and ileus were the reasons for the three other re - operation cases . 
 the most frequent complication was wound infection.table 1postoperative complications after simultaneous resection for sclm according to clavien gradecomplicationsno . 
 of patientsgr igr iigr iiiagr iiibgr ivacolon and rectum  anastomotic leakage18 ( 21%)126  intrapelvic abscess6 ( 7%)141  intraperitoneal abscess5 ( 6%)1031  rectovaginal fistula4 ( 5%)13liver  bile leakage7 ( 8%)61  hepatic abscess7 ( 8%)511  liver failure3 ( 3%)111  postoperative bleeding1 ( 1%)1other organs  wound infection25 ( 29%)232  pleural effusion12 ( 14%)111  wound dehiscence6 ( 7%)321  enteroparesis5 ( 6%)5  postoperative delirium4 ( 5%)13  dysuria4 ( 5%)4  urinary tract infection3 ( 3%)3  pneumonia2 ( 2%)2  others7 ( 8%)142 postoperative complications after simultaneous resection for sclm according to clavien grade the morbidity rate of the 167 patients who underwent hepatectomy for metachronous colorectal liver metastasis during the same period was 19.8% , and that of 1,728 patients who underwent only resection for colorectal cancer was 32.1% . 
 postoperative complications were significantly correlated with presence of diverting stoma ( p  <  0.01 ) , duration of operation greater than 8  h ( p  <  0.01 ) , amount of intraoperative blood loss ( p  <  0.01 ) , and intraoperative blood transfusion ( p  <  0.01 ) . 
 only a greater amount of blood loss had a predictive value for increased occurrence of postoperative complications . 
 then , the correlations between anastomotic leakage and clinicopathological factors were examined to identify risk factors for anastomotic leakage after simultaneous resection for sclm . 
 patients who underwent abdomino - perineal resection ( n  =  4 ) or hartmann 's operation ( n  =  1 ) were excluded from the analysis . anastomotic leakage was significantly correlated with lateral lymph node dissection ( p  <  0.01 ) , primary site of rectum ( p  =  0.01 ) , duration of operation greater than 8  h ( p  <  0.01 ) , and amount of intraoperative blood loss ( p  =  0.02 ) . neither serum levels of tp and alb , steroid usage , nor neoadjuvant therapy showed correlation with occurrence of anastomotic leakage ( data not shown ) . 
 0.01 ) as the only independent predictive factor for anastomotic leakage after simultaneous resection of sclm ( table  2 ) . extent of hepatectomy , timing of anastomosis and hepatectomy , and usage of pringle maneuver did not correlate with occurrence of complication or anastomotic leakage.table 2correlation between anastomotic leakage and clinicopathological factors in patients who underwent simultaneous resection for sclmleakage (  ) ( n  =  63)leakage ( + ) ( n  =  18)univariate analysis p valuemultivariate analysis p value , rr ( 95%ci)patient characteristics  median age ( range ) ( years)59 ( 4085)59 ( 4173)0.81  male / female33/3012/60.42  bmi ( mean    sd)21.9    2.922.5    2.20.44  preoperative comorbidity  absent44120.78  present196primary colorectal tumor  sitecolon4260.01n.s.rectum2112  stenosisabsent5600.34present718  tumor size , mm52.058.00.25  pt stagept34190.25pt4229  pn stagepn01720.22pn+4616  histologywell , mod60150.12poor33liver metastasis  distributionunilobar3890.43bilobar259  number of tumors ( range)2.3 ( 18)2.6 ( 18)0.57  tumor size , mm47330.06operative factors  lateral lymph node dissection  absent5510<0.01n.s .  present88  diverting stoma  absent48110.24  present157  liver resection  partial hx , segmentectomy51160.72 
 lobectomy122  timing of anastomosis  colectomy    anastomosis    hx2040.20  colectomy    hx    anastomosis75  hx    colectomy    anastomosis369  pringle maneuver  absent1010.44  present5317  operation time  < 8  h538<0.01<0.01 , 6.63 ( 2.0920.9 )  8  h1010  blood loss , g ( range)1,345 ( 1626,000)2,487 ( 4306,560)0.02n.s .  transfusion  absent3990.37  present249  blood transfusion , ml3431,2120.05rr relative risk , ci confidence interval , hx hepatectomy , n.s . 
 non - significant ( p  >  0.05 ) correlation between anastomotic leakage and clinicopathological factors in patients who underwent simultaneous resection for sclm rr relative risk , ci confidence interval , hx hepatectomy , n.s . 
 non - significant ( p  >  0.05 ) table  3 showed the rates of complication  iiia and anastomotic leakage according to operative procedures of the primary and hepatic resections which were performed in the same patient . 
 complication  iiia and anastomotic leakage were more frequently observed in patients with rectal resection ; however , extent of hepatectomy did not seem to affect occurrence of complication  iiia or anastomotic leakage.table 3rates of complication  gr iiia and anastomotic leakage according to the site of primary colorectal resection and extent of hepatectomyprimary colorectal resectionhepatectomycomplication  gr iiiaanastomotic leakagecolectomy < lobectomy4/40 ( 10%)5/39 ( 13%)lobectomy0/7 ( 0%)1/7 ( 14%)rectal resection < 
 lobectomy11/32 ( 34%)11/28 ( 39%)lobectomy2/7 ( 29%)1/7 ( 14%)one patient who underwent hartmann 's operation was excluded from the analysisfour patients who underwent abdomino - perineal resection were excluded from the analysis rates of complication  gr iiia and anastomotic leakage according to the site of primary colorectal resection and extent of hepatectomy one patient who underwent hartmann 's operation was excluded from the analysis four patients who underwent abdomino - perineal resection were excluded from the analysis hospitalization was significantly longer in the 55 patients with postoperative morbidity ( 32.2  days ) than in the 31 patients without postoperative morbidity ( 17.6  days ) ( p  <  0.01 ) . in addition , hospitalization was significantly longer in the 18 patients with anastomotic leakage ( 43.5  days ) than in the 63 patients without anastomotic leakage ( 22.2  days ) ( p  <  0.01 ) . 
 the overall survival rate after simultaneous resection for sclm of the 86 patients was 61% at 3  years and 45% at 5  years , with mst of 47  months . 
 for patients with resectable sclm , both primary tumor resection and hepatectomy for liver metastasis could lead to long - term survival , with a 5-year survival rate of 2337% . 
 however , the optimal strategy , including surgical resection and perioperative treatment , remains controversial for resectable sclm . in terms of surgical resection for sclm 
 , it has not been resolved whether simultaneous resection or staged resections would be preferable . 
 a series of neoadjuvant chemotherapy or chemoradiotherapy , resection of primary tumor , chemotherapy between two operations , hepatectomy , and adjuvant chemotherapy could be the maximal total treatment for sclm , while simultaneous resection could simplify and shorten the treatment schedule by eliminating one operation . 
 completion of the two resections and initiation of adjuvant chemotherapy occur earlier with simultaneous resection than with staged resections . 
 considering survival , comparable survival for simultaneous resection was shown in comparison with that for staged resections.13 furthermore , simultaneous resection could relieve patients from a considerable degree of mental and physical stress and decrease total treatment cost by preventing a second resection for hepatic metastases . 
 recent advances in colorectal and hepatic surgery have enabled simultaneous resection to be performed more safely . 
 the overall complication rate was reduced , and length of hospital stay was shortened , with no change in operative mortality.7,8 however , at present , staged resections with initial resection of the primary tumor followed by hepatic resection have been frequently performed in patients with sclm for several reasons.4,5,9,10 first , the perioperative risk of staged resections has been thought to be less than that of simultaneous resection.4,13,14 sheele et al . 
 reported 13 anastomotic leakages of 90 simultaneous procedures in their series , and two of them led to death.4 thelen et al . proposed the criteria for simultaneous liver resection according to the age and extent of liver resection , because death after simultaneous liver resection ( n  =  4 ) occurred after major hepatectomies , and three of these four patients were 70  years of age or older.15 second , staged resections might offer a chance to evaluate liver or extrahepatic metastases between the two operations . 
 lambert et al . reported that staged resections of synchronous hepatic metastases with an interval of 3 to 6  months might allow occult disease to become clinically detectable and could potentially identify patients for whom a hepatic resection would offer no survival benefit.10 fujita recommended an interval resection to assess the metastatic status of the regional lymph nodes , because the presence of six or more lymph node metastases was an independent poor prognostic factor in patients with resected sclm and a relative contraindication for hepatic resection.9 some authors proposed chemotherapy between primary tumor resection and liver resection to select patients that could benefit from hepatectomy.13,16 alternatively , a liver - first approach of doing liver resection first and primary resection second was newly proposed as a strategy for sclm.17,18 the liver - first approach might avoid needless radical colorectal surgery by confirming curability of hepatic metastases first and also might increase resectability compared with the ordinary staged resections especially in patients with progressive hepatic metastases . 
 this study evaluated morbidity , especially anastomotic leakage , after simultaneous resection for sclm in order to assess the safety of simultaneous resection . 
 anastomotic leakage is sometimes fatal and can cause a difficult situation with physical and mental discomfort or pain . 
 the morbidity rate of patients who underwent simultaneous resection for sclm seemed to be higher than that of patients with resected metachronous colorectal hepatic metastasis or that of patients who underwent only resection for colorectal primary cancer . 
 predictive factors for postoperative morbidity and for anastomotic leakage were intraoperative blood loss and operation time greater than 8  h , respectively . the overall morbidity rate and the rate of anastomotic leakage were 91% and 50% , respectively , in patients with operation time greater than 8  h , and 54% and 13% , respectively , in patients with operation time less than or equal to 8  h. blood loss and operation time usually represent the amount of surgical stress . 
 in particular , the average hospitalization of the 18 patients with anastomotic leakage was more than 43  days . 
 retrospective studies have also indicated that the occurrence of anastomotic leakage is associated with increased morbidity , mortality , and prolonged hospital stay . 
 additionally , anastomotic leakage may be associated with an increased risk of local recurrence.19 various risk factors for anastomotic leakage have been analyzed by several investigators . 
 age , sex , obesity , level of anastomosis , smoking , blood transfusion , tumor diameter , preoperative ( chemo ) radiotherapy , physical status , obstruction , and coronary heart disease have been shown to be significant risk factors for leakage.2024 in simultaneous resection for sclm , not only the factors related to the tumor , the patient , or the colorectal operation , but factors related to the hepatectomy could affect the occurrence of anastomotic leakage . however , the extent of hepatic resection , sequence of colectomy , hepatectomy , anastomosis , use of the pringle maneuver , and total time of the pringle maneuver were not predictive factors for anastomotic leakage or postoperative complications in patients with resected sclm . 
 recently , a diverting stoma has been often used to prevent anastomotic leakage in patients who undergo low anterior resection by diverting the fecal stream and keeping the anastomosis free of material.19,25,26 in this study , the presence of a diverting stoma was not a predictive factor for absence of postoperative anastomotic leakage . however , the analysis estimating efficacy of a diverting stoma in this study was not accurate , because a diverting stoma was basically used in patients whose risk for anastomotic leakage was considered to be high by the surgeons . 
 the site of primary tumor that has been reported as a strong predictive factor in previous studies was not a predictive factor for anastomotic leakage in this series . 
 use of diverting stoma might affect the result of analyses of predictive factors for anastomotic leakage . 
 a randomized , controlled trial is needed to elucidate the efficacy of a temporary diverting stoma . 
 although several rationales for the simultaneous resection for sclm are clear , staged resections should be selected to prevent anastomotic leakage or serious complications when the scheduled operation would result in considerable surgical stress , i.e. , predicted operation time greater than 8  h according to the results of the present study . predicted operation time should be calculated by considering various factors , such as characteristics of the patient , primary and metastatic tumor , extent of operation , difficulty of the procedure , and so on . 
 based on the results of this study , we now select staged resections when operation time is expected to be greater than 8  h ; otherwise , we select simultaneous resection . a prospective study of sclm to evaluate the efficacy and safety of the operation time - based decision model is in progress . 
 then , comparison of ratio of patients who could receive adjuvant chemotherapy will be essential when comparing the efficacy of simultaneous resection and that of staged resections in a future study of sclm . 
 furthermore , in staged resections , there is a risk that some patients could not undergo a second resection after the first resection due to tumor progression or complication of first surgery . 
 resection rate of patients who could undergo both primary and hepatic resections should be assessed when comparing simultaneous resection and staged resections in sclm . 
 the limitations of our study are its retrospective design and the relatively small number of patients studied . 
 the morbidity rate and the frequency of anastomotic leakage were high with simultaneous resection for sclm , especially in patients with greater intraoperative blood loss or operation time greater than 8  h. for patients with sclm , staged resections should be considered when simultaneous resection would involve excessive surgical stress . 
 this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .","backgroundthe optimal surgical strategy for resectable , synchronous , colorectal liver metastases remains unclear . 
 the objective of this study was to determine which patients could benefit from staged resections instead of simultaneous resection by identifying predictive factors for postoperative morbidity and anastomotic leakage after simultaneous resection of synchronous , colorectal liver metastases and the primary colorectal tumor.methodsthis study involved 86 patients with synchronous colorectal liver metastases who underwent simultaneous resection of the primary colorectal tumor and the hepatic tumor . 
 postoperative mortality , morbidity , and other surgical outcomes , including survival and hospitalization , were assessed . 
 predictive factors for postoperative morbidity and for anastomotic leakage were evaluated.resultspostoperative morbidity and anastomotic leakage were found in 55 ( 64% ) and 18 ( 21% ) patients . 
 predictive factors for postoperative morbidity and for anastomotic leakage were intraoperative blood loss and operation time > 8  h , respectively . 
 the overall 5-year survival rate was 45%.conclusionsthe frequency of morbidity and that of anastomotic leakage seemed to be high after simultaneous resection for synchronous colorectal liver metastases , especially when intraoperative blood loss or operation time increased greatly 
 . staged resections should be considered in cases in which excessive surgical stress from simultaneous resection of synchronous colorectal liver metastases would be expected .",pubmed
"asthma is a chronic inflammatory disease that causes airflow limitation and affects the performance of activities of daily living.1 pharmacological treatment is essential2 and is aimed at achieving and maintaining clinical asthma control.1 inhalation is the most widely used approach for the treatment of asthma because it allows drugs to reach the lungs in a selective manner , increasing drug concentration in the airways and reducing systemic adverse effects . 
 only half of all asthma patients actually use the prescribed medication and do so correctly . 
 5 low treatment adherence is related to the fact that patients do not know how to use or have difficulty in using inhaler devices correctly.3 asthma education can remedy that , being a key component of asthma management.6 
  incorrect inhaler use can lead to treatment failure by reducing drug concentration in the airways and contribute to treatment nonadherence , making clinical asthma control difficult.9 
  the frequency of incorrect use of metered dose inhalers ( mdis ) ranges from 14% to 90% , with an estimated mean of 50%,10 incorrect mdi use reducing lung drug deposition to less than 20% . in a study of patients using inhaled or oral corticosteroids , increased bronchodilator use , nebulizer use , and hospitalizations were observed in more than 50% of the patients who did not adhere to treatment.11 
  instruments ( or scoring systems ) assessing the difficulties that patients face in using inhaled drugs might be useful to reduce those difficulties . by using such instruments , health professionals can devise an educational program targeting the most common errors made by patients , thus improving treatment adherence . 
 there are some scoring systems that assess inhaler technique in patients with lung disease.12 although some assess inhaler technique errors , there is no one system that is considered the gold standard for this purpose 
 . such an instrument could play an important role in the evaluation of patients using inhalers.13 
  leal13 developed a scoring system to assess the difficulties that patients face in using mdis ; the system scores correct and incorrect mdi use , assessing the errors made during mdi use . 
 on the basis of that instrument , santos et al.3 developed scoring systems for the assessment of dry powder inhaler ( dpi ) and mdi technique . 
 although the aforementioned instruments have yet to be validated for use , they are used in our hospital in order to assess inhaler technique in patients with asthma3 and in those with copd . 
 instrument validation allows determination of the congruence between the scoring system used and the reality being measured , thus increasing the reliability of the instrument.14 the use of validated instruments in order to assess inhaler technique in asthma patients will yield results that are more reliable and will ensure data quality . 
 the objectives of the present study were to validate two scores quantifying the ability of patients to use mdis or dpis ; to identify the most common errors made during their use ; and to identify the patients in need of an educational program for the use of these devices . 
 the inclusion criteria were as follows : having been diagnosed with asthma in accordance with the global initiative for asthma criteria15 ; having been in outpatient treatment for at least two years ; being in the 15- to 65-year age bracket ; having had at least four years of schooling ; having no hearing impairment ; and agreeing to participate in the study by giving written informed consent . 
 patients who had previously participated in educational programs regarding the use of asthma medications were excluded . 
 the study was conducted in three phases : validation of the reliability of the inhaler technique scores ( phase 1 ) ; assessment of the content validity of the two scores ( phase 2 ) ; and assessment of the criterion validity and discriminant validity of the instruments ( phase 3 ) .   in phase 1 , 
 two guest pharmacists ( who were blinded to the study methodology ) simultaneously evaluated inhaler technique . 
 they used the inhaler technique scores developed by santos et al.3 in order to identify the errors made during mdi and dpi use . 
 the instruments provide step - by - step descriptions of mdi and dpi techniques . for each step that is performed correctly , 
 patients receive a score of 1 ; for each step that is performed incorrectly , they receive a score of 0 . for each serious inhaler technique error , 
 the final score determines whether a patient is able or unable to use an mdi or a dpi . 
 the pharmacists were trained in the correct use of the aforementioned scoring systems , in accordance with the guidelines established in the third brazilian consensus on asthma management.16 the following drugs were used in the evaluation : for mdis , 250 g of beclomethasone dipropionate and 100 g of albuterol ; for dpis , 200 g of budesonide and formoterol + budesonide ( 6/200 or 12/400 g ; inhalation capsules ) ; and 50 g of salmeterol xinafoate ( diskus . 
 in phase 2 , an expert panel evaluated the instruments from the previous phase in order to assess their contents . 
 the expert panel comprised three pulmonologists , two pediatric pulmonologists , and two allergists , all of whom had extensive experience in the use of inhalers . 
 the proposed changes were judged in terms of the relevance of the items assessed by the preliminary instruments , the need for additions , deletions , or changes to improve the accuracy of the modified inhaler technique scores being evaluated . 
 after the changes had been systematized by consensus , new mdi and dpi technique scores were developed .   in phase 3 , 
 a new sample of patients using dpis , mdis , or both were randomly selected to participate in the study , the eligibility criteria being the same as those used in the previous phases . 
 they were given placebo - containing inhalers and no instructions on how to use them , being asked to use them in the same manner as they did their own inhalers . 
 each patient was simultaneously evaluated by two other pharmacists , who were blinded to the changes that had been made . 
 they used the instruments derived from phases 1 and 2 and were not allowed to communicate their decisions to one another . 
 after the evaluation , all patients were instructed on how to use inhalers correctly . 
 the median and interquartile range of the scores obtained in phase 3 allowed us to establish cut - off values and divide the patients into two groups : the group of patients who were able to use inhalers and that of those who were not . 
 the latter were then enrolled in our pharmaceutical care program .   in phases 1 and 2 
 , we used a convenience sample based on previous validation studies.14 
 17 
 19 the sample size for phase 3 was calculated on the basis of the hypothesis of an association of at least 60% between the scores given by the pharmacists . considering a power of 0.95 and a type i error of 0.05 
 , we calculated that a sample size of 31 assessments was required for each inhaler type . 
 the results obtained in phases 1 and 2 were described qualitatively . in phase 3 , 
 the mean scores were compared between the observers , agreements and disagreements being evaluated individually . 
 the major errors made during inhaler use ( as assessed by the observers ) were described by frequency .   in phase 3 , descriptive analysis of the absolute values of mdi and dpi technique scores by observer 
 was performed with the statistical package for the social sciences , version 18 ( spss inc . , 
 the degree of correlation between the scores by observer was assessed by cronbach 's alpha coefficient . 
 after assessment of data normality , the scores by observer were compared by means of the mann - whitney rank sum test , their correlation being determined by spearman 's test , with sigmastat software , version 3 ( systat software inc . , 
 the chi - square test was used in order to determine interobserver agreement in the classification of patients as being able or unable to use mdis and dpis . 
 pharmacists together identified the need for changes in the scoring systems and proposed two preliminary changes : removal of the overall score ; and removal of the suggestions section in order to reduce the number of sheets . 
 this resulted in a scoring system consisting of a single table for assessment of inhaler technique , including examples of correct / incorrect inhaler use . 
 interobserver disagreement in phase 1 was found in 19% and 25% of the dpi and mdi scores , respectively , indicating not only inhaler technique errors made by the patients but also interpretation errors made by the observers ( the pharmacists ) . 
 that level of disagreement was considered acceptable and was of relatively little significance , being clarified and resolved in the subsequent phase . 
 the expert panel suggested that two changes be made to the dpi technique score , and both suggestions were accepted : the inclusion of an item regarding patient head position ( at an angle of more than 90 or less than 90 ) , patients receiving 1 point for "" an angle of less than 90 "" and 0 points for "" an angle of more than 90 "" ; and the definition of tlc , which was included at the bottom of the page .   with regard to the mdi technique score 
 , the expert panel suggested that eight changes be made , and all of their suggestions were accepted : the item "" shaking the inhaler "" was changed from "" 3 times "" to "" twice or more "" ; the item "" mode of use "" was changed to "" inhaler use "" ; the score for the item "" in the mouth , with a spacer "" was changed to 1 because it provides a correct example of mdi use , as does the item "" out of the mouth , without a spacer "" ; the item "" time point "" was changed to "" time point ( rapid inhalation ) "" ; the items "" upon actuation "" and "" immediately after actuation "" have the same weight and were therefore merged ; the item "" only sprays it into the mouth and does not inhale "" was changed to "" the device is actuated directly into the mouth , and the patient does not inhale "" ; the item "" not completely "" was changed to "" insufficient "" , whereas the item "" adequately "" was changed to "" sufficient "" ; and the item "" interval "" was changed to "" time between mdi actuations "" . 
 according to all observers and experts , the resulting scoring systems were more practical ( chart 1 ) . 
 although the maximum total score remained the same ( i.e. , nine points ) for the instrument assessing mdi technique , it increased ( to six points ) for the instrument assessing dpi technique . according to the literature , a 20% variation in the total score is acceptable for a patient to be considered able to use an inhaler correctly . 
 for the present study , this means a minimum mdi technique score of 7.2 and a minimum dpi technique score of 4.8 ; however , it is not clear whether the aforementioned variation ( standard deviation ) takes into account whether a given patient received previous training in inhaler use . 
 of those patients , 39 were assessed with the mdi technique score and 50 were assessed with the dpi technique score , a total of 89 assessments being performed . 
 those assessments allowed us to identify the most common difficulties encountered / errors made during inhaler use . 
 the most relevant difficulty encountered during the use of both types of devices was the maintenance of tlc after a deep inhalation . 
 the number of errors made was higher during mdi use than during dpi use ( table 1 ) . 
 errors identified in more than 50% of all tests performed included & lt ; 60 s between mdi actuations , maintenance of tlc for & lt ; 10 s after inhalation , incorrect speed of inhalation , incorrect mdi use without a spacer , and not exhaling prior to inhalation .   among the patients who used a dpi , 
 the most common errors were maintenance of tlc for & lt ; 10 s , not exhaling prior to inhalation , and incorrect speed of inhalation ( in 75% , 62% , and 57% , respectively ) , as shown in table 2 .   of a sample of 72 patients , 60 
 did not hold their breath correctly , 58 inhaled too quickly , and 6 did not synchronize inhaler actuation with inhalation . 
 median mdi technique scores in phase 3 of the study were 4 and 5 for observers 1 and 2 , respectively ( table 3 ) . 
 median dpi technique scores were 4.5 and 5.0 for observers 1 and 2 , respectively . 
 the degree of correlation of the scores by observer was 0.97 , as assessed by cronbach 's alpha coefficient . 
 the correlation was statistically significant ( p & lt ; 0.0001 ) , as assessed by spearman 's correlation coefficient ( figure 1 ) . 
 there were no significant differences between the scores given by each observer , as assessed by the mann - whitney test . 
 all patients whose mdi and dpi technique scores were & lt ; 4 were enrolled in an educational program . 
 with regard to discriminant validity , there was good interobserver agreement in the classification of patients as being able / unable to use a dpi ( 50%/50% and 52%/58% ; p & lt ; 0.01 ) and an mdi ( 49%/51% and 54%/46% ; p & lt ; 0.05 ) . 
 the present study allowed assessment of the content validity , criterion validity , and discriminant validity of the dpi and mdi scores . 
 it also allowed identification of the major difficulties that patients encounter when using inhaler devices for the treatment of asthma . 
 finally , it allowed determination of cut - off scores to classify patients as being able or unable to use their inhalers , the latter patients being enrolled in an educational program .   to the best of our knowledge , 
 there is only one validation study of mdi technique scores ( in patients with asthma)12 and one of dpi technique scores ( in patients with copd).21 in addition , according to basheti et al.,22 although the use of device - specific checklists is the most feasible method for assessing inhaler technique , little evidence is available to assess the relative importance of different criteria . 
 divergence between the scoring systems for the same inhaler device in different studies makes direct comparison of results difficult . in our study , 
 mdi and dpi technique scores were validated in asthma patients , and the results allowed us to identify mdi and dpi technique errors and address them in a personalized manner , meaning that the instructions provided to patients were based on their own inhaler technique errors . 
 chart 1scoring systems modified and approved by an expert panel ( phase 2 ) and validated for use in brazil ( phase 3 ) . 
 evaluation criteriaerrors , % interval of & lt ; 60 s between doses ( patients are instructed to allow 60 s between doses)89maintenance of tlc for & lt ; 10 s after inhalation ( patients are instructed to hold their breath for 10 s after inhalation)83inadequate inspiratory flow rate ( patients inhaled too quickly , shallowly , or both)81incorrect mdi use without a spacer ( for patients who used a spacer)73not exhaling prior to inhalation ( patients did not exhale before using their mdis)59shaking the inhaler only once ( patients are instructed to shake their mdis at least twice)32not shaking the inhaler ( patients are instructed to shake their mdis at least twice)31incomplete inhalation ( patients did not perform a deep inhalation maneuver)18incorrect mdi position ( patients are instructed to sit down in such a way that their legs and the floor form a 90 angle)10lack of synchronization between mdi actuation and inhalation ( the device was actuated before the beginning of inhalation)8nasal breathing ( patients breathed through their nose during inhalation)5the device is actuated directly into the mouth , and patients do not inhale ( patients are instructed to place their mdis at a distance of three fingers ' breadth from their mouth and inhale after actuation)5others9.5mdi : metered dose inhaler . 
 our findings corroborate those of other studies.9 
 23 
 24 the inability to hold their breath for more than 10 s after inhalation was the major difficulty that our patients encountered during mdi and dpi use ( 83% and 75% , respectively ) . 
 manzella et al.12 assessed inhaler technique and reported that 69% of the patients studied did not hold their breath for at least 10 s. a review compiling the results of 12 studies ( including 955 patients ) identified the most common errors made by patients using inhaler devices , less than 10 s of breath - holding after inhalation having been observed in 26% . 
 a period of 10 s of breath - holding increases drug deposition in the lungs by allowing more time for the particles to settle.25 the author of the aforementioned review reported that 50% of the patients were able to hold their breath for 10 s between doses . in our study , less than 10 s of breath - holding after inhalation was the most common error made during mdi use , having been made by 89% of those who used mdis . 
 a period of 10 s of breath - holding is important to ensure that a greater quantity of drug reaches the airways.12 
 
 table 2proportion of inhaler technique errors as assessed by the dry powder inhaler score . 
 evaluation criteriaerrors , % maintenance of tlc for & lt ; 10 s after inhalation ( patients are instructed to hold their breath for 10 s after inhalation)75not exhaling prior to inhalation ( patients did not exhale before using their dpis)62inadequate inspiratory flow rate ( patients inhaled too quickly , shallowly , or both)57incomplete inhalation ( patients did not perform a deep inhalation maneuver)21incorrect dose preparation ( patients did not place the capsule inside their dpis , did not puncture the capsule before inhalation , or both)*18inhaling shallowly ( patients did not perform a deep inhalation maneuver)5exhaling into the device ( patients exhaled while their dpis were in their mouth)3nasal breathing ( patients breathed through their nose during inhalation)2inhaling irregularly ( patients were unable to inhale continuously)2dpi : dry powder inhaler . * for the dpi type used in the present study ( adaptation ) . 
 incorrect speed of inhalation was the third most common mdi technique error in our study and was reported by mcfadden as the third most common error in 12 studies , inappropriately rapid inspiratory flow rates having been observed in 19% of the total of patients . 
 this is a serious error , given that a slower inspiratory flow rate translates to higher lung drug deposition with the use of mdis.25 
  not exhaling prior to inhalation was an error made by 62% of the patients using dpis , being the second most common dpi technique error . 
 the lung volume at the beginning of inhalation interferes with drug deposition , which is why patients should exhale before inhaling.26 
 
 figure 1interobserver agreement . in a , metered dose inhaler ( mdi ) scores . in b , 
 incorrect speed of inhalation was the third most common dpi technique error ( 57% ) . in order to deliver medication , dpis depend on patient inspiratory flow . 
 if the flow rate ( volume / time ) is lower than required , the inhaled doses will be lower , and this contributes to treatment failure.8 
 27 
  sandrini et al.28 analyzed mdi use in a sample of patients , classifying inhaler technique as correct , slightly incorrect , moderately incorrect , or clearly incorrect . 
 the most common errors were placing the inhaler in the mouth ( 68.0% ) , inhaling before actuation ( 15.5% ) , inhaling too quickly ( 11.0% ) , and inhaling through the nose ( 9.0% ) . 
 although our method of evaluation differed from that used in the aforementioned study , our patients made the same errors as did those in that study . 
 dalcin et al.24 and souza et al.29 used checklists or forms in order to determine whether patients were using their inhalers correctly at work . 
 souza et al.29 found that 54.5% of the patients who used dpis did not exhale properly before inhaling the medication . 
 this finding is consistent with those of the present study , because that was the second most common error made by our patients . 
 dalcin et al.24 found that the patients who used mdis made more errors than did those who used other types of inhalers , a finding that is also consistent with those of the present study . 
 scoresdpi techniquemdi techniqueobserver 1observer 2observer 1observer 2median ( ir)4.5 ( 4.0/6.0)5.0 ( 3.0/5.0)4.0 ( 0.5/6.0)5.0 ( 1.0/6.5)minimum / maximum1/71/712/913/10dpi : dry powder inhaler ; mdi : metered dose inhaler ; and ir : interquartile range . 
 dpi : dry powder inhaler ; mdi : metered dose inhaler ; and ir : interquartile range .   in the aforementioned studies 
 , the instruments used allowed the authors to quantify and classify the errors made by patients . 
 we found no technical differences between the mdi and dpi techniques used at our institution and those described in the third brazilian consensus on asthma management6 or in the 2012 brazilian thoracic association guidelines for asthma management.1 however , in addition to providing a step - by - step description of inhaler technique , the scoring systems used in the present study are tools that health professionals can use in order to assess objectively and mathematically the errors that patients make when using inhaler devices . in addition , patients can be classified as able or unable to use their inhalers on the basis of their cut - off scores . 
 this allows health professionals to monitor improvements in inhaler technique in an objective manner . 
 our results show that the proportion of mdi technique errors was higher than that of dpi technique errors . 
 this difference might be related to the fact that the correct dpi technique is more easily understandable to patients than is the correct mdi technique . 
 the results showed that incorrect inhaler use affects drug efficacy , and the authors reported that assessment of inhaler technique is still considered irrelevant by many health professionals.21 
  in a study conducted in 2011 in the state of bahia , brazil , inhaler technique errors were assessed , and the proportion of errors was found to be low . 
 this was attributed to the fact that the patients in that study were monitored at an asthma referral center ; they periodically received instructions and refresher training on inhaler technique from a multidisciplinary team.5 
  one of the limitations of the present study is the lack of follow - up . 
 a follow - up evaluation might have allowed us to determine whether there were improvements in inhaler technique . 
 the present study was biased by the fact that the proportion of mdi technique errors was high , with median scores of 4 and 5 for observers 1 and 2 , respectively ; these scores are significantly different from the score of 7.2 suggested in the literature . given that interobserver agreement was high when a cut - off score of less than 4 was used - a finding that suggests that the mdi score has good accuracy - it can be inferred that many patients were classified as being able to use an mdi despite the fact that they did not obtain the minimum score predicted in theory , i.e. , 20% of a maximum total score of 9 . 
 this underscores the need for reassessing inhaler technique at each visit and for sequential use of the mdi score in a large population sample 
 .   the new mdi and dpi scores will allow the implementation of educational programs proposed in asthma management guidelines and strategies , which recommend that patients receive ongoing training in inhaler technique to ensure correct inhaler use . by providing pharmaceutical care 
 , pharmacists can instruct patients on how to use their inhaler devices correctly . by using our inhaler technique scores , 
 pharmacists can assess inhaler technique errors and determine whether patients are able to achieve an ideal level of technical correctness ( of 80% ) and maintain it throughout the educational program . 
 the present study showed that our inhaler technique scores can reveal exactly what it is that patients are doing incorrectly when using mdis or dpis so that the educational program during follow - up can focus on their errors rather than repeating what they already know . 
 inhalation is the most widely used approach for the treatment of asthma , and correct inhaler technique is directly related to the therapeutic efficacy of the drug . 
 therefore , correct inhaler technique is essential for the efficacy of pharmacological treatment.3 we believe that the present study is clinically relevant because it validated objective mdi and dpi technique scores that can reveal the major difficulties encountered by patients using mdis or dpis and provided cut - off scores that can be used in order to classify patients as being able or unable to use their inhalers . by using the validated scores 
 , health professionals will be able to identify and correct inhaler technique errors during visits and , as a result , improve inhaler use . 
 validar dois escores para medir a habilidade de pacientes em utilizar inaladores pressurizados ( ips ) ou inaladores de p ( ipos ) , verificar os erros mais comuns na sua utilizao e identificar os pacientes que necessitam de um programa educacional para o uso desses dispositivos . 
 este estudo foi realizado em trs etapas : validao da confiabilidade dos escores de uso dos dispositivos inalatrios ; validao do contedo dos escores utilizando - se uma amostra de convenincia ; e realizao de testes para a validao de critrio e a validao discriminante desses instrumentos em pacientes que preenchiam os critrios de incluso do estudo . 
 a amostra de convenincia foi composta por 16 pacientes , e houve discordncia interobservador em 19% e 25% para os escores de ipo e ip , respectivamente . 
 aps a anlise de expertos no assunto , os escores sofreram modificaes e foram aplicados em 72 pacientes . 
 a dificuldade mais relevante no uso de ambos os dispositivos foi a manuteno da capacidade pulmonar total aps inspirao profunda . 
 o grau de correlao dos escores por observador foi de 0,97 ( p & lt ; 0,0001 ) . 
 houve boa concordncia interobservador na classificao dos pacientes como aptos / no aptos para uso de ipo ( 50%/50% e 52%/58% ; p & lt ; 0,01 ) e de ip ( 49%/51% e 54%/46% ; p & lt ; 0,05 ) . 
 os escores validados permitem identificar e corrigir os erros da tcnica inalatria ao longo das consultas e , em consequncia , melhorar o manejo dos dispositivos para inalao . 
 a asma  uma doena crnica inflamatria que limita o fluxo areo e pode impedir que os pacientes exeram suas atividades dirias adequadamente.1 o tratamento farmacolgico  essencial2 e tem como objetivo alcanar e manter o controle clnico da doena.1 a inalao  a via mais utilizada para o tratamento da asma porque permite que os medicamentos alcancem seletivamente os pulmes , com maior concentrao do frmaco nas vias areas , reduzindo os efeitos adversos sistmicos.1 
 3 
 4 
  apenas metade dos asmticos utiliza , de fato e corretamente , a medicao prescrita.5 destaca - se que o baixo ndice de adeso est relacionado ao desconhecimento e / ou  dificuldade de administrao dos medicamentos para inalao.3 tais aspectos podem ser melhorados se a educao em asma ocorrer , sendo ela um componente estratgico para o controle da doena.6 
  a utilizao incorreta dos inaladores pode acarretar o insucesso teraputico por diminuir a concentrao do frmaco nas vias areas e contribuir para a no adeso ao tratamento , dificultando o controle clnico da asma.9 
  a frequncia de uso incorreto de inalador pressurizado ( ip ) varia entre 14% e 90% , com uma mdia estimada de 50%,10 acarretando deposio pulmonar do medicamento inferior a 20% . 
 um estudo com pacientes que faziam uso de corticoterapia inalatria ou oral apontou que mais de 50% dos pacientes que no eram aderentes ao tratamento apresentavam maior uso de broncodilatadores e nebulizaes , alm do aumento no nmero de hospitalizaes.11 
  a utilizao de instrumentos ( ou escores ) que meam e pontuem as dificuldades dos pacientes durante o uso dos seus medicamentos para inalao pode ser til para reduzi - las . 
 ao utilizar esses instrumentos , o profissional de sade pode elaborar um programa educacional direcionado para os principais erros cometidos pelo paciente , revertendo assim a baixa adeso ao tratamento . 
 existem alguns escores que avaliam o uso correto desses inaladores em portadores de molstias pulmonares.12 alguns deles avaliam os erros na utilizao desses dispositivos para inalao ; porm , no existe um escore considerado padro - ouro com essa finalidade . 
 tal instrumento poderia ser um diferencial na avaliao de pacientes que utilizam esses dispositivos.13 
  leal13 desenvolveu um escore para avaliao das dificuldades dos pacientes no uso de ips ; tal escore pontua acertos e erros na utilizao dos dispositivos , promovendo a valorao dos erros . 
 a partir daquele instrumento , santos et al.3 desenvolveram escores para a avaliao de inaladores de p ( ipo ) e ip . 
 esses instrumentos ainda no esto validados e so utilizados em nosso hospital para a mensurao da qualidade da tcnica de uso dos dispositivos para inalao por pacientes asmticos3 e portadores de dpoc .   a validao de um instrumento permite verificar a congruncia entre o escore que  utilizado para realizar uma mensurao e a realidade que  medida , aumentando a confiabilidade do mesmo.14 ao se utilizar instrumentos validados para medir a tcnica de inalao de pacientes asmticos , sero obtidos resultados mais fidedignos e com maior garantia da qualidade dos dados 
 .   os objetivos do presente estudo foram validar os escores para a avaliao da tcnica de uso de ips e ipos em portugus do brasil , verificar quais erros so mais comuns durante a utilizao desses dispositivos e identificar quais pacientes necessitam de sesses de treinamento para essa utilizao . 
 trata - se de um estudo aberto e prospectivo desenvolvido em um hospital universitrio de referncia . 
 os pacientes foram selecionados de acordo com os seguintes critrios de incluso : apresentar diagnstico de asma de acordo com os critrios da global initiative for asthma,15 estar em seguimento ambulatorial por pelo menos dois anos , ter idade entre 15 e 65 anos , ter cursado no mnimo quatro anos do ensino fundamental , ter sua capacidade auditiva preservada e concordar em participar do estudo , assinando o termo de consentimento livre e esclarecido . 
 pacientes que j haviam participado de programas educacionais sobre a utilizao de medicamentos para asma foram excludos . 
 inicialmente , foi realizada a validao da confiabilidade dos escores de uso dos inaladores ( etapa 1 ) e a de seu contedo ( etapa 2 ) . 
 na etapa 3 foram realizados os testes para a validao de critrio e a validao discriminante desses novos instrumentos . 
 na etapa 1 , dois farmacuticos convidados ( cegos em relao  metodologia do estudo ) avaliaram simultaneamente a tcnica de uso dos inaladores . 
 aplicaram os escores para ipo e ip , elaborados por santos et al.,3 para elencar os erros apresentados . 
 esses escores detalham o passo a passo da tcnica para cada tipo de inalador . o paciente recebe um ponto a cada etapa executada de modo correto , e , quando a tcnica  feita de modo incorreto , o paciente no recebe pontos . 
 se o paciente comete erros graves , esses so subtrados . a pontuao determina se o paciente est apto ou no para utilizar o dispositivo para inalao 
 . os farmacuticos foram treinados quanto  correta utilizao dos escores acima referidos , seguindo as diretrizes do iii consenso brasileiro de manejo da asma.16 para essa avaliao foram utilizados os seguintes medicamentos : a ) ip : dipropionato de beclometasona 250 g e salbutamol 100 g ; b ) ipo : budesonida 200 g e formoterol + budesonida ( 6/200 ou 12/400 g ; cpsulas inalatrias ) ; e xinafoato de salmeterol 50 g ( diskus . 
 na etapa 2 , os escores oriundos da etapa anterior foram avaliados por expertos com o objetivo de mensurar o contedo desses instrumentos . o grupo de expertos foi constitudo por trs pneumologistas , dois pneumopediatras e dois alergistas , todos com slida experincia no uso de inaladores .   as modificaes propostas foram 
 julgadas quanto  pertinncia dos itens medidos pelos escores preliminares , avaliando - se a necessidade de incluses , excluses ou alteraes para melhorar a acurcia dos escores modificados . 
 depois que as alteraes foram sistematizadas por consenso , procedeu - se  elaborao de novos escores para ips e ipos . 
 na etapa 3 , novos pacientes utilizando ipo , ip ou ambos foram convidados a participar do estudo , de forma aleatria , para uma avaliao individual , obedecidos os mesmos critrios de elegibilidade das etapas anteriores . 
 foi - lhes solicitado , sem nenhuma orientao prvia , que utilizassem inaladores contendo placebo e que realizassem a mesma tcnica da sua rotina de tratamento . 
 cada paciente foi avaliado simultaneamente por outros dois farmacuticos , cegos em relao s modificaes realizadas , utilizando os escores derivados das etapas 1 e 2 , sendo vetado qualquer tipo de contato entre os avaliadores sobre os valores emitidos . 
 aps as avaliaes , todos os pacientes foram orientados sobre o modo correto do uso dos inaladores . 
 a mediana e o intervalo interquartil dos escores obtidos nessa fase permitiram a determinao dos valores de corte e a diviso dos pacientes em dois grupos : pacientes aptos e pacientes no aptos a utilizar os dispositivos para inalao . 
 os pacientes no aptos foram ento encaminhados ao programa de ateno farmacutica existente em nossa instituio . 
 para as etapas 1 e 2 foi utilizada uma amostra de convenincia baseada em estudos de validao anteriores . 
 para a etapa 3 calculamos a amostra baseados na hiptese de que a associao entre os escores pontuados pelos farmacuticos seria de pelo menos 60% . 
 considerando um poder de 0,95 e um erro tipo i de 0,05 , a amostra indicada 
 na etapa 3 , os valores mdios dos escores foram comparados entre os observadores , avaliando - se as concordncias e divergncias individualmente . 
 na etapa 3 , a anlise descritiva dos valores absolutos dos escores para cada dispositivo , por observador , foi realizada usando o programa statistical package for the social sciences , verso 18 ( spss inc . , chicago , il , eua ) . 
 os graus de correlao entre os escores e por observador foram avaliados utilizando - se o coeficiente alfa de cronbach . 
 aps a avaliao de normalidade , a comparao entre os escores por observador foi realizada atravs do teste de mann - whitney ( rank sum ) e sua correlao pelo teste de spearman , utilizando o programa sigmastat , verso 3 ( systat software inc . , san jose , ca , eua ) . 
 o teste do qui - quadrado foi utilizado para avaliar a concordncia dos grupos de pacientes ( aptos ou no aptos para utilizao dos inaladores ) entre os avaliadores . 
 farmacuticos em conjunto identificaram a necessidade de alteraes em determinados campos dos escores e propuseram duas modificaes preliminares : a retirada da pontuao geral dada ao paciente e a retirada do campo de sugestes para diminuir o nmero de folhas do escore . 
 foi criado um escore com uma nica tabela para a avaliao da tcnica do uso dos inaladores , com os acertos / erros que os pacientes poderiam cometer . a discordncia entre os avaliadores na etapa 1 ocorreu em 19% e 25% em relao aos escores para ipo e ip , respectivamente , indicando no somente erros 
 essas discordncias foram consideradas aceitveis na aplicao dos escores e proporcionalmente pouco significativas , sendo esclarecidas e dirimidas na etapa seguinte . 
 na avaliao dos escores pelos expertos , foram sugeridas e acatadas duas modificaes no escore para ipo : acrescentar um campo sobre a posio da cabea do paciente ( ngulo maior ou menor que 90 ) , sendo atribudo um ponto para "" ngulo menor que 90 "" e zero para "" ngulo maior que 90 "" ; e o significado da sigla cpt , adicionado ao final do escore . 
 em relao ao escore para ip , os especialistas fizeram oito sugestes de modificaes que foram acatadas : o item "" agitao do dispositivo "" foi alterado de "" 3 vezes "" para "" 2 vezes ou mais "" ; o campo "" modo "" foi alterado para "" modo de uso "" ; a pontuao do item "" dentro da boca com espaador "" foi alterada para um ponto por ser to correto quanto  forma de utilizao do inalador no item "" fora da boca sem espaador "" ; no campo "" momento "" , foi adicionado o item "" inspirao rpida "" ; os itens "" junto ao acionamento "" e "" logo depois do acionamento "" possuem o mesmo peso e foram unificados ; o item "" s jogar na boca ou no inspirar "" foi alterado para "" acionamento direto na boca e no inspirar "" ; no campo "" profundidade "" o item "" no completamente "" foi modificado para "" insuficiente "" , enquanto o item "" adequadamente "" foi alterado para "" suficiente "" ; e o campo "" intervalo "" foi alterado para "" intervalo entre os acionamentos "" . 
 os escores resultantes foram considerados mais prticos por avaliadores e expertos ( quadro 1 ) . o novo escore para ip manteve a pontuao total mxima ( nove pontos ) , mas houve um aumento na pontuao do escore novo para ipo ( seis pontos ) . 
 de acordo com a literatura , para se considerar que um paciente conhece e domina a utilizao correta do tipo de inalador , o escore obtido por ele pode apresentar uma variao aceitvel de 20% . 
 isso significa uma pontuao mnima de 7,2 e de 4,8 para os escores para ip e ipo , respectivamente ; entretanto , no est definido se essa variao ( desvio - padro ) leva em considerao se o paciente foi treinado anteriormente para o uso do inalador . 
 participaram da etapa 3 do estudo 72 pacientes , com mdia de idade de 41 anos , sendo 82% do sexo feminino . 
 esses pacientes geraram 89 avaliaes , sendo 39 pacientes avaliados com o escore para a tcnica de uso de ip e 50 pacientes com o escore para a de ipo . 
 essas avaliaes permitiram elencar as principais dificuldades / erros dos pacientes no uso de dispositivos para inalao . 
 o erro mais relevante entre os pacientes avaliados quanto ao uso de ambos os tipos de inaladores foi a falta de manuteno da cpt aps a inspirao profunda . 
 observou - se um maior nmero de erros entre os pacientes com o uso do ip ( tabela 1 ) . 
 o intervalo entre as doses & lt ; 60 s , a manuteno da cpt & lt ; 10 s aps a inalao , a velocidade da inspirao inadequada , o uso inadequado do ip sem espaador e a ausncia de realizao de expirao antes da inalao foram erros cometidos em mais de 50% dos testes realizados . 
 entre os pacientes que utilizaram ipo , os erros mais comuns foram a manuteno da cpt & lt ; 10 s , a ausncia de expirao antes da inalao e a velocidade inadequada da inspirao ( em 75% , 62% e 57% , respectivamente ) , conforme demonstrado na tabela 2 
 . de uma amostra de 72 pacientes , 60 pacientes no realizaram a pausa inspiratria de maneira correta ; 58 pacientes realizaram a inspirao muito rpida ; e 6 pacientes no sincronizaram o incio da inspirao com o acionamento do dispositivo . 
 nas mensuraes para o escore para ip realizadas na etapa 3 , os valores encontrados por avaliador , em mediana , foram 4 e 5 pontos para os observadores 1 e 2 , respectivamente , conforme mostrado na tabela 3 . 
 o grau de correlao entre os escores avaliados por observador , utilizando - se o coeficiente alfa de cronbach , foi de 0,97 . 
 essa correlao foi estatisticamente significativa usando - se a correlao de spearman ( p & lt ; 0,0001 ) , como se observa na figura 1 . 
 o teste de mann - whitney confirmou que os valores atribudos aos escores pelos dois avaliadores no foram estatisticamente diferentes . 
 foram encaminhados ao programa de educao pacientes com valor de escore & lt ; 4 pontos tanto para os escores para ip quanto para ipo . 
 esse foi o menor valor inteiro da mediana de avaliao concordante entre os dois observadores . 
 para a validao discriminante , encontrou - se uma concordncia entre os grupos de pacientes aptos e de no aptos ( necessitando de um programa de educao ) indicados pelo observador 1 e pelo observador 2 , tanto para o escore para ipo ( 50%/50% e 52%/58% ; p & lt ; 0,01 ) quanto para o escore para ip ( 49%/51% e 54%/46% ; p & lt ; 0,05 ) . 
 o presente estudo possibilitou a validao de contedo , de critrio e de discriminante dos escores para ipo e ip , permitindo tambm elencar as maiores dificuldades encontradas pelos pacientes no uso dos dispositivos para inalao no tratamento da asma , alm de determinar uma nota de corte , classificando os pacientes como aptos a utilizar seus inaladores ou identificar aqueles que necessitavam ser encaminhados para um programa educacional . 
 no melhor do nosso conhecimento atual , encontramos na literatura pesquisada um estudo que validou escores para ip para pacientes asmticos12 e , no caso de escores para ipo , somente um estudo com pacientes com dpoc.21 alm disso , segundo basheti et al.22 apesar de o uso de check - lists para um inalador especifico ser o mtodo mais vivel de avaliao , existem poucas evidncias para avaliar a importncia relativa dos diferentes critrios . as divergncias dos sistemas de pontuao para o mesmo dispositivo para inalao em diferentes estudos dificultam a comparao direta dos resultados . 
 considerando que o nosso estudo validou escores tanto para ip quanto para ipo para pacientes asmticos , os resultados obtidos nos permitiram pontuar erros em ambos e trat - los de forma personalizada , de modo que todos os pacientes puderam ser orientados sobre o modo correto de utilizao desses dispositivos com base em seus erros de execuo da tcnica . 
 quadro 1escores modificados e aprovados pelos expertos ( etapa 2 ) e validados para uso em portugus do brasil ( etapa 3 ) . 
 tabela 1proporo de erros realizados pelos pacientes na avaliao do escore da tcnica do inalador pressurizado.critrios avaliadoserros , % intervalo entre as doses & lt ; 60 s ( paciente orientado a aguardar 60 s entre uma dose e outra)89manuteno da cpt & lt ; 10 s aps a inalao ( paciente orientado a prender a respirao por 10 s aps a inalao)83fluxo inspiratrio inadequado ( inspirao rpida e / ou curta)81uso inadequado do ip sem o espaador ( para os pacientes que utilizavam o espaador)73ausncia de expirao antes da inalao ( no expirar antes do uso do ip)59agitao do inalador apenas uma vez ( paciente orientado a agitar o ip no mnimo 2 vezes)32no agitar o inalador ( paciente orientado a agitar o ip no mnimo 2 vezes)31manobra inspiratria incompleta ( paciente no realiza uma inspirao profunda)18posio do ip inadequada ( paciente orientado a sentar - se de modo que suas pernas formem um ngulo de 90 com o cho)10ausncia de sincronizao entre o acionamento do ip e incio da inspirao ( paciente no sincroniza o disparo do ip com o incio da inspirao)8respirao nasal ( paciente realiza a respirao nasal durante a inalao)5acionamento do inalador direto na boca e ausncia de inspirao ( paciente orientado a distanciar o ip trs dedos da boca e realizar a inspirao aps o acionamento)5outros9,5ip : inalador pressurizado 
 
 tabela 2proporo de erros realizados pelos pacientes na avaliao do escore da tcnica do inalador de p.critrios avaliadoserros , % manuteno da cpt & lt;10 s ( paciente orientado a prender a respirao por 10 s aps a inalao)75ausncia de expirao antes da inalao ( no expirar antes do uso do ipo)62fluxo inspiratrio inadequado ( inspirao rpida e / ou curta)57profundidade incompleta ( paciente no realiza uma inspirao profunda)21preparo da dose incorreta ( paciente no coloca a cpsula dentro do ipo e / ou no fura a cpsula antes de realizar a inalao)*18inspirar levemente ( paciente no realiza uma inspirao profunda)5expirar dentro do ipo ( paciente realiza a expirao com o ipo na boca)3respirao nasal ( paciente realiza a respirao nasal durante a inalao)2inspirar irregularmente ( paciente no consegue realizar a inspirao de modo contnuo)2ipo : inalador de p . * relativo ao tipo de ipo utilizado no estudo ( adaptao ) . 
 a capacidade de prender a respirao por mais de 10 s aps a inalao foi a dificuldade mais importante entre os pacientes do nosso estudo , tanto para ip como para ipo ( 83% e 75% , respectivamente ) . 
 manzella et al.12 avaliaram a tcnica inalatria e relataram que 69% dos pacientes avaliados no prendiam a respirao por no mnimo 10 s. uma reviso que compilou resultados de 12 estudos ( 955 pacientes includos ) classificou os erros mais cometidos pelos pacientes , sendo que a dificuldade de prender a respirao por 10 s aps a inalao ocorria em 26% da populao . 
 esse tempo determinado de prender a respirao  importante para que ocorra uma maior sedimentao das partculas do medicamento nos pulmes.25 
  o autor da reviso tambm relatou que 50% dos pacientes faziam a pausa de tempo necessria entre as doses . 
 no nosso estudo , esse foi o erro mais frequentemente ( 89% ) encontrado em relao ao escore para ip . 
 esse passo da tcnica  importante para garantir que uma maior quantidade do medicamento alcance as vias areas.12 
 
 tabela 3valores dos escores encontrados por instrumento de acordo com o observador.valoresipoipavaliador 1avaliador 2avaliador 1avaliador 2mediana ( ii)4,5 ( 4,0/6,0)5,0 ( 3,0/5,0)4,0 ( 0,5/6,0)5,0 ( 1,0/6,5)mnimo / mximo1/71/712/913/10ipo : inalador de p ; ip : inalador pressurizado ; e ii : intervalo interquartil . 
 ipo : inalador de p ; ip : inalador pressurizado ; e ii : intervalo interquartil . 
 a velocidade de inalao inadequada , terceira dificuldade mais comumente encontrada em nosso estudo para ip , foi igualmente relatada por mcfadden como a terceira dificuldade mais frequentemente observada em 12 estudos , sendo que , no total , 19% dos pacientes realizavam a inalao de forma muito rpida . 
 esse erro  grave , pois quanto mais devagar  a taxa de medicamento inalado , maior ser a deposio pulmonar desse medicamento com o uso de ip.25 
  em relao ao ipo , a ausncia de expirao antes da inalao foi verificada em 62% dos casos , sendo a segunda dificuldade mais frequente para esse tipo de inalador . 
 o volume pulmonar existente no incio da inspirao interfere na deposio da droga e  por isso que se recomenda a realizao da expirao antes do incio da inalao.26 
  a velocidade de inspirao inadequada foi o terceiro erro mais comumente encontrado ( 57% ) no uso de ipo . 
 no caso desses dispositivos para inalao , a sada do medicamento  decorrente do fluxo inspiratrio que o paciente realiza . 
 se o fluxo ( volume / tempo ) for menor que o necessrio , as doses inaladas sero reduzidas , e esse  um fator importante para o insucesso teraputico . 
 sandrini et al.28 analisaram o uso de ip em uma amostra de pacientes , classificando a tcnica de inalao em correta , pouco errada , com erro intermedirio e muito errada . 
 os autores classificaram a tcnica de 48% dos pacientes como no correta . os erros mais encontrados foram colocao do inalador dentro da boca ( 68,0% ) ; inspirao antes de acionar o jato ( 15,5% ) ; inspirao rpida ( 11,0% ) ; e inspirao pelo nariz ( 9,0% ) . 
 esses erros tambm foram encontrados em nossos pacientes , mesmo com o modo de avaliao diferente do utilizado naquele estudo . 
 dalcin et al.24 e souza et al.29 utilizaram listas ou fichas de controle para verificar a utilizao adequada dos inaladores pelos pacientes em seus trabalhos . 
 souza et al.29 encontraram que 54,5% dos pacientes avaliados que faziam uso de ipo no realizavam a expirao antes da inalao de forma adequada . 
 esse achado corrobora o nosso , pois esse foi o segundo erro mais comumente encontrado na avaliao de nossos pacientes . 
 j dalcin et al.24 encontraram um maior nmero de erros nos pacientes que utilizavam ip quando comparados aos que usavam outros inaladores , sendo esse achado tambm igual ao nosso . 
 fato que , nos trabalhos acima citados , os instrumentos utilizados permitiram aos autores quantificar e classificar os erros cometidos pelos pacientes . 
 comparando as tcnicas de inalao para o uso de ip e ipo publicadas no iii consenso de asma16 e nas diretrizes para o manejo de asma de 2012,1 verifica - se que as orientaes no diferem tecnicamente das utilizadas em nossa instituio . 
 entretanto , verificamos que os escores utilizados no presente estudo , alm de especificarem o passo a passo da tcnica de inalao , so uma ferramenta para que o profissional de sade possa mensurar , de forma objetiva e matemtica , os erros cometidos pelos pacientes . 
 alm disso , eles possibilitam verificar se o paciente est apto ou no a utilizar seu inalador atravs da especificao de uma nota de corte . 
 isso tambm permite que o profissional de sade acompanhe objetivamente a evoluo do paciente quanto a sua tcnica de inalao . 
 nossos resultados demonstram que a proporo de erros no escore para ip foi maior do que no para ipo . 
 essa diferena na proporo de erro entre os dispositivos pode estar associada  maior facilidade na compreenso da tcnica de uso de ipo em detrimento quela de ip por parte dos pacientes . 
 destacamos que lavorini et al.21 realizaram uma reviso da literatura com 47 artigos que analisavam a tcnica do uso do ipo . 
 os resultados demonstram que o uso incorreto de inaladores influencia a eficcia da droga , e os autores destacam que a avaliao do uso correto dos inaladores ainda  considerada irrelevante por muitos profissionais de sade.21 
  em um estudo realizado em 2011 na bahia , foram avaliados os erros que os pacientes cometiam ao utilizar os seus inaladores ; encontrou - se baixa proporo de erros na tcnica inalatria . 
 tais resultados foram associados ao acompanhamento em um centro de referncia em asma que os pacientes daquele estudo recebiam ; eles participavam de peridicas orientaes e revises acerca da tcnica inalatria , realizadas por uma equipe multiprofissional.5 
  como limitao do estudo , pode - se indicar a falta de seguimento dos pacientes avaliados aps o mesmo . 
 tal acompanhamento poderia permitir que se determinasse a evoluo da tcnica do uso dos inaladores pelos pacientes . 
 um vis do presente estudo ocorreu porque os pacientes apresentaram um alto ndice de erros ao utilizarem ip ( mediana de 4 e 5 pontos para os avaliadores 1 e 2 , respectivamente ) , distantes dos valores tericos de 7,2 pontos sugeridos pela literatura . 
 como os resultados obtidos pelos dois avaliadores foram altamente concordantes usando uma nota de corte menor que 4 pontos , sugerindo boa acurcia do escore , pode - se inferir que os observadores classificaram muitos pacientes como aptos para o uso do ip apesar de no terem atingido a pontuao mnima prevista em teoria ( 20% de 9 pontos mximos ) . 
 isso refora a necessidade de reavaliao do uso do inalador a cada consulta e a utilizao sequencial desse escore em uma grande amostra populacional . 
 estes novos escores permitiro a execuo de programas educacionais , propostos em diretrizes e estratgias para o tratamento da asma . 
 segundo esses documentos , os pacientes precisam receber treinamento contnuo sobre a tcnica de inalao para que se possa garantir a utilizao correta do inalador . o farmacutico , por meio da ateno farmacutica , pode 
 orientar o paciente quanto ao manejo dos dispositivos para inalao e , ao utilizar nossos escores , pode quantificar os acertos / erros e mensurar se o paciente alcana e mantm o ideal de 80% de correo na tcnica ao longo do programa de educao . os escores no presente estudo demonstraram ser capazes de especificar em qual passo os pacientes esto errando , permitindo direcionar o programa educativo durante o acompanhamento sem repetir o que paciente j sabe . 
 a inalao  a via mais utilizada para o tratamento da asma , e a realizao de uma tcnica correta na utilizao dos dispositivos est diretamente relacionada  eficcia teraputica do medicamento . 
 sendo assim , a execuo correta da tcnica de inalao  essencial para que o tratamento farmacolgico seja eficaz.3 acreditamos que o presente estudo , ao validar escores objetivos para o uso de ip e de ipo que especificam as maiores dificuldades encontradas pelos pacientes no uso dos dispositivos e uma nota de corte , assim como classifica os pacientes como aptos a utilizar seus inaladores ou no , pode ser de relevncia clnica . 
 o profissional de sade ser capaz , por meio desses escores validados , de identificar e corrigir os erros do paciente ao longo das consultas e , consequentemente , melhorar o manejo dos inaladores .","abstractobjective : to validate two scores quantifying the ability of patients to use metered dose inhalers ( mdis ) or dry powder inhalers ( dpis ) ; to identify the most common errors made during their use ; and to identify the patients in need of an educational program for the use of these devices . 
 methods : this study was conducted in three phases : validation of the reliability of the inhaler technique scores ; validation of the contents of the two scores using a convenience sample ; and testing for criterion validation and discriminant validation of these instruments in patients who met the inclusion criteria . 
 results : the convenience sample comprised 16 patients . 
 interobserver disagreement was found in 19% and 25% of the dpi and mdi scores , respectively . 
 after expert analysis on the subject , the scores were modified and were applied in 72 patients . 
 the most relevant difficulty encountered during the use of both types of devices was the maintenance of total lung capacity after a deep inhalation . 
 the degree of correlation of the scores by observer was 0.97 ( p & lt ; 0.0001 ) . 
 there was good interobserver agreement in the classification of patients as able / not able to use a dpi ( 50%/50% and 52%/58% ; p & lt ; 0.01 ) and an mdi ( 49%/51% and 54%/46% ; p & lt ; 0.05 ) . 
 conclusions : the validated scores allow the identification and correction of inhaler technique errors during consultations and , as a result , improvement in the management of inhalation devices .",pubmed
"bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria that deserve considerable interest for their use as natural and nontoxic food preservatives as well as for their potential use in human and veterinary applications , and in the animal production field . in 1976 
 tagg et al . defined bacteriocins as proteinaceous compounds that kill closely related bacteria but it is now recognized that bacteriocins can also have activity across genera ( broad spectrum )     [ 24 ] . the antimicrobial sensitivity of a target strain to any given bacteriocin may depend on the ecological conditions under which it is grown as variations in salt concentration , ph , the presence of membrane disrupting molecules , or inducing cultures , and a large number of other environmental parameters can have a significant impact [ 5 , 6 ] . 
 the classification of gram - positive bacteriocins is rendered difficult because of their heterogeneity and thus , as the number of gram - positive bacteriocins identified has continued to increase , classification schemes have had to continuously evolve . 
 klaenhammer   suggested four classes of bacteriocins including class i or lantibiotics , which are small membrane - active peptides ( <  5  kda ) containing the unusual amino acids lanthionine or -methyl lanthionine ( hence the name lantibiotics ) and dehydrated residues . 
 class ii is defined as small heat - stable nonlanthionine containing membrane active peptides characterized by the presence of a gly - gly processing site in the bacteriocins precursor , the presence of amphiphilic helices with varying amounts of hydrophobicity and moderate to high - heat stability . 
 these were further subdivided into three subgroups named subclass iia , which are listeria active peptides and ygngv box in the n - terminal ; subclass iib : poration complexes consisting of two proteinaceous peptides for activity      and subclass iic , which contains thiol activated peptides requiring reduced cysteyl residues for activity . 
 class iii is large heat labile proteins , often with enzymatic activity and finally class iv , which is composed of complex proteins . 
 many enterococcus strains from various ecosystems were characterized as antagonists of broad range of foodborne pathogens     [ 12 , 13 ] . 
 the bacteriocins produced by enterococcal strains displayed heterogeneous structures and differences in their mode of action . 
 most of bacteriocin producers enterococcal strains were isolated not only from different types of food , but also from broilers     [ 2 , 4 ] and animal secretions . 
 as reported in the literature , some of these bacteriocins exhibited high activities against broad spectrum of bacteria including gram - positive bacteria , like staphylococcus and streptococcus and gram - negative bacteria like campylobacter or salmonella , strains of medical interest     [ 2 , 4 , 16 ] . 
 the ability to produce such a biocompound may play a role in providing an ecological advantage on nonbacteriocin producer strains . 
 previously , we have isolated a strain of e. faecalis from chicken drop   which appeared to be a bacteriocin producer . in this work , 
 we have undertaken the purification and characterization of the mode of action of this peptide named enterocin s37 . 
 e. faecalis s37 was isolated from poultry feces collected in a french farm located in ancenis ( west of france ) . 
 e. faecalis s37 showed in first instance an antagonism directed against strains belonging to listeria and campylobacter species . 
 the antibacterial activity of enterocin s37 was assessed against the gram - negative and gram - positive bacteria listed in table 1 . 
 the cell - free supernatant ( cfs ) and partially purified enterocin s37 , by ammonium sulfate precipitation and two successive reversed phase - high performance liquid chromatography ( rp - hplc ) steps , were used in order to assess the antagonism toward the target strains listed in table 1 . 
 e. faecalis s37 , e. faecalis jh2 - 2 , lactobacillus brevis f1.114 , lb . 
 enterica serotype enteritidis cip 81.3 , staphylococcus epidermidis cip 68.21 , bacillus cereus cip 78.3,and leuconostoc mesenteroides subsp . 
 mesenteroides dsm 20240 were grown on brain heart infusion broth ( bhi ) medium ( aes , bruz france ) , whilst l. monocytogenes egde and l. innocua f were grown on elliker medium ( aes ) . 
 for this purpose , 100  l from overnight culture of targeted strains was added to 20  ml of elliker or bhi soft agar medium already containing 0.8% ( w / v ) of agar ( biokar diagnostics , beauvais , france ) . 
 wells were performed in solid agar and 50  l of each sample tested was poured into the wells . 
 the petri dishes were left at room temperature , in sterile conditions , for 1  h before incubation for 18  h at adequate temperature according to the target strain to be tested . 
 after this period of incubation , the antibacterial activity was detected by observing the inhibition zones around the well containing the cfs . 
 it should be noted that supernatant was obtained by centrifuging at 15000  g during 30  min at 4c , an overnight culture of e. faecalis grown at 37c for 18 to 24  h , on de man rogosa sharpe ( mrs ) medium   ( aes ) . 
 the cfs was neutralized by addition of 1  n naoh and filtered through 0.2  m membrane ( sartorius , goettingen , germany ) . 
 serial two fold dilution of samples was performed with phosphate buffer ( 25  mm k2hpo4 , 25  mm kh2po4 , ph 7 ) filtered through 0.2  m membrane ( sartorius ) . 
 these samples were tested by agar diffusion test on l. monocytogenes egde strain , as described previously . 
 one arbitrary unit was defined as the reciprocal of the lowest dilution that did not show growth of the target strain . 
 the cfs neutralized and filtered ( 400  ml ) was precipitated with ammonium sulfate to 80% saturated solution . 
 three distinct fractions were recovered and were resuspended in 40  ml of phosphate buffer ( 25  mm k2hpo4 , 25  mm kh2po4 , ph 7 ) ; the active one was applied onto rp - hplc column ( atoll 15 mp3 ; 250  mm  10  mm interchim , montluon , france ) previously equilibrated with solvent a ( 0.05% , v / v , trifluoroacetic acid [ tfa ] ) . 
 elution was performed by using a gradient from 0% solvent b ( 0.045% , v / v , tfa , 80% acetonitrile ) to 100% solvent b in 45  min . 
 the flow rate was adjusted to 3  ml.min   and the absorbance was recorded at 220 and 280  nm 
 the acetonitrile was removed from the active fractions by speed - vac concentrator ( sc110a , savant ) and ph was adjusted to 6.8 using phosphate buffer ( 50  mm ; ph 7 ) . 
 the active fractions were pooled and the purification of the bacteriocin was continued by rp - hplc on a waters alliance apparatus with millennium software ( millford , ma , usa ) . concentrated bacteriocin ( 80  l ) was injected into an analytical c18 column ( symmetry 300 ; 5 m ; spherical 300  ; 150  4.6  mm , waters , guyancourt , france ) equilibrated in 0% solvent c ( 0.045% tfa in h2o ) and 50% solvent d ( 80% acetonitrile , 20% h2o , 0.05% tfa ) . the elution was performed with a linear gradient from 30% to 100% solvent d in 30  min . 
 the eluted peaks were detected by spectrophotometry measuring the absorbance between 210 and 300  nm with a photo diode array detector ( pda 996 ; waters ) and collected manually . 
 the effect of -chymotrypsin ( 1  mg      ml ) , papain ( 1  mg      ml ) , proteinase k ( 0.5  mg / ml ) , lysozyme ( 2  mg      ml ) , and lipase ( 1  mg      ml ) ( all enzymes were from sigma - aldrich , germany ) on the antibacterial activity of enterocin s37 was checked by the agar diffusion assay as described by batdorj et al . . 
 thus , samples ( cfs ) of 10 ml were adjusted to ph values ranging from 3 to 10 with 1  n hcl or 1  n naoh . 
 the obtained fractions were incubated for 2  h at 37c . after this period of incubation , 
 the antibacterial activity of each treated sample was determined by the agar well diffusion assay . 
 the stability of enterocin s37 was studied at high temperature by heating 100  l of supernatant at 80c for 15  min , 80c for 1  h and 90c for 15  min . after cooling in ice , 
 each sample was tested for its antagonism by agar well diffusion assay . in order to determine the apparent molecular mass of enterocin s37 
 the part of the gel containing the molecular markers and the samples were stained by silver nitrate coloration method , and the remaining part , containing only samples , was extensively washed with regularly replaced sterile milliq water . 
 second part of the gel was used for direct detection of antimicrobial activity by overlaying with soft agar ( 0.8% ) seeded with indicator strain l. monocytogenes egde and incubated overnight at  37c . 
 resistant variants to enterocin s37 were developed by stepwise culture of strain of l. monocytogenes egde at 30c in tryptic soy broth ( biokar diagnostics ) supplemented with 0.6% ( w / v ) yeast extract ( organotechnie sa , la courneuve , france ) and called tsye broth ( tsyeb ) . 
 enterocin s37 was added at 2- 4- 6- 8- and 10-fold the minimal inhibitory concentration ( mic ) . 
 the mic was determined for l. monocytogenes egde according to naghmouchi et al .    using microplate reader elx808 ( biotek instruments , bad friedrichshall , germany ) . 
 after growth of 50 generations in tsyeb at 30c , the strain was inoculated in the tsyeb containing enterocin s37 at 10-fold mic . 
 l. monocytogenes egde and its bacteriocin - resistant variant were grown at 30c for 24  h in tsyeb in the presence of enterocin s37 at 5  g      mland k channel modulators such as pinacidil ( pi ) at 10  g      ml , cromakalim ( cro ) at 10  g      ml and glipizide ( gli ) at 10  g      ml combination of enterocin s37 with each of k channel modulator was also realized in similar conditions . 
 growth was monitored by measuring the optical density at 595  nm ( od595 ) of 250  l of freshly inoculated medium placed into microplate wells . the inhibitory activity ( ia ) of enterocin s37 in presence or absence of each k channel modulator was calculated as described by naghmouchi et al . 
 clearly , a percentage of ia = 100  100 ( od595(x)/od595(i ) ) ( during exponential growth ) . 
 the value ( x ) was the culture containing inhibitor and ( i ) was the uninhibited control culture . 
 data were expressed as the percent change in ia obtained with the enterocin s37/k channel modulator . 
 cfs of e. faecalis s37 displayed activities against e. faecalis jh2 - 2 , lb . 
 brevis f145 , l. monocytogenes egde , and l. innocua f. however , none antibacterial activity was detected against the remaining strains listed in table 1 . 
 surprisingly , the anti - campylobacter activity previously observed   was not recovered in this study arguing on instability trait ( table 1 ) . 
 furthermore , the activity observed against strains of enterococcus genus is interesting in order to generate novel knowledge on immunity proteins and cross - resistance within this genus . 
 cfs of e. faecalis s37 displayed activity against the target strains e. faecalis jh2 - 2 , lb . 
 brevis f145 , l. monocytogenes egde and l. innocua f even after treatments with lipase ( 1  mg  ml ) and lysozyme ( 2  mg  ml ) . the antibacterial activity observed upon lipase and lysozyme treatments was similar to that observed with the untreated supernatant used as positive controls . however , treatment with proteolytic enzymes , such as -chymotrypsin , papain , and proteinase k resulted in the loss of the bacteriocin activity ( table 2 ) . 
 the stability of antibacterial activity of enterocin s37 remained intact after heating treatments ( 80c for 1  h and 90c for 15  min ) and at ph values ranging from 4 to 9 ( table 2 ) . 
 attempts to reduce putative disulfide bridge ( s ) of enterocin s37 were performed with dithiothreitol ( sigma , germany ) ; the experiment was not conclusive because of toxicity of this compound , even at low concentrations , towards the target strains . 
 the bacteriocin was purified by a three - step method including ammonium sulfate precipitation and two rp - hplc . the first stage consisted in an ammonium sulfate precipitation at 80% , which allowed concentrating the active fraction 10-fold ( v / v ) ; subsequently , 
 active fractions ( those exhibiting antibacterial activity ) were eluted at 60% of elution buffer containing 80% acetonitrile , 20% h2o , and 0.05% tfa . 
 the purity of each active fraction collected after each step was checked on sds - page ( table 3 , figure 1 ) . 
 bacteriocin appeared to have a molecular weight comprised between 4 and 6  kda ( figure 1(a ) ) , and fitting thereof with the inhibition zone observed on sds - page ( figure 1(b ) ) . 
 for this purpose , the electrophoresis gel obtained after migration under nondenaturing conditions was plated directly on elliker soft agar ( 0.9% ) inoculated with l. monocytogenes egde with 1% volume transfer ( v / v ) . 
 the antimicrobial activity of each sample , obtained during purification process , was also checked by agar diffusion test on petri dish ( figure 1(c ) ) both k channel activators ( pinacidil and cromakalim ) and inhibitor ( glipizide ) affected differently the growth of l. monocytogenes egde and its bacteriocin - resistant variant to enterocin s37 obtained in this research . as shown in figure 2 , the highest effect was attributed to the combination of enterocin s37 ( 5  g      ml ) and pinacidil ( 10  g      ml ) . 
 effect of the combinations of enterocin s37 with cromakalim ( 10  g      ml ) and glipizide ( 10  g      ml ) was less important on l. monocytogenes egde bacteriocin - resistant variant . 
 the predominant habitat is the gastrointestinal tract of animals and humans where they can be found in numbers as high as 10  cfu / g of feces     . 
 enterococci were found in foods of animal origin ( milk , cheese , and fermented sausages ) , vegetables and plant materials     . 
 many enterococci were reported to produce bacteriocins called enterocins , which are various , having great diversity in their structure , and active against numerous microorganisms , especially foodborne pathogens and against microorganisms of environmental and medical interests     [ 2 , 4 , 14 , 23 ] . 
 have studied a set of 139 strains of e. faecium and e. faecalis for their capabilities to produce enterocins and they concluded that 51% of isolates were producers of enterocins and 46% of them were able to produce more than one enterocin . 
 the strains producing enterocins were carrying at least one structural gene coding for the most known enterocins ( enta , entp , entl50ab and cyll ) . 
 other studies pointed out the importance of e. faecalis as sources of enterocins     [ 24 , 25 ] . taken together , these studies indicate the distribution and importance of enterococcal strains . it should be pointed out that most of enterocins so far studied exhibited activity towards gram - negative and gram - positive bacteria . we have undertaken isolation and characterization of enterocin named enterocin s37 , which is produced by e. faecalis recently isolated from chicken feces . 
 enterocin s37 appeared to be interesting regarding its activities against the food - borne pathogen l. monocytogenes egde and casually against campylobacter jejuni nctc 11168 ( table 1 ) . 
 however the anti - campylobacter activity remained unstable and less important than that observed for other bacteriocins recently reported in the literature     [ 24 ] . 
 the data gathered from this study let us think of the possibility of enterocin s37 to be a class iia bacteriocin , as ( i ) this peptide was anti - listeria , ( ii ) has a molecular weight < 10  kda , ( iii ) was sensitive to proteases , and ( iv ) was stable at different ph and high temperatures . 
 the activity increased several folds ( from 400  au      ml to 51,200  au      ml ) during the purification process ( table 3 ) , in parallel with an increase of specific activity , which reaches 1,557.17 au      g . this phenomenon was largely described previously as the result of removing inhibitory compounds     [ 2628 ] . 
 the spectrum of partially purified enterocin s37 was the same to that observed with cfs , but with stronger activities ( data not shown ) . for the first time , the effect of k channel modulators on enterocin activity was revealed in this research . to this aim , 
 we have utilized two k channel activators ( cromakalim and pinacidil ) and one k channel blocker ( glipizide ) . 
 cromakalim was shown to act on atp - sensitive potassium channels and caused membrane hyper polarization , pulls their membrane potential away from the threshold . in the pharmacology sector , the use of cromakalim to treating hypertension was reported . 
 finally , glipizide was described to affect the cell by partially blocking the k channels , reducing k conductance and causing depolarization of the membrane , which leads to ca ions influx through voltage - sensitive ca channels , causing rising of the intracellular concentrations of ca ions     [ 31 , 32 ] . 
 furthermore , the impact of cromakalim and glipizide on growth of sensitive and resistant l. monocytogenes strains was studied and resulted to be overall weak . 
 thus , the inhibition of wildtype ( sensitive phenotype ) to cromakalim and glipizide was estimated to 14.29%  1.38 and 17.14%  4.08 while the inhibition of the mutant strain ( resistant phenotype ) was evaluated to 4.46%  3.53 and 8.93%  1.06 . 
 pinacidil showed high - inhibitory activity on the wildtype ( 86.79%  1.78 ) and interestingly not on the mutant strain ( 19.64%  4.02 ) . 
 combinations of enterocin s37 and different k channel modulators suggested an implication of enterocin s37 in a depletion of intracellular k level , by an action on katp channels rather than kv channels . 
 these data are in good agreement with those obtained formerly with nisin a , whose antibacterial activity was connected to katp channels with concomitant loss of all intracellular k and atp . as for enterocin as-48 and enterocin p     [ 33 , 34 ] , 
 enterocin s37 is certainly responsible of uncontrolled efflux of intracellular k and affecting sensitive cells growth . 
 the resistant variant was able to grow but was slightly affected by the combination of enterocin s37/k modulators , except for pinacidil , where the inhibitory effect has reached 49.79%  3.36 , instead of 23.84%  7.73 and 24.11%  7.07 when cromakalim and glipizide were added , respectively ( figure 2 ) . 
 it was also well established that many bacteriocins , including enterocins , interact with membrane lipids leading to the formation of pores and subsequently to the loss of intracellular components , resulting thereof in the cell death     [ 3539 ] . 
 the lipid composition of membrane appeared to play an important role in the efficiency of bacteriocin activity     [ 9 , 35 , 36 , 38 ] . 
 enterocin s37 alone did not affect the growth of bacteriocin - resistant variant of l. monocytogenes egde . 
 however , the combination of enterocin s37/k channel modulators impacted strongly the growth of resistant variant . otherwise , the k channel modulators enhanced the inhibitory activity of enterocin s37 on sensitive strain of l. monocytogenes egde , with values reaching , 92.86%  5.90 and 94.29%  4.55 when enterocin s37 was combined with glipizide and pinacidil , respectively . 
 combination of cromakalim and enterocin s37 resulted in 84.29%   2.5 inhibition of the growth of sensitive strain ; this data is equal to that obtained with enterocin s37 alone 84.21%  5.87 ( figure 2 ) . 
 however , in the case of resistant strain , when enterocin s37 was combined to pinacidil , a high - inhibitory activity ( 49.79%  3.36 ) was observed as compared to combinations of cromakalim + enterocin s37 ( 23.84%  7.73 ) and enterocin s37 + glipizide ( 24.11%  7.07 ) ( figure 2 ) . 
 the mode of action of enterocin s37 could be triggered by potential interaction with cell membrane .","the aim of this research was to purify and characterize the mode of action of enterocin s37 , a bacteriocin produced by enterococcus faecalis s37 , a strain recently isolated from the chicken feces . 
 enterocin s37 has a molecular weight comprised between 4 and 5  kda . 
 it remained active after 1  h at 80oc and at ph values ranging from 4.0 to 9.0 . 
 furthermore , cell - free supernatant of enterococcus faecalis s37 and purified enterocin s37 were active against gram - positive bacteria including listeria monocytogenes egde , l. innocua f , enterococcus faecalis jh2 - 2 , and lactobacillus brevis f145 . 
 the purification of enterocin s37 was performed by ammonium sulfate precipitation followed up by hydrophobic - interaction chromatography procedures . 
 treatment of enterocin s37 with proteinase k , -chymotrypsin , and papain confirmed its proteinaceous nature , while its treatment with lysozyme and lipase resulted in no alteration of activity . 
 enterocin s37 is hydrophobic , anti - listeria and likely acting by depletion of intracellular k+ ions upon action on katp channels . 
 this study contributed to gain more insights into the mode of action of enterocins .",pubmed
"these trials were conducted in compliance with the declaration of helsinki , the international conference on harmonisation good clinical practice guidelines , the european union clinical trials directive , and local regulatory requirements . 
 approval for the study protocols and all amendments were obtained from columbia university medical center institutional review board ( approval # aaai6758 and # aaak5458 ) . 
 written informed consent and assent ( if applicable ) were obtained before any evaluations were conducted for eligibility . 
 the phase 1 study was a first - in - human , open - label , escalating - dose study to assess the safety , tolerability , and pharmacokinetics of a single intrathecal dose ( 1 , 3 , 6 , or 9 mg ) of nusinersen in children with spinal muscular atrophy . 
 each dose cohort had 6 to 10 participants ( n = 28 ) . upon completion , 
 all participants had the opportunity to enroll in a subsequent extension study and receive additional dosing with nusinersen . 
 the methods and results of the phase 1 study and its extension are detailed elsewhere . 
 briefly , medically stable spinal muscular atrophy participants 2 to 14 years of age were enrolled in 4 sites in the united states ( boston children s hospital , boston , ma ; columbia university medical center , new york , ny ; ut southwestern medical center  children s medical center dallas , dallas , tx ; and university of utah school of medicine , salt lake city , ut ) . 
 eligible participants had to be able to complete all study procedures , meet age - appropriate institutional guidelines for lumbar puncture procedures , and have a life expectancy of > 2 years . 
 participants were excluded for serious respiratory insufficiency , hospitalization for surgery or pulmonary event within the past 2 months , active infection at screening , history of brain or spinal cord disease or bacterial meningitis , presence of implanted cerebrospinal fluid drainage shunt , clinically significant laboratory abnormalities , any ongoing medical condition that would interfere with the conduct and assessments of the study , or treatment with another investigational drug within 1 month of screening . 
 patients with scoliosis were allowed to participate if , in the opinion of the investigator , a lumbar puncture could be performed safely . 
 a total of 3 lumbar punctures were scheduled during the 2 trials for drug delivery and/or follow - up collection of cerebrospinal fluid for safety and pharmacokinetic analyses . 
 drug was administered via intrathecal injection of a 5-ml bolus over 1 to 3 minutes . 
 the protocol recommended a 22- to 25-gauge spinal anesthesia needle ( 21-gauge needle allowed if participant s weight or condition dictated ) and that the lumbar punctures were performed at the l3-l4 disc space or 1 level above or 1 to 2 levels below , as needed . in all cases , 
 anesthesia and/or sedation and fluoroscopy or ultrasonography were permitted to facilitate the procedure and varied by institution at the discretion of the investigators at each site . 
 participants underwent the first lumbar puncture on day 1 for cerebrospinal fluid collection and nusinersen dosing , the second lumbar puncture on day 8 or day 29 for cerebrospinal fluid collection , and the third lumbar puncture during the extension study for cerebrospinal fluid collection and redosing with nusinersen 9 to 14 months after the initial lumbar puncture . in the phase 1 single - dose study , participants were initially monitored for safety and tolerability for 29 days ( 1-mg and 3-mg cohorts ) or 85 days ( 6-mg and 9-mg cohorts ) post dosing . 
 safety reporting included adverse events related to lumbar puncture , and a subgroup analysis was performed to compare reported lumbar puncture  related adverse events by needle size , participant age , and spinal muscular atrophy type . 
 these trials were conducted in compliance with the declaration of helsinki , the international conference on harmonisation good clinical practice guidelines , the european union clinical trials directive , and local regulatory requirements . 
 approval for the study protocols and all amendments were obtained from columbia university medical center institutional review board ( approval # aaai6758 and # aaak5458 ) . 
 written informed consent and assent ( if applicable ) were obtained before any evaluations were conducted for eligibility . 
 the phase 1 study was a first - in - human , open - label , escalating - dose study to assess the safety , tolerability , and pharmacokinetics of a single intrathecal dose ( 1 , 3 , 6 , or 9 mg ) of nusinersen in children with spinal muscular atrophy . 
 each dose cohort had 6 to 10 participants ( n = 28 ) . upon completion , 
 all participants had the opportunity to enroll in a subsequent extension study and receive additional dosing with nusinersen . 
 the methods and results of the phase 1 study and its extension are detailed elsewhere . 
 briefly , medically stable spinal muscular atrophy participants 2 to 14 years of age were enrolled in 4 sites in the united states ( boston children s hospital , boston , ma ; columbia university medical center , new york , ny ; ut southwestern medical center  children s medical center dallas , dallas , tx ; and university of utah school of medicine , salt lake city , ut ) . 
 eligible participants had to be able to complete all study procedures , meet age - appropriate institutional guidelines for lumbar puncture procedures , and have a life expectancy of > 2 years . 
 participants were excluded for serious respiratory insufficiency , hospitalization for surgery or pulmonary event within the past 2 months , active infection at screening , history of brain or spinal cord disease or bacterial meningitis , presence of implanted cerebrospinal fluid drainage shunt , clinically significant laboratory abnormalities , any ongoing medical condition that would interfere with the conduct and assessments of the study , or treatment with another investigational drug within 1 month of screening . 
 patients with scoliosis were allowed to participate if , in the opinion of the investigator , a lumbar puncture could be performed safely . 
 a total of 3 lumbar punctures were scheduled during the 2 trials for drug delivery and/or follow - up collection of cerebrospinal fluid for safety and pharmacokinetic analyses . 
 drug was administered via intrathecal injection of a 5-ml bolus over 1 to 3 minutes . 
 the protocol recommended a 22- to 25-gauge spinal anesthesia needle ( 21-gauge needle allowed if participant s weight or condition dictated ) and that the lumbar punctures were performed at the l3-l4 disc space or 1 level above or 1 to 2 levels below , as needed . in all cases , 
 anesthesia and/or sedation and fluoroscopy or ultrasonography were permitted to facilitate the procedure and varied by institution at the discretion of the investigators at each site . 
 participants underwent the first lumbar puncture on day 1 for cerebrospinal fluid collection and nusinersen dosing , the second lumbar puncture on day 8 or day 29 for cerebrospinal fluid collection , and the third lumbar puncture during the extension study for cerebrospinal fluid collection and redosing with nusinersen 9 to 14 months after the initial lumbar puncture . 
 in the phase 1 single - dose study , participants were initially monitored for safety and tolerability for 29 days ( 1-mg and 3-mg cohorts ) or 85 days ( 6-mg and 9-mg cohorts ) post dosing . 
 safety reporting included adverse events related to lumbar puncture , and a subgroup analysis was performed to compare reported lumbar puncture  related adverse events by needle size , participant age , and spinal muscular atrophy type . 
 a total of 28 children were enrolled and received dosing in the phase 1 study ; 15 children had spinal muscular atrophy type 2 and 13 had spinal muscular atrophy type 3 ( table 1 ) . at baseline , 
 participant mean ( range ) age was 6.1 ( 2.0 - 14.0 ) years , 17 were female , 10 were ambulatory , and 13 had scoliosis . 
 one child had vertical expandable prosthetic titanium rods inserted and the pedicle screws were inserted into t2 , t3 , and t4 with fixation of the growing rods to the pelvis , leaving the lumbar spine area spared . 
 the first and second lumbar punctures were performed on 28 and 27 children in the parent study , respectively , and the third on 18 children who re - enrolled in the extension study . 
 of the 10 children not re - enrolling , 6 re - enrolled in a multiple - dose study of nusinersen and 4 decided not to enroll . 
 of these , 1 child did not re - enroll for reasons related to the lumbar puncture procedure . 
 this was a 12-year - old girl with spinal muscular atrophy type 2 who had severe scoliosis at baseline and in whom the second fluoroscopically guided lumbar puncture procedure was not performed successfully at day 8 in the phase 1 study . 
 participants were enrolled and treated in 4 sites ; the institutional lumbar puncture procedures used in each site are reported in table 2 . 
 both cutting - tip and atraumatic ( pencil - point ) spinal needles were used and the general practice was to perform the procedure in the lateral decubitus or prone position by pediatric anesthesiologists , neurologists , or neuroradiologists in either inpatient or outpatient settings . 
 all sites performed the procedure under intravenous ( midazolam , ketamine , fentanyl , remifentanil , and/or propofol ) or inhaled anesthesia / sedation ( sevoflurane and/or nitrous oxide ) . 
 some sites also reported the use of topical anesthesia ( emla cream ) or locally injected lidocaine ( table 2 ) . 
 21 , 22 , 25 , or 27 gauge whitacre 3.0-inch spinal needle whitacre spinal needles sprotte 35-mm 24-gauge spinal needle quincke 25-mm 22-gauge spinal needle quincke 1.5- to 3-in . 
 spinal needle inhalational anesthesia : sevoflurane  nitrous oxide iv sedation : remifentanil , midazolam , propofol , fentanyl inhalational anesthesia : sevoflurane  nitrous oxide iv sedation : midazolam  ketamine inhalational anesthesia : sevoflurane and/or nitrous oxide iv sedation : propofol inhalational anesthesia : sevoflurane and nitrous oxide iv sedation : propofol neuroradiologist ( lumbar puncture ) neurologist ( drug administration ) pediatric anesthesiologist ( anesthesia / sedation ) neurologist ( lumbar puncture / drug administration ) anesthesiologist , nurse practitioner , or sedation nurses ( anesthesia / sedation ) pediatric anesthesiologist ( entire procedure ) pediatric anesthesiologist ( entire procedure ) prone position on the angiography table left lateral decubitus position or supporting sitting lateral decubitus position left lateral position interventional diagnostic suite ( part of the department of radiology ) 
  rapid treatment unit or outpatient clinic suite within the hospital setting ecg , pulse oximetry , non - invasive blood pressure , end - tidal co2 , temperature ecg , oxygen saturation , and expired co2 
  oxygen saturation and heart rate monitoring 
  ecg , pulse oximetry , oscillometric blood pressure , and end - tidal co2 
  ventilation was spontaneous or assisted ecg , pulse oximetry , blood pressure , temperature , and end - tidal co2 
  abbreviations : ecg , electrocardiogram ; iv , intravenous . 
 n s indicate the number of lumbar punctures performed at each site ( total 73 ) . 
 fluoroscopy was used routinely per institutional practice . 
 only in some participants . 
 in participants with severe scoliosis only . 
 for children receiving lumbar puncture . 
 for children receiving iv sedation 
 the majority of lumbar punctures were carried out using either a 22- ( 48% ) or 25-gauge ( 37% ) needle inserted in the l3-l4 ( 44% ) or l4-l5 ( 29% ) space 
 . nearly half ( 44% ) of the lumbar punctures were guided using fluoroscopy ( table 3 ) . 
 participants were treated for post  lumbar puncture headache with acetaminophen , ibuprofen , and/or caffeine citrate . 
 of the 73 lumbar punctures performed , the majority ( n = 50 ; 68% ) had no complications ; lumbar puncture  related adverse events were reported in 23 ( 32% ; table 4 ) . 
 the most common adverse events were headache ( 9 events ) , back pain ( 9 events ) , and post  lumbar puncture syndrome ( 8 events ; post  dural puncture headache with or without vomiting ) , and all events resolved without long - term complications . 
 the timing of the adverse events varied ; 67% ( n = 6 ) of the headaches occurred 12 to 72 hours post lumbar puncture , whereas back pain was reported 0 to 48 hours post procedure ( figure 1 ) . 
 fifty percent ( n = 4 ) of the post  lumbar puncture syndrome events occurred between 12 and 48 hours , with the remaining occurring after 72 hours ( figure 1 ) . 
 all 8 incidences of post  lumbar puncture syndrome were managed with acetaminophen , ibuprofen , and/or caffeine citrate for headache . 
 all 8 cases of post  lumbar puncture syndrome resolved with conservative therapy , and none of the 7 participants needed an epidural blood patch . in 1 case , 
 a blood patch was performed prophylactically during the second lumbar puncture procedure in a patient who had experienced a post  dural puncture headache following the first procedure . 
 resolution of the post  lumbar puncture syndrome occurred between 4 hours and 5 days , with the majority of the events ( 50% of cases ) lasting 1 to 2 days . 
 participants reporting > 1 adverse event after each lumbar puncture were counted only once for the lumbar puncture . 
 time of onset of headache , back pain , and post  lumbar puncture syndrome . 
 most common lumbar puncture  associated adverse events ( n = 73 ) and their time of onset are shown . 
 a subgroup analysis compared lumbar puncture complications with needle size , participant age , and spinal muscular atrophy type and demonstrated that headache , back pain , and post  lumbar puncture syndrome were observed more frequently when a 21- or 22-gauge needle was used , in older children ( 8 - 14 years of age ) , and in children with spinal muscular atrophy type 3 ( table 5 ) . 
 lumbar puncture  related adverse events are well documented in children and infants , with the most common complications being back pain and headache with an incidence of 11% to 40% and 12% to 33% , respectively . although the incidence of post  lumbar puncture syndrome in children is not well reported , 4% to 11% of the reported headaches are classified as post  dural puncture headaches . 
 in addition to headache , post  lumbar puncture syndrome can also include transient effects of backache , dizziness , nausea with or without vomiting , numbness , and lower extremity weakness . in rare cases , 
 more severe symptoms have been reported , such as intracranial hypotension , epidural hematoma , and cauda equina syndrome . in this phase 1 study and its extension , 
 73 lumbar punctures were performed for cerebrospinal fluid collection and/or intrathecal drug administration in children with spinal muscular atrophy . 
 approximately one - third of the procedures were associated with adverse events , most commonly with headache , back pain , and post  lumbar puncture syndrome , and the majority occurred within 72 hours after the procedure . 
 a subgroup analysis demonstrated that the highest incidence of adverse events was reported in older children , children with spinal muscular atrophy type 3 , and when a larger 21- or 22-gauge needle was used . using a 24-gauge needle or smaller atraumatic needle inserted with the 
 bevel parallel to the dura fibers had been suggested to considerably reduce damage to the dura and consequently decrease the risk for cerebrospinal fluid leak after lumbar puncture , including in children . 
 needle type ( atraumatic or traumatic ) may have a greater impact on the reported incidence of post  dural puncture headache than needle size . 
 turnbull et al found the incidence of post  dural puncture headache varied from 0.6% to 4% ( 22 gauge ) and 0% to 14.5% ( 25 gauge ) with an atraumatic ( whitacre ) needle versus 36% ( 22 gauge ) and 3% to 25% ( 25-gauge ) with a traumatic cutting - tip ( quincke ) needle , respectively . 
 although using an atraumatic needle may reduce the incidence of post  lumbar puncture syndrome , some studies have identified increased failure rate and paresthesia when using an atraumatic needle versus a cutting - tip spinal needle . 
 nearly half of the lumbar punctures were successfully carried out in children with spinal muscular atrophy using a 24-gauge spinal needle or smaller and , consequently , were associated with reduced incidence of headache and post  lumbar puncture syndrome compared with procedures performed using a 21- or 22-gauge needle . 
 after initial experiences , all investigational sites switched to using a smaller needle size , except when use of a larger needle size was required ( eg , scoliosis ) 
 . the higher incidence of adverse events , particularly headaches , observed among older children and/or in children with spinal muscular atrophy type 3 were likely related to the use of larger bore / gauge spinal needles , cutting - tip needles ( eg , quincke type ) , multiple attempts , and/or due to technical difficulties resulting from increased body weight and the presence of scoliosis or excessive lumbar lordosis . in most cases , post 
 dural puncture headache can be successfully treated with conservative therapy , consisting of bed rest in prone or lateral position , hydration , oral or intravenous caffeine , anti - nausea or antiemetic therapy , and/or analgesics . 
 however , in children with post  dural puncture headaches persisting > 48 hours or that worsen despite the use of conservative therapy , a therapeutic epidural blood patch may be indicated . 
 although there is conflicting evidence , some studies also support the use of a prophylactic blood patch to prevent post 
 dural puncture headache ; however , none of these studies included children . in this study , all post  dural puncture headaches resolved with conservative treatment of bed rest , adequate hydration , and administration of caffeine and other analgesics , and therapeutic epidural blood patches proved unnecessary . other considerations that may facilitate lumbar puncture success in children include patient positioning and the use of spinal ultrasound . 
 few studies in children and infants have compared the feasibility of lumbar puncture in upright versus lateral recumbent position . both the seated and lateral decubitus position with hip flexion increased the interspinous space compared with hip neutral positions and may increase lumbar puncture success rate . 
 the use of spinal ultrasound may facilitate the lumbar puncture procedure , and is favored over fluoroscopy in children , particularly if repeated procedures are planned , to minimize radiation exposure and cost . in this study , 
 lumbar punctures were performed in the lateral decubitus or prone position , and nearly half of the lumbar punctures were successfully done without ultrasound and/or fluoroscopy . however , in patients with spinal muscular atrophy with scoliosis , spinal rods , or other hardware , the use of imaging may be warranted to facilitate the procedure and increase the success of intrathecal medication delivery . 
 lumbar punctures were successfully performed in the participants with scoliosis ( 13 in the first procedure , 12 in the second procedure , and 11 in the third procedure ) and in the one child who had spinal rods . 
 the second fluoroscopy - guided procedure was unsuccessful in only 1 patient because of scoliosis . 
 this suggests that in some cases scoliosis might hinder lumbar puncture success in children with spinal muscular atrophy , but the frequency needs to be confirmed in larger studies . 
 general anesthesia and/or sedation are routinely performed in children to reduce procedure - related anxiety , pain , and distress , and to increase the overall success rate of lumbar puncture . however , increasing concerns about the impact of repeated anesthesia exposure on the developing nervous system in infants and young children must be carefully considered when repeated procedures are planned . 
 all 73 lumbar punctures performed in this study were performed using either intravenous ( midazolam , ketamine , fentanyl , remifentanil , and/or propofol ) or inhaled anesthesia / sedation ( sevoflurane , nitrous oxide ) , with no associated complications reported . 
 children with spinal muscular atrophy who have moderate to severe muscle weakness and/or severe scoliosis are at higher risk for hypoventilation and respiratory compromise . 
 thus , if deep sedation or general anesthesia is used , assisted ventilation may be required by either non - invasive or invasive means . 
 the use of local topical or subcutaneous anesthesia may help decrease the requirements for sedation . 
 based on our experience in this study , we recommend the most minimal use of sedative medications or anesthesia to permit safe and effective completion of the procedure . 
 the limitations of this study include a small sample size and the limited number of lumbar puncture procedures performed . 
 further experiences with lumbar puncture in children and infants with spinal muscular atrophy are needed to add to this knowledge . 
 in summary , repeated lumbar punctures were successfully performed in children with spinal muscular atrophy in the initial nusinersen clinical studies . 
 the frequency of lumbar puncture  related adverse events in children with spinal muscular atrophy was similar to that previously reported in children and infants , and were mainly limited to headache , back pain , and post  lumbar puncture syndrome . a 24-gauge needle or smaller was successfully used in children with spinal muscular atrophy to perform lumbar puncture with lower incidence of complications , suggesting that using a 24-gauge or smaller needle would likely reduce the chance of adverse events . bed rest , 
 adequate hydration , oral caffeine , and/or analgesics were sufficient to resolve post  lumbar puncture headache / syndrome without the need of a therapeutic blood patch in all patients . provisions for ultrasound guidance may be warranted in future research protocols given the potential benefit of reduced radiation exposure compared to fluoroscopy , especially in cases of serial procedures for repeated drug delivery . 
 overall , we conclude that intrathecal delivery of medication is feasible , safe , and well tolerated . 
 our experience may prove useful for guiding the development of best practice strategies for safe and effective intrathecal delivery of nusinersen and/or other promising emerging therapies for spinal muscular atrophy .","nusinersen ( isis - smnrx or isis 396443 ) is an antisense oligonucleotide drug administered intrathecally to treat spinal muscular atrophy . 
 we summarize lumbar puncture experience in children with spinal muscular atrophy during a phase 1 open - label study of nusinersen and its extension . during the studies , 
 73 lumbar punctures were performed in 28 patients 2 to 14 years of age with type 2/3 spinal muscular atrophy . 
 no complications occurred in 50 ( 68% ) lumbar punctures ; in 23 ( 32% ) procedures , adverse events were attributed to lumbar puncture . 
 most common adverse events were headache ( n = 9 ) , back pain ( n = 9 ) , and post  lumbar puncture syndrome ( n = 8) . in a subgroup analysis , adverse events were more frequent in older children , children with type 3 spinal muscular atrophy , and with a 21- or 22-gauge needle compared to a 24-gauge needle or smaller . 
 lumbar punctures were successfully performed in children with spinal muscular atrophy ; lumbar puncture  related adverse event frequency was similar to that previously reported in children .",pubmed
"the main concern is to ensure the highest possible standard for the services provided and to meet the needs of individual service users and communities ( 1 ) . in 1997 , 
 the uk department of health introduced clinical governance ( cg ) as a strategy for improving quality of health care services ( 2 ) . 
 the classic definition of cg is provided by scally and donaldson as  a system through which [ health ] organizations are accountable for continuously improving the quality of their services and safeguarding high standards of care by creating an environment in which excellence in clinical care will flourish  ( 2 , 3 ) . 
 the iranian ministry of health and medical education ( mohme ) has applied cg as a framework for improving quality and safety in all hospitals since 2009 . 
 mohme used the definition cited above as a guide to implement the policy . the cg model developed in iran 
 consists of seven interlocking components including : clinical effectiveness , clinical audit , risk management , patient and public involvement , education and training , staff and staff management , and use of information ( 4 ) . 
 systems awareness , leadership , ownership , teamwork , and communication are considered as a foundation of this model . 
 mohme required curative deputy of medical universities and hospital managers to work together to implement such initiatives in iranian hospitals . 
 the deputies for curative affairs in each medical university have the role of leadership in planning , implementing , monitoring and following up ministry of health policies particularly in quality improvement programs including cg ( 5 ) . 
 a number of studies have assessed the implementation of cg in different health systems and health care settings ( 611 ) . 
 insufficient knowledge and attitude toward cg , lack of resources , inadequate information technology systems , resistance to change , necessity of cultural change and professional boundaries were the main factors explored by lathman et al . 
 another study using qualitative research identified some barriers such as speed of cg implementation , workload and earmarked funding in cg implementation ( 7 ) . 
 ( 2002 ) considered lack of adequate senior management support as well as resources , structural and cultural issues as obstacles ( 8) . 
 the scarcity of resources was one of the most important barriers in implementation of cg noted by walsh et al . 
 a number of factors were identified which ultimately could affect the success of quality improvement activities . raising awareness of cg among managers , supportive culture and sufficient resources 
 declared that state level accountability in clinical governance implementation could be addressed by allocating proper resources and empowering policy implementers with proper performance control system ( 11 ) . 
 previous literatures mainly provide inadequate understanding about senior managers viewpoint toward facilitators and barriers in implementation of cg . in the current study an effort was done to capture both facilitators and barriers in cg implementation from the viewpoint of curative deputies in iranian medical universities . 
 to obtain a comprehensive understanding about senior managers viewpoint toward cg barriers and facilitators , a qualitative research was employed . 
 to do so , two main information sources were used : face to face interviews and relevant document reviews . 
 deputies for curative affairs of all types of iranian medical universities were purposefully selected to maximize the sample diversity and provide a comprehensive view toward cg implementation . the sampling continued until reaching data saturation . finally , forty three deputies were interviewed . in the first step , 
 an interview topic guide was developed on the basis of findings of literature review and expert opinions ( table 1 ) . 
 it covered the concept of clinical governance , key factors relating to clinical governance implementation process , facilitators and barriers that hospitals were experiencing . 
 some relevant documents were also analyzed such as cg annual reports , audit reports and minutes of meetings . 
 the qualitative thematic framework analysis was used to analyze the data with the assistance of the atlas - ti , qualitative data analysis software . 
 data analysis process includes five stages : familiarization , developing a thematic framework , indexing , charting , and mapping and interpretation ( 12 ) . to increase the validity , 
 it helped to ensure that the findings were congruent with participants perceptions , beliefs and opinions ( 13 ) . 
 the five main themes were explored and presented according with their sub - themes in ( table 2 ) . 
 most senior managers accepted that improving quality of health care should be integral to their role and essential to safeguard patient care . 
 they believed that quality improvement is a strategic goal , to achieve central government targets . 
 the findings demonstrated the willingness to support and positive attitude towards clinical governance .. senior managers also declared that having adequate knowledge about clinical governance also positive attitude toward the necessity of the program are the main factors affecting clinical governance implementation . 
 the level of enthusiasm expressed by senior managers seemed to be related in particular to their knowledge and attitude about cg concept and components . 
 those managers who had gained such knowledge were more optimistic toward success of the program . 
 continuous training about clinical governance concept and principles can make a positive attitude toward the program  . 
 main themes of senior managers viewpoint toward cg barriers and facilitators it was stated that such quality improvement program may facilitates the development of a culture focusing on continuous improvement . 
 most interviewees declared that appropriate culture for improving quality in the organization ; team work and readiness toward change are the main factors which influence cg implementation . 
 culture of openness in which staffs are willing to bring ideas related to service quality development was the other important factor mentioned by participants . 
 it was believed that culture which promotes alignment of clinical governance goals at both managerial and staff level should be developed . 
 they also stated that clinical governance components needed to be embedded in day to day work .  at first , we should culturalise cg in our work place  . 
 interviewees believed that adequate organizational commitment toward clinical governance should be created in order to increase the likelihood of program progress . 
 when continuous meetings are hold in high managerial levels of medical university about reporting our progress in implementing cg , it makes me sure that there is an organizational commitment .  effective organizational interactions both within and between departments was the other organizational key factor highlighted by senior managers . 
 they emphasized that clinical governance activities would be more effective if all departments in organization accept the importance of cg and participate in the implementation process .  everywhere in our organization you can see something about cg definition , process , implementation and so on . 
 they stated that most of staff working in this program does not have official position in the organizational chart which negatively affects their work . 
 they suggested that creation of such position can guarantee stability and legitimacy of staff in their workplace and have positive effect . 
 senior managers accepted that allocating adequate staff to undertake the responsibilities required by program had a critical role in pushing forward the program . 
 the other important factor recognized as an organizational factor was the necessity for development and dissemination of national standards . 
 interviewees believe that establishment of such standards and presence of guidelines is prerequisites of such huge quality improvement program . 
 in addition , it can help to conduct evaluation against determined standards which shows gaps in delivery of services . 
 one of the factors was managerial commitment toward clinical governance and their executive ability in implementing the program . 
 most of senior managers mentioned themselves ultimately accountable for clinical activities and quality of care in their organization . 
 they also believed that successful outcome of quality activities depends on managers participation in quality procedures and the level of their commitment . 
 interviewees added that senior managers themselves should demonstrate executive capabilities in order to motivate departments to implement clinical governance principles , provide adequate resources , facilitate staff training and remove any obstacle in the way . 
 i feel positive about the future of clinical governance because of my involvement with the program  . 
 furthermore , managers should use appropriate incentive mechanisms to sustain staff motivation and participation . 
 the matter is that how top managers will motivate us in implementing quality programs besides doing our regular organizational task . 
 interviewees highlighted that stability in managerial and executive positions is crucial to maintain the consistency and continuity of the program . 
 such unnecessary managerial position changes not only waste money and reduce the program progress , but also , ruin the managers motivation . 
 major changes in top managerial positions threaten the stability of cg and other quality improvement programs . 
 most of the senior managers agreed that shortage of staff and limited dedicated resources to implement clinical governance were main barriers in implementing clinical governance . 
 they believed that such barriers leave many managers feel beleaguered and faced with problems to effectively perform the program . 
 i think a major challenge in implementation of cg is lack of resources especially staff and money . 
 senior managers mostly emphasized that fostering a sense of engagement among medical staff especially physicians is important . 
 one of the reasons for resistance was that the staff is overwhelmed by high workload and different responsibilities . 
 they are seeing the program as being imposed and as policing their performance , rather than supporting quality improvement . 
 there is somehow resistance to clinical governance among some physicians and medical staff which i think it is because they are overwhelmed with the responsibilities they have regarding to care giving  . 
 some managers had a concern that this program might be temporary and act as a wave . 
 in addition some issues such as lack of support from physicians and medical staff , legal challenges , parallel quality improvement models running in the hospitals , increased workload , parallel functions in different domains of curative deputy and inadequate supporting systems developed for the staff in the way of clinical governance implementation were mentioned as barriers . 
 much of work i perform on clinical governance is done on my own limited time competing with other activities on my time . 
 i have lots of responsibilities to undertake and this encounters me with lack of time  . 
 some activities were also addressed by senior managers in order to mitigate problems in five afore - mentioned domains . 
 most senior managers accepted that improving quality of health care should be integral to their role and essential to safeguard patient care . 
 they believed that quality improvement is a strategic goal , to achieve central government targets . 
 the findings demonstrated the willingness to support and positive attitude towards clinical governance .. senior managers also declared that having adequate knowledge about clinical governance also positive attitude toward the necessity of the program are the main factors affecting clinical governance implementation . 
 the level of enthusiasm expressed by senior managers seemed to be related in particular to their knowledge and attitude about cg concept and components . 
 those managers who had gained such knowledge were more optimistic toward success of the program . 
 continuous training about clinical governance concept and principles can make a positive attitude toward the program  . 
 it was stated that such quality improvement program may facilitates the development of a culture focusing on continuous improvement . 
 most interviewees declared that appropriate culture for improving quality in the organization ; team work and readiness toward change are the main factors which influence cg implementation . 
 culture of openness in which staffs are willing to bring ideas related to service quality development was the other important factor mentioned by participants . 
 it was believed that culture which promotes alignment of clinical governance goals at both managerial and staff level should be developed . 
 they also stated that clinical governance components needed to be embedded in day to day work .  at first , we should culturalise cg in our work place  . 
 interviewees believed that adequate organizational commitment toward clinical governance should be created in order to increase the likelihood of program progress . 
 when continuous meetings are hold in high managerial levels of medical university about reporting our progress in implementing cg , it makes me sure that there is an organizational commitment .  effective organizational interactions both within and between departments was the other organizational key factor highlighted by senior managers . 
 they emphasized that clinical governance activities would be more effective if all departments in organization accept the importance of cg and participate in the implementation process .  everywhere in our organization you can see something about cg definition , process , implementation and so on . 
 they stated that most of staff working in this program does not have official position in the organizational chart which negatively affects their work . 
 they suggested that creation of such position can guarantee stability and legitimacy of staff in their workplace and have positive effect . 
 senior managers accepted that allocating adequate staff to undertake the responsibilities required by program had a critical role in pushing forward the program . 
 the other important factor recognized as an organizational factor was the necessity for development and dissemination of national standards . 
 interviewees believe that establishment of such standards and presence of guidelines is prerequisites of such huge quality improvement program . 
 in addition , it can help to conduct evaluation against determined standards which shows gaps in delivery of services . 
 one of the factors was managerial commitment toward clinical governance and their executive ability in implementing the program . 
 most of senior managers mentioned themselves ultimately accountable for clinical activities and quality of care in their organization . 
 they also believed that successful outcome of quality activities depends on managers participation in quality procedures and the level of their commitment . 
 interviewees added that senior managers themselves should demonstrate executive capabilities in order to motivate departments to implement clinical governance principles , provide adequate resources , facilitate staff training and remove any obstacle in the way . 
 i feel positive about the future of clinical governance because of my involvement with the program  . 
 furthermore , managers should use appropriate incentive mechanisms to sustain staff motivation and participation . 
 the matter is that how top managers will motivate us in implementing quality programs besides doing our regular organizational task . 
 interviewees highlighted that stability in managerial and executive positions is crucial to maintain the consistency and continuity of the program . 
 such unnecessary managerial position changes not only waste money and reduce the program progress , but also , ruin the managers motivation . 
 major changes in top managerial positions threaten the stability of cg and other quality improvement programs . 
 most of the senior managers agreed that shortage of staff and limited dedicated resources to implement clinical governance were main barriers in implementing clinical governance . 
 they believed that such barriers leave many managers feel beleaguered and faced with problems to effectively perform the program . 
 i think a major challenge in implementation of cg is lack of resources especially staff and money . 
 senior managers mostly emphasized that fostering a sense of engagement among medical staff especially physicians is important . 
 one of the reasons for resistance was that the staff is overwhelmed by high workload and different responsibilities . 
 they are seeing the program as being imposed and as policing their performance , rather than supporting quality improvement . 
 there is somehow resistance to clinical governance among some physicians and medical staff which i think it is because they are overwhelmed with the responsibilities they have regarding to care giving  . 
 some managers had a concern that this program might be temporary and act as a wave . 
 in addition some issues such as lack of support from physicians and medical staff , legal challenges , parallel quality improvement models running in the hospitals , increased workload , parallel functions in different domains of curative deputy and inadequate supporting systems developed for the staff in the way of clinical governance implementation were mentioned as barriers . 
 much of work i perform on clinical governance is done on my own limited time competing with other activities on my time . 
 i have lots of responsibilities to undertake and this encounters me with lack of time  . 
 some activities were also addressed by senior managers in order to mitigate problems in five afore - mentioned domains . 
 in the present study , we tried to explore senior managers viewpoint about the facilitators and barriers in cg implementation . 
 we found that sufficient knowledge and clear understanding about the principles and practice of cg have major roles in achieving desired improvement in service quality and patient safety in health care settings . 
 this study has also highlighted the importance of supporting culture , appropriate organizational structure and managerial commitment as perceived facilitators in cg implementation . 
 there was also a strongly accepted view that staff at all levels should be consulted , involved in planning and implementation of cg programs . 
 the main identified obstacles were lack of adequate managerial support as well as resource , structural and cultural issues and professional boundaries . 
 our results are parallel with the findings of many other studies . a model developed by o brien et al 
 the model outlines four dimensions in successfully implementation of clinical governance : cultural , technical , structural and strategic . 
 the cultural dimension related to beliefs , values , norms and behaviors in the organization which either suppress or support quality improvement activities . 
 an organization with strong and clear vision and goals , stable managerial leadership , supportive structures for team work , effective interactions and inter professional relationships and continuous learning culture is more likely to successfully implement clinical governance ( 14 ) . in our study 
 it is clear that senior managers perception about the important factors in implementing clinical governance were classified in five main domains . 
 the domains were knowledge and attitude , culture , organizational factors , managerial factors and barriers . 
 most of the senior managers believed in knowledge and attitude toward clinical governance and culture as the most important factors in cg implementation . 
 this was thought to be a major factor in achieving improvement in service quality and patient safety mentioning in another study . 
 similar to o brien study , our findings showed that culture is also an important factor and many organizations expend much effort to shape their culture in a way to improve quality . 
 a sense of ownership toward quality improvement and a positive attitude to contribute new ideas also provide an organizational climate necessary to allow alignment of attitudes and values with a continuous quality improvement . in hogan study about 
 consultants attitudes to clinical governance  , quality improvement was considered as an integral part of consultants role and they accepted that maintaining service standards , monitoring and improving outcomes for patients were activities they should undertake . 
 there was also recognition about the importance of team based approaches to quality improvement ( 15 ) . 
 this supports the findings of our study which focuses on the importance of being involved in quality improvement activities by all staff especially physicians and medical staff . 
 these include structures and processes with clear vision and goals toward quality improvement , involving staff in the process of change , rewarding positive behaviors , improving the effectiveness of communication across the organization and providing opportunity for team work . 
 campbell study on  the role of cg as a strategy for quality improvement in primary care  found significant barriers in the way of cg implementation . 
 these included in appropriate culture , too few staff , limited resources , disengagement by some practices and staff , lack of time to perform quality activities ( 8) . our study supported the above findings and declared that some senior managers felt powerless with the volume of work and shortage of resources . 
 meaningful engagement and commitment at all levels of managers and staff has been highlighted as a major factor in implementing cg . 
 the managerial level needed to match its commitment to a program of change with realistic timetables to secure the cultural and organizational changes needed to improve quality of care . the need for top management support is the most frequently cited imperative for success of any program . 
 wilkinson and witcher in an examination of factors important in successfully implementation of clinical governance stressed on the importance of quality committed senior managers and staff effectively involved in all levels of organization ( 16 ) . 
 fenton o creevy suggests that the most consistently barrier to the success of every quality improvement program is resistance from managers ( 17 ) . 
 dawson found that one of the major problems encountered in implementing quality program is lack of commitment at the middle and supervisory management . 
 they suggest that many of the problems of survivor syndrome arise from the breakdown of traditional psychological contract where managers promised job security ( 18 ) . in our study , the necessity of physicians and medical staff participation in clinical governance program has been emphasized . 
 lawler , mohrman and ledford have demonstrated the close relationship between success of quality programs and employee involvement initiatives ( 19 ) . 
 another issue is the importance of having an employee recognition and rewards system with supporting mechanisms providing adequate salaries for the staff being involved in the implementation of clinical governance program . 
 encouraging workers to become involved in continuous improvement activities is relatively an important factor ( 20 ) . 
 wilkins and witcher suggest that employees who are highly skilled , with adequate salaries and incentives are typically more likely to accept the program ( 16 ) . 
 master produced a list of eight barriers in the way of implementing a quality improvement program includinglack of management commitment , lack of training , inability to adopt organizational culture suitable for quality improvement , lack of employee involvement , lack of resources , improper planning , in compatible organizational structure and inadequate use of team work ( 21 ) . 
 he declares that quality improvement activities , team work , effective communication and supporting the quality program in all organizational levels are of great importance ( 22 ) . 
 sohal , samson and ramsay also investigated the barriers of successful implementation of quality plan from the viewpoint of organizational management . 
 they categorized the barriers in a number of groups : organizational culture ( top management support and effective involvement , changing values and culture to align with quality improvement requirements ) , strategic planning issues ( lack of planning for quality , inappropriate organizational structural ) , resource management issues ( lack of resources , inadequate number of personnel and additions to normal working load ) ( 23 ) . 
 another study conducted by terziovski , sohal and moss showed that a successful quality organization would include the following characteristics : managers and staff with positive attitude toward quality , employment of quality management practices , dissemination of responsibility of quality to all staff at all levels , , leadership commitment , having strategic planning , providing adequate resources , focusing on training and adoption of appropriate culture ( 24 ) . in our study , senior managers stated some recommendations for implementing clinical governance more effectively such as : creating a suitable culture for implementing quality programs , evaluating the quality of organization , determining the existing deficiencies , setting up appropriate strategic and functional plan to achieve determined objectives , following the programs , evaluating the results and encouraging quality improvement activities continuously ( table 3 ) . 
 the interviews reflect only top managerial perspectives at medical university level and not managers working in hospitals who should play important role in cg implementation . 
 in addition , due to the nature of qualitative studies the results can not be generalized although we are looking for theoretical generalization . 
 although this study has provided the first evaluation of senior managers viewpoint about facilitators and barriers in cg implementation in iran , further research is required to track the progress of the cg policy as it unfolds over time . 
 this qualitative paper explores main facilitators and barriers perceived by deputies in curative affairs of iranian medical universities . 
 identifying facilitators and barriers from the viewpoint of senior managers can have an effective role in successful progress of cg program . 
 the reason is that these managers are directly responsible for piloting such quality programs and are the most familiar with challenges existing in the way of implementing cg . 
 the authors conclude that one of the possible solutions is developing educational courses and workshops with the purpose of raising staff awareness toward cg concept and practice . developing a supportive culture , having all levels of staff commitment and involvement , effective communication , developing clinical guidelines , 
 using incentive tools and overcoming legal challenges are other resolutions mentioned in this regard . by successfully implementing the program , patients will benefit from quality services . 
 also health professionals take an advantage of working in a safer and more supportive system . 
 evidence suggests that governance needs to match its commitment to a program of change with realistic timetables to secure the cultural and organizational changes needed to improve quality of care . 
 ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .","abstractbackgroundhealth care systems should assign quality improvement as their main mission . clinical governance ( cg ) 
 is a key strategy to improve quality of health care services . 
 the iranian ministry of health and medical education ( mohme ) has promoted cg as a framework for safeguarding quality and safety in all hospitals since 2009 . 
 the purpose of this study was to explore perceived facilitators and barriers to implementing cg by deputies for curative affairs of iranian medical universities.methodsa qualitative study was conducted using face to face interviews with a purposeful sample of 43 deputies for curative affairs of iranian medical universities and documents review . 
 thematic analysis was used to analyze the dataresultsfive themes were explored including : knowledge and attitude toward cg , culture , organizational factors , managerial factors and barriers . 
 the main perceived facilitating factors were adequate knowledge and positive attitude toward cg , supporting culture , managers commitment , effective communication and well designed incentives . 
 pe rceived barriers were the reverse of facilitators noted above in addition to insufficient resources , legal challenges , workload and parallel quality programs.conclusionssuccessful implementation of cg in iran will require identifying barriers and challenges existing in the way of cg implementation and try to mitigate them by using appropriate facilitators .",pubmed
"hydatid cyst disease is one of the most important zoonotic diseases , which occur , in larval stage of echinococcus granulosus parasite . in this disease , 
 canines such as dog , wolf , fox , and jackal ( the final host ) are infected by eating contaminated viscera , and small adult worms with size of 27 mm are formed in their intestines and begin to spawn . 
 canines repel the eggs of adult worms along with feces and infect the soil , water , and vegetables . 
 herbivores as the main intermediate host are infected by eating forage contaminated with the larvae of this parasite . besides , human as the intermediate host 
 is infected accidentally either by eating contaminated water and vegetables , or direct contact with contaminated dogs is infected to parasite egg of this disease ; and hydatid cyst is usually involved the lung and liver in human ( 1 , 2 ) . epidemiological , clinical , and pathological symptoms of this disease depend on some factors such as age , gender , infection severity , cyst size , and the involved organ in the body ( 1 , 2 ) . 
 this disease is common in most parts of the world especially in countries where animal husbandry is current . 
 contamination to this parasite is spreading in most tropical regions of the world and it is globally distributed in many countries such as argentina , peru , east africa , central asia , china , and south america ( 3 ) . in endemic regions , the incidence rate of disease can be reached over 50 per 100,000 person - years with the prevalence rate of 5%10% in human ( 3 , 4 ) . on the other hand , hydatidosis is among the most important human diseases with the high health and economic burden ( 5 , 6 ) . 
 iran is one of the areas that has been considered as hyper - endemic area by who in terms of close relationship of a high proportion of society with animals , traditional animal husbandry , and then contact with the sources of infection ( 7 , 8) . 
 the prevalence rate of this disease in the intermediate host has reported as 11.5%34.6% ( 9 ) , and the prevalence rate of human hydatid cyst has been reported from 1.1% to 13.7% in different parts of iran ( 1012 ) . 
 assessment of the epidemiological and clinical aspects of the disease can help health policy - makers to focus on the main public health problems in the community and to assess the efficacy of control and preventive programs to limit the spread and transmission of the parasite in humans . 
 hence , the present study aimed to determine the epidemiological and clinical status of human hydatidosis in khorasan razavi province during 201114 . 
 this cross - sectional study was conducted in khorasan razavi province , the northeast of iran , in 2014 . 
 the study population was all cases with hydatid cyst who diagnosed in governmental and private laboratories , hospitals and health centers in khorasan razavi province during 201114 . 
 the data collection was performed using a checklist including age , sex , nationality , place of residence , occupation , infected organ , the number of cysts , the type of diagnosis ( ct scan , mri , radiology , sonography , immunofluorescence , other ) , clinical symptom , and having risk factors such as how to wash vegetables and contact with dog . 
 descriptive statistics including frequency tables and diagrams as well as analytical statistics including chi square and t - student tests were used for data analysis . 
 all statistical analyses were performed at 0.05 significance levels using stata software , version 12 ( stata corp , college station , tx , usa ) . 
 in addition , mapping the percentage of hydatid cyst cases was created using gis software by cities . for considering ethical issues in data collection 
 , it should be noted that all information of the patients has been confidential and the information has been recorded with code and without mentioning name . 
 in total , 357 hydatid cyst cases had been registered in the health centers during the periods 201114 in all parts of province . 
 the most percentage of cases was related to the cities mashhad ( 51.8% ) , ghoochan ( 7.8% ) , neishabour ( 7.0% ) , and fariman ( 4.8% ) , respectively . 
 percentage of hydatid cyst cases by county in khorasan razavi province in 201114 demographic characteristics of studied patients by sex are shown in table 1 . 
 of 357 cases , 54.9% were women , 40.3% rural , 45.8% housewives , and 3.4% were afghan . 
 the mean age of women was higher than that of men ( 39.1318.9 compared to 34.717.9 , respectively , p - value=0.025 ) . 
 demographic characteristics of patients with hydatid cyst by sex in khorasan razavi province in 201114 data are presented as number ( % ) . 
 the most common symptom of cases was related to the cities mashhad ( 51.8% ) , ghoochan ( 7.8% ) , neishabour ( 7.0% ) , and fariman ( 4.8% ) , respectively . moreover , 45.9% of patients had domestic dog and hygiene principles of washing the vegetables was adhered by 6.7% of patients and 64.4% were washed the vegetables with water alone ( table 2 ) . 
 risk factors of hydatid cysts in the patients in khorasan razavi province in 201114   the majority of patients ( 70.5% ) were infected with a single cyst , and 24.5% of them had multiple cysts . 
 one hundred fifty one ( 42.3% ) of patients were diagnosed by radiology method and 37.8% by ct scan method . 
 the most common clinical symptom in the hydatidosis patients was abdominal pain ( 42.3% ) , after that hepatomegaly ( 27.4% ) , chest pain ( 21.6% ) , and cough ( 8.7% ) were other common symptoms . 
 liver involvement was reported in 59.4% of patients , and 8.4% had multiple organ involvement ( fig . 
 2 ) . status of organ involvement in the patients with hydatid cysts in khorasan razavi province during 201114 
 previous studies conducted from different parts of iran found the annual incidence of 1.33 in hamadan ( 13 ) , and 0.54 in kermanshah ( 14 ) per 100,000 population . 
 in addition , the rate of infection varied from 1.79% in ardabil ( 11 ) to 13.8% in khozestan ( 12 ) . 
 our findings showed that the highest proportion of hydatidosis ( 39.2% ) in the age group of 2140 years old . according to some factors such as geographical region , culture , and workforce ; age can play an important role on the occurrence of the infection . in this 
 regard , previous studies have reported the highest proportion of hydatidosis in the different age groups . 
 for example , the age group of 6069 in isfahan ( 10 ) , 4049 year in ardabil ( 11 ) , and 3060 year in qom ( 15 ) . based on a review study , 
 the range of 2040 years old as the age group of the highest proportion has been reported ( 7 ) . 
 the findings of this study showed the most percentage of infection in women than men ( 54.9% compared with 45.1% , respectively ) . 
 this might be due to more contribution of women to agricultural and animal husbandry in this region . 
 the high frequency of the disease in women than men is consistent with the obtained results of other iranian studies ( 16 , 17 ) and other countries ( 18 , 19 ) . 
 however , some studies reported the higher rate of infection in men ( 10 , 11 , 15 ) . 
 moreover , as stated in the previous studies conducted in iran ( 7 , 20 ) , the housewives had the highest proportion of infection . 
 it may be due to the more likely to contact with the source of contamination especially vegetables contaminated to parasitic eggs . in our study , 
 for example , the most percentage of the province s population is urbanite and another might be a good - quality disease reporting . 
 there are conflicting results in this regard ; some studies reported the same result with our study ( 11 , 15 , 20 , 21 ) , and other reported inconsistent result ( 8 , 22 ) . 
 we found that abdominal pain was the main symptom with a proportion of 42.3% in the patients . 
 studies conducted in turkey ( 23 ) and iran ( 24 ) has reported a proportion of 77.6% and 66% for this symptom , respectively . in our study in line with previous reports 
 ( 24 , 25 ) , liver was the most common localization of hydatid cyst . 
 about 8.4% of the patients in this study had multiple organs involved and it was 12.3% in another study in iran ( 24 ) . 
 the common diagnosis methods of the disease were radiology ( 42.3% ) followed by ct scan ( 37.8% ) . in a study to determine the pattern of hydatid cyst during 200010 in tehran revealed that ultrasonography and ct scan were the most used imaging methods ( 24 ) . 
 our findings demonstrate that these methods are the available tools for detecting hydatid cyst in iran . 
 concerning risk factors of infection with hydatidosis , our results found that 45.9% of individuals were in contact with dog , 64.4% of them have washed the vegetables with the water alone and 6.7% have washed the vegetables by hygiene principles of washing . 
 consequently , focus of improvement in education level of the society could be better . in bulgaria ( 25 ) 90% of the affected children 
 the prevalence of human hydatidosis , as a most important neglected disease , should be considered by health policy - makers in public health domain . in addition 
 , educational programs to better recognition of the disease symptoms , and to identify the infection sources are needed in high risk group of population .","background : this study aimed to investigate the epidemiological and clinical aspects of patients with hydatid cyst during 2011 to 2014.methods:this cross - sectional study was conducted in khorasan razavi province , the northeast of iran , from 2011 to 2014 . 
 the study population was all cases with hydatid cyst who diagnosed in governmental and private laboratories , hospitals and health centers ( hc ) in khorasan razavi province during 201114.results:the prevalence rate of hydatidosis was 1.44 per 100000 individuals . 
 of 357 cases , 54.9% were women , 40.3% rural , 45.8% housewives , and 3.4% were afghan . 
 the mean age of women was higher than that of men ( 39.1318.9 compared to 34.717.9 yr , respectively , p - value=0.025 ) . 
 the highest proportion of cases ( 39.2% ) was in the age group of 2140 yr old . 
 abdominal pain was reported in 42.3% of cases . 
 liver involvement was the most common localization of hydatid cyst reported in 59.4% of patients , and 8.4% had multiple organ involvement . 
 the common diagnosis methods of the disease were radiology ( 42.3% ) followed by ct scan ( 37.8% ) . 
 45.9% of patients had domestic dog and hygiene principles of washing the vegetables was adhered by 6.7% of patients.conclusion:the prevalence of human hydatidosis , as a most important neglected disease , should be considered by health policy - makers in public health domain . 
 in addition , educational programs to better recognition of the disease symptoms , and to identify the infection sources are needed in high risk group of population .",pubmed
"friederich is credited with describing the first case of a ciliated hepatic foregut cyst ( chfc ) in 1857 and positing their congenital origin . 
 wheeler and edmonson were the first to use the term  chfc  to describe the lesion . 
 they also identified specific characteristics , which differentiate it from other hepatic cysts , such as the presence of four typical layers : a pseudostratified columnar epithelium layer with interspersed mucus cells , a sub - epithelial connective tissue layer , a smooth muscle layer and an outer fibrous capsule . 
 this lesion is thought to be the result of evagination of the foregut during embryonogenesis . in the last 150 years 
 , approximately 103 cases have been reported in the literature world - wide , only four of which describe laparoscopic excision . 
 we report 5 case of a patient who recently underwent successful laparoscopic excision of a chfc and the rationale for attempting laparoscopic removal of these lesions and review of the literatures . 
 this paper reports a case of a 32-year - old woman presented with a 6-month history of right upper quadrant pain . 
 the ca 19 - 9 serum level was 18 iu / l ( normal 0 - 37 
 an abdominal ultrasound scan reported thin walled 10 cm  7.6 cm  5.8 cm cyst in the superior part of the right lobe of liver raising a suspicion of a simple cyst of liver . 
 the cystic lesion did not enhance postcontrast and appeared to be well circumscribed without evidence of hepatic invasion suggestive of a simple cyst . 
 therefore , a laparoscopic deroofing of the cyst was planned . at the time of surgery , 
 port placement was initiated with a 10-mm infraumbilical trocar followed by two 5-mm ports inferior to the right costal margin along the anterior and midaxillary lines . 
 intraoperatively [ figure 2 ] , the 10 cm  8 cm cyst was found adherent to the superior surface of liver compressing segments vii and viii . on further dissection 
 , it was noted that the cyst was actually free from the liver parenchyma and was adherent to the liver by a small pedicle . 
 since there was only a pedicle , it was decided to do a complete excision instead of a de - roofing . 
 pathologic review of the cyst showed an inner layer of ciliated pseudo stratified mucin secreting columnar epithelium with scattered goblet cells surrounded by loose connective tissue , a discontinuous smooth muscle layer and an outer fibrous capsule these findings were consistent with a ciliated foregut cyst of hepatic origin without evidence of epithelial dysplasia or invasive malignancy . 
 contrast enhanced computed tomography shows non - enhancing cystic lesion in segment viii of right lobe of liver laparoscopic view of ciliated hepatic foregut cyst in segment viii 
 chfcs are rare , with only 103 cases reported since first described as a congenital malformation by friederich in 1857 . 
 the majority ( 55 ) of these cases have been reported in the last 25 years as imaging techniques have improved . 
 the term chfc was coined to indicate the origin of this lesion as being a detached hepatic diverticulum or detachment and migration of buds from the oesophageal and bronchial regions of the foregut and subsequent entrapment by the liver during the early embryonic development of the foregut . 
 it is found most commonly in segment iv of the left lobe just beneath the hepatic capsule . 
 the cyst size ranges from < 1 cm to 24 cm in greatest diameter , the mean greatest dimension is 4 cm and the median is 3 cm . 
 the average age of presentation is 52 years , ranging from 3 months to 82 years , with a slight female predominance . 
 nearly 40% of the reported cases have been found incidentally on imaging studies , 26% incidentally at autopsy , 6% incidentally at surgery and 22% presented with abdominal symptoms . chfc are most frequently located superficially in the median segments of the liver ( segments iv , v , viii ) , are rarely multi - loculated or septated and are mostly asymptomatic . 
 they also identified specific characteristics , which differentiate it from other hepatic cysts , such as the presence of four typical layers : a pseudostratified columnar epithelium layer with interspersed mucus cells , a sub - epithelial connective tissue layer , a smooth muscle layer and an outer fibrous capsule . 
 friederich initially suggested a congenital origin , but currently it is suspected that the chfc is a detached hepatic diverticulum or abnormal tracheobronchial bud that may have migrated caudally to be included with the liver during the early embryological development of the foregut . 
 although , sludge - like bile and viscid mucoid content of the cyst have been described , no communication with the biliary tree could be demonstrated in any reported cases . 
 the imaging appearances of chfc 's are variable and appear as anechoic or hypoechoic cysts with spotty hyperechoic areas on ultrasound . 
 ct findings are of a non - enhancing , rounded lesion of varying density depending on the contents of the cyst , which can include calcium crystals and cholesterol . 
 typical magnetic resonance imaging features are a hyperintense cyst on t2-weighted images and a variable appearance on t1-weighted images . 
 pathologically , the cysts are typically solitary and unilocular , and have four layers : an outer fibrous rim , a layer of smooth muscle ( often incomplete ) , subepithelial connective tissue and a lining of ciliated , pseudo - stratified , mucinsecreting columnar epithelium .. presence of unique histological features indicates chfc with a reasonable degree of confidence , although a rare morphologic variant of chfc without smooth muscle layer has reported . 
 histologically , the differential diagnosis of a hepatic cyst included simple cyst , hydatid cyst , biliary cystadenoma , cystadenocarcinoma and bronchogenic cyst . 
 the main differential diagnosis can be bronchogenic cyst because both cystic lesions share a common embryonic origin . 
 simple cysts are usually located in the right hepatic lobe and are more prevalent in women . 
 serologic tumour markers can be confounding , as elevated ca19 - 9 levels have been found in association with benign chfc . 
 an aspirate of hepatocytes admixed with ciliated cells and mucus cells distinguish chfc from other radiologically benign - appearing entities among many of the considered lesions , which are easily differentiated by their typical epithelial lining , cyst . 
 of concern are reports of chfc harbouring squamous cell carcinoma ( 4.4% in reported cases ) . 
 there is also a single report of portal vein compression secondary to the mass - effect of the chfc . until date , 
 the recommended treatment of chfc described in the literature has varied from observation to aspiration to surgical excision however , given the recent cases of chfc malignant transformation , serial imaging may be required for those patients undergoing cyst sclerosis . 
 most of the authors agree that chfcs should be surgically excised for cysts larger than 4 cm to 5 cm , symptomatic or enlarging lesions and asymptomatic lesions with wall abnormalities on imaging or in patients with otherwise unexplained abnormal liver function tests . until date 
 several authors have also noted that laparoscopic approach of aspiration and injection of a sclerosing agent can be utilised effectively to stop cyst growth , but the definitive approach is excision of cyst but minimally invasive approach . 
 this technique is best suited to lesions in segments ii to vi , the anterolateral portion of the liver where visualisation and access are easiest . 
 lesions located centrally or in the posterior aspects of segments vi , vii , or iva are less accessible and render laparoscopic therapy more challenging . in general , 
 the patient is positioned in lithotomy , allowing the surgeon to stand between the legs with assistants at the sides . 
 the cyst wall is excised with harmonic scalpel ; hemostasis may be obtained with electrocautery . 
 for patients with lesions in segment vii or viii , positioning and port placement similar to that used for laparoscopic right adrenalectomy may afford improved exposure . 
 first , the small size and anterior subcapsular location allow for easy access to the cyst with minimal dissection to isolate the lesion . 
 second , the benign nature of the process allows the cyst to be enucleated from is hepatic bed with little concern for adequate circumferential margins . 
 third , the chfc traditionally has a thick cyst wall that permits easy handling with laparoscopic instruments . 
 the subcapsular location and the presence of thick outer fibrous layer in the cyst wall makes it amenable to laparoscopic excision with minimal morbidity . 
 the rarity of chfc underscores the importance of re - porting individual cases , as illustrated by the case presented here , which may be few reported cases of chfc presenting with abdominal pain in her early 30 s. it should be noted that although the size and location of the cystic mass were characteristic of chfc , this patient 's cyst demonstrated imaging features that are less common for a chfc , leading to the decision to excise the cyst . by utilising a minimally invasive approach 
 to excise the chfc , the patient benefited from an effective ambulatory procedure for the removal of a lesion with risk for malignant transformation . 
 chfc should be considered as a diagnosis for a superficially located unilocular hepatic cystic mass that presents incidentally or asymptomatically and laparoscopic excision should be considered as a first - line treatment to remove this benign cyst .","ciliated hepatic foregut cysts ( chfcs ) are rare congenital cysts of the liver that originate from the embryologic foregut . 
 despite an increase in incidence , they remain rare and several key characteristics remain poorly understood , including the range of presentation and the risk of malignant conversion . 
 large , symptomatic cysts and small asymptomatic , atypical cysts should be resected . 
 we present a patient who recently underwent laparoscopic excision of a chfc , review the literature and propose the rationale for attempting removal of these cysts through a laparoscopic approach .",pubmed
"oral route is one of the most preferred routes of drug administration due to its safety , ease of administration , and acceptability by patients . 
 about 60% of conventional dosage forms are available as the oral solid dosage forms . the low bioavailability , longer onset of action , and dysphasia patients turned the manufacturer towards the parenterals and liquid dosage forms . 
 but the liquid dosage forms ( syrup , suspension , emulsion , etc . ) have the problem of accurate dosing and parenterals are painful drug delivery systems , so they result in patient incompliance . 
 the most popular oral dosage forms are tablets and capsules ; one major drawback of these dosage forms is the difficulty to swallow . drinking water plays an important role in the swallowing of oral dosage forms . 
 people experience inconvenience in swallowing tablet dosage forms when water is not available particularly in the case of traveling ( motion sickness ) and sudden episodes of coughing during the common cold , allergic condition , and bronchitis . under such circumstances , tablets that can rapidly dissolve or disintegrate in the oral cavity known as fast dissolving tablets have attracted a great deal of attention . fast dissolving tablets 
 are also known as mouth - dissolving tablets , orodispersible tablets , rapidmelts , and porous tablets . fast dissolving tablets 
 dissolve or disintegrate within 60 seconds when placed in the mouth without drinking water or chewing . 
 the active ingredients are absorbed through mucous membranes in the mouth and git and enter the blood stream . 
 but due to certain disadvantages like their physical solid form , psychological fear of swallowing , chewing , or chocking , friability of wafer like porous and low pressure moulded tablet , and expensive packaging cost of these dosage forms to protect them , a new technology was developed as orally dissolving strip . orally dissolving strips are the most advanced form of oral solid dosage form due to more flexibility and comfort . 
 it improves the efficacy of apis by dissolving within a minute in oral cavity after the contact with saliva without chewing and need of water for administration . 
 it gives quick absorption and instant bioavailability of drugs due to high blood flow and permeability of oral mucosa which is 41000 times greater than that of skin . 
 orally dissolving strips are useful in patients such as pediatrics , geriatrics , bedridden , and emetic patients and conditions such as sudden episodes of allergic attacks or coughing . 
 there is an increasing interest in the development of orally dissolving strips as an alternative to fast dissolving tablets , due to their faster dissolution rate , higher flexibility , and better patient compliance . 
 presently , research work on the use of orally dissolving strips as promising carriers for the delivery of multiple active pharmaceutical ingredients has emerged [ 611 ] . 
 marketed orally dissolving strips products have also become available including listerine , chloraseptic , triaminic , and multivitamins . 
 the backbone of an orally dissolving strip is generally formed of a plasticizer and film forming polymer or a mixture of polymers that provide the necessary elasticity and shape to the film . 
 examples of polymers that have been used in the formulation of orally dissolving strips include hydrocolloids or povidone k-90 , maltodextrin ( mdx ) , hydroxypropyl ethylcellulose ( hpmc - e15 , 5 ) , pectin , sodium alginate , or blends of polymers . 
 orally dissolving strips can be prepared using a solvent - casting , rolling , hot melt extrusion , or solid dispersion methods . 
 epileptic seizures can occur in nonepileptic patients subjected to a variety of stresses and stimuli . 
 epilepsy is a neurological disorder which requires quick management of seizures in order to avoid the risk of permanent brain damage . 
 management of epilepsy differs from the treatment of other diseased conditions in that a single epileptic attack has a major negative effect on quality of life . 
 clobazam is a newer 1 , 5-benzodiazepine derivative which is a well tolerated , safe , and very effective antiepileptic drug having a broad spectrum of antiepileptic activity and minimal side effects and being relatively inexpensive . 
 thus , to control the epileptic seizures in the shortest possible time , an attempt has been made to develop , evaluate , and optimize orally dissolving strips of clobazam with improved bioavailability and palatability . 
 clobazam is an ideal drug candidate for an orally dissolving strip formulation because of its indication in children and its low - dose requirement . 
 the formulation of clobazam as an orally dissolving strip , required to be placed on the patient 's tongue without swallowing for dose administration , would significantly facilitate dose administration , with subsequent improvement in patient compliance . 
 thus , the aim of this work was to design , characterize , and optimize orally dissolving strip of clobazam using two polymers : ssg ( disintegrant ) and pva ( film former ) . 
 a 3 factorial design was used to evaluate the influence of film forming polymer pva and disintegrating agent ssg on the film 's mechanical properties , disintegration time , and dissolution rate . 
 this study also assessed the in vivo performance and ivivc of the optimum formulation by administration to healthy rabbits . 
 sodium starch glycolate , peg-400 , and directly compressible mannitol were received from loba chemie pvt . 
 the pure drug clobazam and physical mixture of clobazam and polymers were mixed with ir grade kbr pellets in the ratio of 100  :  1 and corresponding pellets were prepared in a hydraulic press . 
 the pellets were scanned over a wave number range of 4000500  cm in using perkin emler spectrum 400usa , ftir instrument . 
 differential scanning calorimetry ( dsc ) analysis was undertaken to visualize the changes , if any , observed during the preparation of the orally dissolving strip using mettler toledo model dsc 821e instrument . 
 dsc of clobazam ( pure drug ) , physical blend of pva , clobazam and ssg ( polymer ) , and optimized film formulation f6 were carried out over a temperature range of 30 to 300c at a scanning rate of 5c / min . 
 solubility studies were carried out by taking   an excess amount of drug in 10  ml of different solvents and ph 6.8 buffers in conical flask , closed with aluminum foil and constantly agitated at room temperature for 24  hrs , using orbital shaking incubator ( remi instruments , c-24 bl , and mumbai , india ) . 
 further , the solutions were filtered and the amount of drug solubilised was estimated at a wave length of 232  nm by using systronics pc based double beam spectrophotometer 2202 , mumbai , india . the orally dissolving strips of clobazam using film forming polyvinyl acetate ( pva ) and peg 400 as plasticizer were prepared by solvent - casting method . 
 an aqueous solution of the polymer was prepared in warm distilled water and was kept aside for 4 hours for swelling of polymer . 
 clobazam was added to the aqueous polymeric solution after levigation with required volume of peg 400 . 
 this was followed by the addition of mannitol as a sweetener as well as a solubilizer and sodium starch glycolate as a superdisintegrant . 
 the solution was casted on a plastic petri dish and dried at room temperature for 24  hr . 
 the strip was carefully removed from the petri dish , checked for any imperfections , and cut into the required size ( 2  2  cm ) to deliver the equivalent dose of 5  mg per strip . 
 preliminary trials were undertaken for designing the orally dissolving strips where the effect of various concentrations of the film forming agent and superdisintegrants on the characteristics of the strips was noted . 
 in addition , the prepared strips were also checked for surface perfection , smoothness , and ease of removal from petri dish without rupturing , folding , or cracking . 
 a 3 full factorial design was employed to study the effect of independent variables x1 ( pva ) and x2 ( ssg ) over the dependent variables like tensile strength ( n / cm ) , disintegration time ( sec ) , and in vitro drug release ( % ) as shown in design layout tables 1 and 2 . in this design 
 , two factors were evaluated each at three levels ( 1 , 0 , + 1 ) and all possible nine experimental batches were formulated . 
 composition of all nine possible combinations of orally dissolving strip of clobazam using 3 full factorial designs is shown in table 3 . 
 the data was subjected to contour and 3d response surface plot using design - expert software version 8.0.7.1 . 
 a multiple linear regression equation incorporating interactive and polynomial terms was used to calculate the response as follows : 
 ( 1)y = b0+b1x1+b2x2+b12x1x2+b11x12+b22x22 , 
 where y is the dependent , that is , response variable , namely , disintegration time , tensile strength , and in vitro drug release ; b0 is the arithmetic mean response of the nine runs ; and b1 and b2 are the estimated coefficients for the factors x1 and x2 , respectively . 
 the main effects ( x1 and x2 ) represent the average result of changing one factor at a time from its low to high value . the interaction term ( x1x2 ) shows how the response changes when two factors are simultaneously changed . 
 the polynomial equations can be used to draw conclusions after considering the magnitude of the coefficient and the mathematical sign it carries ( i.e. , positive or negative ) . 
 the high values of the correlation coefficients for the dependent variables indicate a good fit . 
 the thickness of each strip was measured at five different locations ( centre and four corners ) using calibrated digital vernier caliper ( mituotoyo , japan ) . 
 a strip of ( 2  2  cm ) was cut and repeatedly folded at the same place till it broke . 
 the number of times the strip could be folded at the same place without breaking was taken as a measure of folding endurance . 
 the oral strip of size 4  cm was dissolved in 10  ml of phosphate buffer ph 6.8 and solution was filtered and drug content was estimated at 232  nm using double beam uv / visible spectrophotometer ( systronics 2202 , mumbai , india ) . 
 the experiments were carried out in triplicate for the strips of all formulations and average values were recorded . 
 the surface ph of orally dissolving strips was determined in order to investigate the possibility of any side effects in vivo . as 
 an acidic or alkaline ph may cause irritation to the oral mucosa , it is determined to keep the surface ph as close to neutral as possible . 
 the ph was measured by bringing the electrode in contact with the surface of the oral strip . 
 this study was performed on three strips of each formulation and mean  sd calculated . 
 in vitro disintegration time 
 was measured by placing the film ( 2  2  cm ) on stainless steel wire mesh placed in a petri dish containing 10  ml of phosphate buffer ph 6.8 . 
 time required for the oral strip to break was noted as in vitro disintegration time . 
 the test was performed on three strips of each formulation batch and mean  sd was calculated . 
 in vitro dissolution test 
 was performed according to the usp type ii paddle apparatus ( labindia ds 8000 , mumbai , india ) . 
 test was performed by fixing the oral strip ( 2  2  cm ) to rectangular glass plates so as to prevent it from floating and it was placed at the bottom of dissolution vessel containing 900  ml of phosphate buffer ph 6.8 at 37c with a rotation speed of 50  rpm . 
 a 5  ml of sample was taken at time intervals from 1 to 30  min , and the same volume was replenished with fresh buffer solution maintained at 37c . 
 the samples were filtered and analyzed at 232  nm using double beam uv / visible spectrophotometer ( systronics 2202 , mumbai , india ) ; the content of drug was calculated using equation generated from standard calibration curve of clobazam . 
 the release mechanism of clobazam from oral strip was also determined by fitting the release data to different kinetic models , zero order , first order , and higuchi . 
 the surface morphology of the optimized orally dissolving strip formulation was observed with scanning electron microscope ( hitachi s-3400n type ii model , japan ) . 
 pictures were taken at an excitation voltage of 1.0  kv and a magnification of 1000x . 
 films were cut into 2  2  cm square strips ( 4  cm ) . 
 the moisture uptake by the strip ( n = 3 ) was determined by exposing them to an environment of 75% relative humidity ( rh ) at room temperature ( 25  2c ) for 1 week [ 26 , 27 ] . 
 the uptake of moisture by the strips was measured and calculated as percent increase in weight . 
 tensile strength testing was determined at central institute of post harvesting engineering and technology ( ciphet ) , ludhiana , india , using a texture analyzer tahdi ( stable microsystem ) , equipped with a 5  kg load cell . 
 the strip was cut into 100  12.5  mm strips and equilibrated at 25c for 1 week . 
 initial grip separation was 60  mm and crosshead speed was 50  mm  min . 
 tensile strength , elongation at break , was computed to evaluate the tensile properties of the strips . 
 tensile strength ( ts ) was calculated by dividing the maximum load by the original cross - sectional area of the strip and it was expressed as ( n / cm ) . 
 percent elongation at break ( e% ) was calculated by dividing the length at the time of break of the strip by the initial length of the strip and multiplying by 100 using the following equation : 
 ( 2)e%=ll0l0100 , 
 where l0 is the initial length of the strip and l is the length at the time of break . 
 the calibration curve was performed with standards of the final concentrations of 5 , 10 , 25 , 50 , and 100  ng / ml in rabbit plasma . 
 the intraday , interday precisions and accuracy of the method were determined with three replicates spiked plasma samples at different concentrations of clobazam . 
 the intraday and interday variation was calculated in terms of percent relative to standard deviation . 
 six rabbits of either sex ( weighed 2.5  0.2  kg ) were selected for the study . 
 all the rabbits were fasted overnight before the administration of the selected fast dissolving film and marketed formulation but had free access to water . 
 the rabbits were randomly divided into two groups with each group containing three rabbits ( n = 3 ) . 
 the rabbits were positioned on a table with lower jaw supported in a horizontal position and orally dissolving strip was carefully placed on the rabbit tongue in one group as shown in figure 12 . 
 blood samples for pharmacokinetic analysis were collected from marginal ear veins of rabbits immediately before drug administration and at 5 , 10 , 15 , 30 , 60 , 120 , 180  min , 6  hrs , 12 , and 24  hrs interval . 
 the plasma samples were prepared by mixing 0.1  ml of plasma with 0.05  ml ibuprofen as internal standard ( from the stock 6000  ng / ml ) in a clean eppendorf polypropylene tube and then extracting with 1.5  ml of acetonitrile after vertical agitation ( 1  min ) and centrifugation at 1000  rpm , for 5  min . 
 the hplc analysis was performed using agilent 1200 series system by using a mobile phase composed of water ph 3.5 , adjusted with orthophosphoric acid and acetonitrile ( 55  :  45  v / v ) , and binary eluted at a flow rate of 1.5  ml  min . 
 protocols for all the animal studies were approved by institutional ethical committee [ iaec / ccp/12/pr-007 ] . 
 maximum plasma concentration cmax and the time tmax were obtained directly from the individual plasma concentration versus time curves . 
 the terminal half - life , t1/2 , was obtained from log linear regression analysis of the plasma concentration time curves in the terminal phase . 
 the area under plasma concentration time curve ( auc0t , and auc0 ) was determined by linear trapezoidal method . for 90% confidence interval , 
 the auc and cmax values were transformed into their respective logarithms and analysis of variance was calculated using software kinetica 5.0 . 
 the in vivo percentage of the drug absorbed was plotted against the in vitro percentage of the drug released to determine the correlation coefficient . 
 the fraction of the drug absorbed was determined using the wagner nelson method , using the following equation : 
 ( 3)fa=[(ct+keauc0t)keauc0]100 , 
 where fa is the fraction of drug absorbed , ct is the plasma drug concentration at time t , ke is the overall elimination rate constant , and auc0t and auc0 are areas under the curve between time zero and time t and between time zero and infinity , respectively . 
 the optimized orally dissolving formulation of clobazam was subjected to stability studies by packing the individual strip in aluminum foil and loading the formulation on remi stability chamber sc-10 plus ( remi elektrotechnik ltd . , 
 vasai , india ) as per ich guidelines for 180 days at 40c  2c/75% rh  5% . 
 samples were withdrawn at regular intervals and evaluated for in vitro drug release , disintegration time , and tensile strength . 
 ftir spectra of pure drug and physical mixture of pva and clobazam as shown in figures 1(a ) , 1(b ) , and 1(c ) indicate that there was no interaction between drug and film forming polymer used . 
 pure clobazam displays a peak characteristic of c = o stretching vibration at 1694.7  cm , aromatic ch stretching at 3075.4  cm , c  c stretching at 1493.5  cm , c 
 n stretching at 11001200  cm , ch bending at 600800  cm , and ch3 bending at 1371  cm ; the spectra of drug with pva showed all characteristics peaks of drug indicating that the drug is compatible with pva . 
 dsc thermograms of clobazam ( pure drug ) , pva ( polymer ) , clobazam and ssg , and clobazam loaded orally dissolving strip as shown in figures 2(a ) , 2(b ) , and 2(c ) illustrated a sharp endothermic peak corresponding to the melting of crystalline clobazam at 180c . 
 the melting endothermic peak of clobazam was not observed in the drug loaded pva orally dissolving strip . 
 this indicates that clobazam was uniformly dispersed and present in an amorphous state in the polymeric matrix . 
 physicochemical characterization of clobazam films as given in table 4 indicated that all the strips prepared with different polymer concentrations were flexible , smooth , transparent , nonsticky , and homogeneous . 
 it was observed that there was no significant difference in the thickness among the strips , which indicated that the orally dissolving strips were uniform . 
 however , the folding endurance of the oral strip f7f9 was found to decrease with increasing content of pva . 
 thus , it appears that the increasing amount of pva decreases the flexibility of the strip . 
 since the surface ph of orally dissolving strips was found to be around neutral ph , there will not be any kind of irritation to the mucosal lining of the oral cavity . 
 all formulations were found to contain uniform quantities of drug ranging between 97.93% and 99.96% , as indicated by content uniformity studies . 
 in vitro disintegration time of the formulated orally dissolving strips 
 increase in the concentration of superdisintegrant decreases the disintegration time of films , which was observed in f1 , f2 , f3 , and f4 ; as the amount of pva increases , the disintegration time increases because of the increasing thickness of the strip but again decreases with increasing amount of ssg and formulation with f6 exhibited minimum disintegration time . 
 mathematical relationship generated using multiple linear regression analysis for the studied variable is expressed as follows : 
 ( 4)disintegration  time=28.32222 + 1.56667x1  4.33333x20.075000x1x2  0.33333x12 + 1.66667x22 . 
 the model f - value of 42.62 implies that the model is significant . 
 in vitro drug release was found to decrease with increase in the film forming polymer concentration which may be due to increase in the thickness of the oral strip and due to increase in the time required for wetting and dissolving the drug molecule present in the polymeric matrix but increase with increase in the concentration of the disintegrant . 
 formulations f1 , f2 , and f3 showed drug release up to 79.1% , 85% , and 89% , respectively , as shown in figures 4(a ) , 4(b ) , and 4(c ) at the end of 30  min . 
 film formed with higher quantity of polymer had shown slower dissolution rate ; this might be due to the increase level of pva that results in formation of high viscosity gel layer due to more intimate contact between the particles of polymer resulting in decrease in the mobility of drug particles from the swollen matrix , which leads to a decrease in the release rate . 
 the order of drug release in each set of formulation can be given as 
 ( 5)f1<f2<f3,f4<f5<f6,f7<f8<f9 . 
 the formulation f6 showed a maximum percentage drug release of 96.6% in 30  min as shown in figure 4(b ) . 
 reflection point at 30  min in the curve might be due to the complete solubilization of pva in the medium which improve the wettability and solubilize the drug and provide the fast dissolution rate . 
 further , drop in the percent of drug release after 30  min might be due to the availability of less number of drug molecules in the free void spaces and a decrease in mobility through polymeric matrix . 
 mathematical relationship generated using multiple linear regression analysis for the response variable is expressed as follows : 
 ( 6)%  drug  release=81.701115.16833x1  + 5.1683x2 + 1.00250x1x2  4.5016x12  + 1.99833x22 . 
 the model f - value of 77.23 implies that the model is significant . tensile strength and % 
 elongation were found to increase with the increasing content of pva which may be due the increase in the elasticity nature of the film forming polymer . 
 mathematical relationship generated using multiple linear regression analysis for the studied variable is expressed as follows : 
 ( 7)tensile  strength=2.39111 + 0.56500x1  + 0.31167x20.01500x1x2  0.1916x12 + 0.12833x22 . 
 addition of ssg affects tensile strength and % elongation ; higher concentration of ssg increases thickness and crystallinity of the film , which causes decrease in the tensile strength and % elongation . 
 there was a good linear correlation ( r = 0.9896  0.9999 ) obtained by plotting the percent of clobazam released from all the orally dissolving strip formulations against the square root of time . 
 thus , it was concluded that the release of clobazam from orally dissolving strip formulations followed a diffusion - controlled drug release profile and is in agreement with the higuchi model . 
 the results of multiple linear regression analysis are shown in table 6 , indicating that for all response variables which are disintegration time , tensile strength , and % drug release , the amount of pva ( x1 ) had a negative effect while the concentration of ssg ( x2 ) had a positive effect ; it means that as the amount of ssg increases the tensile strength , disintegration time decreases and there is increase in % drug release , while as the amount of pva is increased , both the tensile strength and disintegration time increase [ 3436 ] . 
 therefore , high level of ssg and medium level of pva should be selected for the rapid disintegration and a faster in vitro drug release of the film . 
 the observed values are in good agreement with the predicted values for the optimized formulation , which demonstrate the feasibility of surface response method in the formulation of oral fast dissolving films . 
 the comparison of observed values and predicted values with % prediction error is shown in table 7 . the data of the response surface plot as shown in figures 5 , 6 , and 7 demonstrated that both x1 and x2 affect the disintegration time , tensile strength , and % release of drug . 
 the surface morphology as shown in figure 8 using scanning electron microscopy of the optimized orally dissolving strip formulation of clobazam depicted smooth surface with some little pores , which is an indication of uniform distribution of drug particles . based on the responses the formulation f6 showing the highest dissolution rate , in vitro disintegration time suitable for fast - dissolving dosage form , and satisfactory tensile strength properties 
 was chosen for subsequent comparative study relative to a marketed clobazam formulation as shown in table 8 . 
 comparative results of in vitro drug release of f6 formulation and marketed formulation ( frisium5 ) as shown in figure 9 indicate that the optimized formulation was comparable to the marketed formulation . 
 the results from the hplc method validation in the rabbit plasma are shown in table 9 . 
 the within day and between day precision as given in terms of % relative standard deviation ranged from 0.5 to 6.2% and from 1.1 to 1.6% , respectively . 
 the mean clobazam plasma concentration time profiles for the prepared orally dissolving strip and the marketed tablets are shown in figure 10 . 
 the statistical comparison of cmax , auc0t , and auc0 indicated no significant difference between the two treatments ( test and reference marketed tablets ) . 
 the 90% confidence intervals for the mean ratio ( test / reference ) of cmax , auc0t , and auc0 were 95.87% , 98.12% , and 99.21% , respectively , and are shown in table 10 , while the acceptable range is 80125% for auc0t and auc0 , and 70143% for cmax , as proposed by the fda . 
 in vitro - in vivo correlation as shown in figure 11 of percentage drug absorbed and percentage drug released showed good in vitro - in vivo correlation for optimized film formulation f6 with r value 0.994 . 
 it was concluded that there was no significant change observed in the weight of orally dissolving strip , tensile strength , and % in vitro drug release . 
 the average clobazam content of formulation f6 after 6-month storage was 95.1  0.17  mg ( ranging from 91.88 to 96.80% ) . 
 therefore , the prepared formulation was stable up to 6 months at 40c ( 75%rh ) . 
 the orally dissolving strips of clobazam prepared using pva as film forming material and ssg as disintegrant by the solvent - casting method showed satisfactory drug dissolution and acceptable physicomechanical characteristics . 3 factorial design was used for the optimization , amongst nine formulations prepared as per the design layout , which indicates that the film prepared using 100  mg of pva and 6% of ssg ( f6 ) showed the highest dissolution rate , suitable in vitro disintegration time , and satisfactory tensile strength and was selected as the optimized formulation . in vivo studies also indicated absence of significant difference between f6 and frisium5 marketed tablets and both exhibited comparable drug plasma level - time profiles . accelerated stability studies results showed that prepared formulation was stable enough for the period of at least 6 months . 
 therefore , the present orodispersible film formulation containing clobazam considered is potentially useful for the treatment of epileptic attack where improved patient compliance and convenience are expected and can be used as an alternative to the fast dissolving tablet formulation .","clobazam orally dissolving strips were prepared by solvent casting method . 
 a full 32 factorial design was applied for optimization using different concentration of film forming polymer and disintegrating agent as independent variable and disintegration time , % cumulative drug release , and tensile strength as dependent variable . in addition 
 the prepared films were also evaluated for surface 
 ph , folding endurance , and content uniformity . 
 the optimized film formulation showing the maximum in vitro drug release , satisfactory in vitro disintegration time , and tensile strength was selected for bioavailability study and compared with a reference marketed product ( frisium5 tablets ) in rabbits . formulation ( f6 ) was selected by the design - expert software which exhibited dt ( 24  sec ) , ts ( 2.85  n / cm2 ) , and in vitro drug release ( 96.6% ) . 
 statistical evaluation revealed no significant difference between the bioavailability parameters of the test film ( f6 ) and the reference product . 
 the mean ratio values ( test / reference ) of cmax ( 95.87% ) , tmax ( 71.42% ) , auc0t ( 98.125% ) , and auc0 ( 99.213% ) indicated that the two formulae exhibited comparable plasma level - time profiles .",pubmed
"stroke is a major cause of death and disability world - wide . by 2020 stroke , together with coronary - artery disease , 
 stroke represents an often devastating disruption to life , the majority of survivors experiencing some degree of impairment requiring additional care or support 1 year post - stroke . 
 stroke is an acute event , but may result in significant long - term impact for the individual , such as social isolation , mood disturbance , communication difficulties and reduction in mobility and life roles . 
 recovery following stroke is complex and multidimensional , encompassing bio - medical , psychological and sociological elements [ 911 ] . 
 engagement in self - management practices by individuals with long - term conditions has been suggested as key to promoting recovery   and is cited as a means of empowerment and facilitator of improved health outcomes . 
 self - management is a prominent issue in uk health policy [ 1517 ] and has been identified as a key priority for health by organisations independent of the uk government . 
 self - management can be defined as the  active management by individuals of their treatment , symptoms , lifestyle , physical and psychological consequences inherent with living with a chronic condition  . 
 self - management is an attractive initiative in managing the increasing burden on health and social care resources and reducing associated costs ; the assumption being that effective self - management by an individual reduces their healthcare utilisation [ 2123 ] . 
 self - management interventions ( smis ) are designed to enable people to manage their health more effectively . 
 evaluation therefore must consider two key areas ; firstly whether people develop the skills to manage their own health and secondly , if this consequently results in better health . 
 is complex , not least due to variation in delivery , culture of the sponsoring healthcare organisation and anticipated goals and outcomes . 
 the uk medical research council advocates establishing the theoretical basis of an intervention as a first step in estimating its possible outcomes . 
 currently , evidence suggests that the mediators of change and theoretical premises in smis are unclear [ 2830 ] . 
 firstly , doubt exists regarding the appropriate outcome(s ) to monitor to assist evaluation of the intervention and aid determination of cost - effectiveness and clinical impact . 
 secondly , if the theoretical premises underpinning the intervention are uncertain , intervention fidelity is difficult to monitor and maintain . questions then exist regarding what influences change and how this can be appropriately measured and smis evaluated . 
 smis may be evaluated by examining the effect on health outcomes that potentially change as a consequence of better self - management . using patient 
 reported outcome measures ( proms ) ( e.g. functional status , symptom control , mood and health - related quality of life ) is an important way of to ensuring evaluation considers outcomes important to patients . 
 preliminary investigation of self - management suggests that effective self - management corresponds with positive changes in health behaviour . 
 more recently there has been a focus upon measuring attitudes since these are thought to modify health behaviour [ 2931 ] . 
 proms endeavor to capture information that is not directly observable and unmediated by healthcare professionals ; consequently accurate measurement is contingent on the extent that the prom is an accurate reflection of the variable in question . 
 therefore , it is vital to evaluate whether the measures adopted in smi studies provide legitimate information to evaluate self - management , both the process and obtaining of skills to better manage health and subsequent potential improvements in health . before using an outcome measure in research or clinical practice , 
 it should be assessed and considered to possess adequate psychometric properties . despite the recognised value of reliable and valid outcome measures and the increasing importance of identifying effective smis in stroke , we know of no review that has systematically evaluated international research for the quality of outcome measures used in stroke self - management . 
 the purpose of this article is to systematically review outcome measures used in stroke self - smis , with the aim of informing researchers , healthcare professionals and policy - makers and making recommendations for the design of future outcome measures suitable for use in stroke self - management . 
 this review seeks to systematically examine the outcome measures adopted in stroke smis in terms of the methodology adopted in their development and subsequent strength of their psychometric properties for use with stroke populations . 
 often the methodology adopted in reviews of outcome measures , use differing assessment standards , creating confusion for researchers and clinicians . a recent international delphi study of 57 experts ( 63% response rate ) resulted in a tool to assess the methodological quality of studies on measurement properties , referred to as the consensus - based standards for the selection of health status measurement instruments ( cosmin ) checklist . 
 the cosmin list has good inter - rater agreement and reliability   and represents the first critical appraisal tool that is based on the consensus of experts in psychometric theory . 
 cosmin has been used in other systematic reviews examining the measurement properties of outcome measures in a range of health conditions [ 3941 ] . the delphi study consisted of four rounds and sought to reach consensus on the terminology and definitions to be adopted with regard to psychometrics . 
 this consensus offers both researchers and clinicians guidance with regard to some of the complexities of measurement properties . 
 cosmin also addresses modern psychometric theory methodology , such as item response theory , as well as classical test theory . 
 further information on cosmin can be accessed via www.cosmin.nl 
  the properties examined in this systematic review are defined by cosmin   and consist of nine items : internal consistency ; reliability ; measurement error ; content validity ; structural validity ; hypothesis testing ; cross - cultural validity ; criterion validity and responsiveness . 
 the purpose of the review is not to make a judgement on the quality of the smi studies , or to synthesize findings to answer questions regarding the effectiveness of the interventions in the review . 
 instead the review focuses upon the value of the outcome measures adopted within stroke smi studies according to their methodological quality , reliability and validity for stroke populations as outlined by cosmin .this is vital since judgments about the results and impact attributed to smis are dependent on valid and reliable measurement . in order to examine the properties of the outcome measures used in stroke smis , it was first necessary to identify which measures were used to evaluate the smis . 
 stroke self - management literature was systematically searched on the following electronic databases by one author ( e.j.b . ) : medline , psychinfo , science direct , web of science and cinahl . 
 the following terms were used to identify existing stroke smi studies : search terms were chosen to represent concepts often linked to self - management ( education , rehabilitation ) , however , studies were excluded unless they specifically stated their purpose was to enhance self - management . 
 article reference lists , website of uk government health department , generic internet search engines , and stroke - specific organisations were also searched . 
 dissertations and conference abstracts were excluded , however , searches for publications by dissertation or conference abstract authors were conducted . 
 selected articles described either ( 1 ) stroke - smi development and/or implementation or ( 2 ) presented outcomes of stroke smis . identified interventions and associated outcome measures were extracted and tabulated . 
 authors screened each abstract to eliminate articles that were not relevant , based on the following inclusion criteria : the study was published in english ; the article addressed self - management specific to stroke ; and was published between january 1990 and june 2011 , to examine the current and most relevant evidence for practice . 
 one reviewer ( e.j.b . ) assessed all relevant full text articles obtained and a second reviewer ( s.d . ) assessed 10% to check reliability . 
 outcome measures included in studies were then identified and recorded ( the version adopted by the smi was selected for review ) . a second literature search then sought to find evidence for the measurement properties ( as outlined by cosmin ) of those outcome measures identified . 
 the following electronic databases were used : medline , psychinfo , science direct , web of science and cinahl . 
 the following search terms were used in conjunction with the title of the identified outcome measure : for example , for a search on the validity of the geriatric depression scale , the search terms  stroke  and  validity  and  geriatric depression scale  was performed . 
 search terms were chosen to represent the properties of outcome measure quality as outlined by the cosmin checklist . 
 no time limitations were set as advocated by cosmin , since older literature on measurement properties is still relevant . 
 this is crucial since a measure s reliability and validity are on - going properties , dependent upon the context and population with which it is used . 
 for example , a measure developed to assess quality of life with a traumatic brain injury population will not necessarily possess acceptable content validity for stroke populations , since the issues faced by both populations may have similarities and differences . 
 article reference lists from the originating studies and generic internet search engines were also searched . 
 discussion between the authors sought to clarify any issues regarding terminology and interpretation of the cosmin checklist and preceded scoring of the identified measurement tools . 
 studies were excluded if they investigated postal or proxy reliability and validity unless this was how they were used in the smis . for outcome measures with more than one result per cosmin criteria , the article stating the most robust results was reviewed . where it was not clear that the study populations were specifically stroke , articles were excluded . 
 one reviewer ( e.j.b . ) assessed all relevant full text articles using a standard data extraction form advocated by cosmin . to ensure consistency of interpretation and scoring , a second reviewer ( s.d . ) independently scored a random 10% of the articles , with discussion between the two reviewers regarding the scores attained . 
 paper authors were contacted for further details where the study reported on early phases , or cited unpublished work . 
 ten items are used to assess whether a study meets the standard for good methodological quality . 
 two items contain general requirements for articles in which item response theory ( irt ) methods and general requirements for the generalisability of the results are applied . where a published paper does not report on a cosmin item 
 for example , if the responsiveness of the measure has yet to be determined , this item is not scored . 
 each item is rated as excellent ( + + + + ) , good ( + + + ) , fair ( + + ) or poor ( + ) . 
 43 abstracts were identified as potentially relevant studies and were screened ( 46 duplicate records were excluded ) . from these , 
 19 articles were retrieved and reviewed for inclusion criteria and data extraction ( studies were excluded because they did not meet the detailed criteria or if they reported on an earlier phase of the same study ) . 
 outcome measures within each study were then identified and grouped conceptually into different themes , using content analysis . 
 three studies of stroke - smis originated from the uk ; three from australia ; two from canada ; two from the usa ; two from sweden and one from hong kong . 
 nine studies reported upon interventions aimed at community - dwelling participants ; two upon the acute recovery phase ( <3 months post - stroke ) ; one upon recovery for care home residents and one study did not report details of the setting . 
 four studies delivered individualised interventions ; three utilised workbook interventions ; four tested group smis designed specifically for stroke and two tested existing self - management programs adapted for stroke . 
 interventions were delivered primarily ; by allied health professionals ( six ) ; nurse specialists ( four ) ; researcher ( two ) and lay experts ( one ) . 
 four studies identified primary outcomes ; health - related quality of life ; self - efficacy ; physical functioning   and feasibility . 
 although all studies focused upon stroke self - management , none measured stroke self - management as a discrete concept . instead , a range of concepts were measured which presumably were selected to reflect the expected outcomes or process of self - management . 
 evaluation relies upon judgements concerning the process of the smi and the outcome expected following participation in the smi . 
 the majority of measures used sought to measure health outcomes e.g. physical functioning , mood , quality of life ( figure 1 ) . 
 attitudes were also measured which could be considered to more readily reflect the process of self - management e.g. healthcare utilization , medication compliance although the theoretical mechanisms linking self - management to these concepts was not elucidated by the authors . 
 the term  unreported  is used in this review to describe an outcome measure that has not , at time of writing , been published in peer reviewed publicly available media . 
 unreported measures relate to those developed either by the study authors or through modifications made to existing measures without examination to ensure such assumptions or modifications were valid . 
 therefore , it is not possible to determine if unreported measures meet any of the cosmin criteria . 
 six studies adopted unreported measures of concepts presumably ( although the theoretical links were not explicitly stated by the authors ) , relating to the process of self - management , that had unknown reliability , validity , responsiveness . 
 allen et al .   included an unreported measure to assess condition management and patient and carer satisfaction with the intervention . 
 two studies used 10 point visual analogue rating scales ( vass ) to assess usefulness and intelligibility   and satisfaction   related to the intervention . whilst vass are brief and simple to administer and minimal in terms of respondent burden , without established reliability or validity relating to the underlying construct purported to be measured , they remain of limited value . 
 ljungberg and colleagues   designed four questions to assess life satisfaction pre- and post - participation in the smi and sit and colleagues   modified an existing stroke knowledge scale ; details of the modifications were absent in the paper . 
 marsden and colleagues   used a measure of stroke knowledge test , but stated clearly they were not basing inferences from the data obtained using this measure . 
 forty - three different outcome measures were adopted by studies in this review of measures used in stroke smis . of these , 
 21 measures ( 49% ) demonstrated some properties in stroke populations , according to the cosmin checklist   ( table ii ) . 
 for the remaining measures no evidence could be found for any of the cosmin properties in stroke populations ( n = 16 , 39% ) , or the measures were observer - based assessments ( n = 5 , 12% ) . 
 cosmin property scores in stroke populations of outcome measures used in stroke self - management interventions . 
 a summary of how the measures included in the stroke smi studies scored according to cosmin , when examined for their measurement properties with stroke populations , is shown in figure 2 . 
 none scored in every category of cosmin . where more than one paper addressed a cosmin category , the article which stated the most robust results was scored . the majority of measures scored either 
 excellent  rating was content validity . three measures scored  excellent  in this category as follows ; the stroke adapted sickness impact profile ( sa - sip30 ) ; the stroke self - efficacy questionnaire ( sseq ) and the subjective index of physical and social outcome ( sipso ) . 
 43 abstracts were identified as potentially relevant studies and were screened ( 46 duplicate records were excluded ) . from these , 
 19 articles were retrieved and reviewed for inclusion criteria and data extraction ( studies were excluded because they did not meet the detailed criteria or if they reported on an earlier phase of the same study ) . 
 outcome measures within each study were then identified and grouped conceptually into different themes , using content analysis . 
 . three studies of stroke - smis originated from the uk ; three from australia ; two from canada ; two from the usa ; two from sweden and one from hong kong . 
 nine studies reported upon interventions aimed at community - dwelling participants ; two upon the acute recovery phase ( <3 months post - stroke ) ; one upon recovery for care home residents and one study did not report details of the setting . 
 four studies delivered individualised interventions ; three utilised workbook interventions ; four tested group smis designed specifically for stroke and two tested existing self - management programs adapted for stroke . 
 interventions were delivered primarily ; by allied health professionals ( six ) ; nurse specialists ( four ) ; researcher ( two ) and lay experts ( one ) . 
 four studies identified primary outcomes ; health - related quality of life ; self - efficacy ; physical functioning   and feasibility . although all studies focused upon stroke self - management , none measured stroke self - management as a discrete concept . instead 
 , a range of concepts were measured which presumably were selected to reflect the expected outcomes or process of self - management . 
 evaluation relies upon judgements concerning the process of the smi and the outcome expected following participation in the smi . 
 the majority of measures used sought to measure health outcomes e.g. physical functioning , mood , quality of life ( figure 1 ) . 
 attitudes were also measured which could be considered to more readily reflect the process of self - management e.g. healthcare utilization , medication compliance although the theoretical mechanisms linking self - management to these concepts was not elucidated by the authors . 
 the term  unreported  is used in this review to describe an outcome measure that has not , at time of writing , been published in peer reviewed publicly available media . 
 unreported measures relate to those developed either by the study authors or through modifications made to existing measures without examination to ensure such assumptions or modifications were valid . 
 therefore , it is not possible to determine if unreported measures meet any of the cosmin criteria . 
 six studies adopted unreported measures of concepts presumably ( although the theoretical links were not explicitly stated by the authors ) , relating to the process of self - management , that had unknown reliability , validity , responsiveness . 
 allen et al .   included an unreported measure to assess condition management and patient and carer satisfaction with the intervention . 
 two studies used 10 point visual analogue rating scales ( vass ) to assess usefulness and intelligibility   and satisfaction   related to the intervention . whilst vass are brief and simple to administer and minimal in terms of respondent burden , without established reliability or validity relating to the underlying construct purported to be measured , they remain of limited value . 
 ljungberg and colleagues   designed four questions to assess life satisfaction pre- and post - participation in the smi and sit and colleagues   modified an existing stroke knowledge scale ; details of the modifications were absent in the paper . 
 marsden and colleagues   used a measure of stroke knowledge test , but stated clearly they were not basing inferences from the data obtained using this measure . 
 forty - three different outcome measures were adopted by studies in this review of measures used in stroke smis . of these , 21 measures ( 49% ) demonstrated some properties in stroke populations , according to the cosmin checklist   ( table ii ) . for the remaining measures no evidence could be found for any of the cosmin properties in stroke populations ( n = 16 , 39% ) , or the measures were observer - based assessments ( n = 5 , 12% ) . 
 cosmin property scores in stroke populations of outcome measures used in stroke self - management interventions . 
 a summary of how the measures included in the stroke smi studies scored according to cosmin , when examined for their measurement properties with stroke populations , is shown in figure 2 . 
 none scored in every category of cosmin . where more than one paper addressed a cosmin category , the article which stated the most robust results was scored . the majority of measures scored either 
 three measures scored  excellent  in this category as follows ; the stroke adapted sickness impact profile ( sa - sip30 ) ; the stroke self - efficacy questionnaire ( sseq ) and the subjective index of physical and social outcome ( sipso ) . 
 this review examined the methodological quality of studies determining the psychometric properties of outcome measures , used in stroke smis according to criteria outlined by the cosmin checklist . 
 consistent with measurement theory we explored the validity and reliability of these measures for use in people with stroke , not their general use in broader populations . to our knowledge , this is the first review to systematically appraise and summarize the evidence on the quality of outcome measures used in stroke smis . 
 since no study adopted a measure of stroke self - management attitudes or behaviours , the theoretical concepts utilised by studies in the review to measure self - management will first be addressed . 
 the range and number of different published outcome measures adopted by studies in this review   may suggest a current lack of consensus regarding the appropriate measures to assist evaluation of stroke smis . alternatively , the use of heterogeneous measures may be reflective of recognition by researchers that self - management embraces a range of differing concepts . 
 the current absence of consensus may in part reflect an underlying lack of consensus about the concept and operation of self - management in stroke . 
 in addition , most smis have been developed for generic audiences , which may partly explain the lack of specific measures developed for stroke self - management . 
 an argument exists for research to investigate the conceptual properties of stroke self - management , to examine which measurement concepts currently being used , if any , are appropriate . 
 some captured health outcomes , such as physical functioning , which the study authors anticipated may be affected by the smi ; others attempted to capture behaviours , such as resource utilisation or attitudes , such as changes in self - efficacy thought to be associated with self - management processes ( figure 2 ) . 
 however , how the concepts measured align with the patient experience of stroke self - management remain unknown . physical function ( pf ) was most often used as an indicator of effective self - management . of the 21 measures possessing at least one property of the cosmin checklist in this review , 11 related to pf ( 52% ) . 
 this is potentially suggestive of an assumption that effective self - management results in improved pf , or that improved pf is a desired outcome . 
 pf appears to remain a dominant concept within stroke rehabilitation , despite increasing evidence of the role of psychosocial factors in recovery [ 5355 ] . 
 for example , the measurement of pf is of limited value in studies that target speech disorder , depression , social participation or cognitive function , debatably all factors in effective stroke self - management . 
 questions regarding the differing priorities of rehabilitation between healthcare professionals and those affected by stroke have been raised before . 
 effective self - management extends beyond the ability to perform certain tasks , encompassing decision making and choices regarding health and behaviour . 
 the role of pf in stroke self - management requires further clarification before it can be adopted as a robust indicator of effective self - management . 
 . however , issues of potential greater importance to patients , for example a change in confidence or increased awareness about how to manage fatigue , may not be captured in measures focused upon management of health behaviours or resource utilisation . 
 there is a need to further conceptualise stroke self - management to ensure that self - management strategies pertinent to people recovering from stroke are captured in existing or new outcome measures . 
 eight of the studies in this review explicitly cited a theoretical basis to the intervention adopted in the study ( table i ) . 
 the most commonly cited theory was psychologist albert bandura s social cognition theory and the concept of self - efficacy ( n = 4 studies ) . 
 self - efficacy can be described as the belief in one s capabilities to organise and execute the course of action required , to produce given achievements . 
 the validity of outcome measures is contingent upon using them for the purpose they were intended for . 
 of the four studies citing self - efficacy as a theoretical premise underpinning the intervention , only two studies utilised outcome measures to reflect change attributed to this theoretical concept in the interventions . the measure adopted by kendall and colleagues , the self - efficacy scale , 
 has unknown psychometric properties in stroke populations , and therefore requires further examination to establish its validity for use in these populations . 
 the stroke self - efficacy scale adopted by jones and colleagues   was developed with stroke populations . 
 data were generated with people a relatively short time frame since stroke ( mean duration was 4.2 weeks and 16 days post - stroke for two of the development phases ) . 
 this may not represent sufficient time since stroke for individuals to adequately appraise their situation , especially as some were still in hospital . 
 the relationship to self - management of any of the measures in this review was not explicitly stated by any of the study authors . 
 this suggests that further clarification is required to determine the extent to which they reflect the process or outcomes of self - management . 
 whilst potential theoretical bases for the self - management of long - term conditions , such as self - efficacy , have gained increasing acknowledgement , the role in stroke self - management remains unclear . 
 this is in part due to a lack of robust outcome measures and , in addition , a lack of clarity regarding the purported theoretical foundations of stroke self - management . a paucity of measures scored  excellent  or  good  for quality according to the criteria outlined by cosmin ( figure 2 ) . 
 the cosmin checklist does not advocate summarising the quality criteria into one overall quality score , as is often the case in other systematic reviews . 
 since measurement properties are in part affected by the context in which they have been determined , this approach would be misleading . 
 for example in our review , no measure was scored on cross - cultural validity , since the purpose of the review was not to assess how well a measure had been developed and validated in other languages or cultures . 
 outcome measures should be developed with involvement of the target population to identify what is meaningful from their perspective and hence enhance content validity and clinical utility . 
 three measures scored  excellent  in the content validity category ( sseq ; sipso ; sa - sip30 ) . 
 measures that did not include involvement of users in the development of the measure scored  poor  on the cosmin checklist , regardless of other aspects of the content validity process which may have been classed  fair 
 this is partly as a result of the cosmins scoring method in which the lowest score in any given category counts , but is also indicative of the importance of involving potential users in measurement development . 
 arguably , measures developed without user - involvement have questionable meaning and other types of validity , since without steps in the design to capture the experience of the population to be measured , the context of the measure remains largely that of the measure developers . 
 more recently techniques such as cognitive interviewing , have been used by researchers   to ensure the content validity of new measures is optimal . 
 difficulty exists in determining which measures used in the stroke smis in this review reflect self - management with validity since most measures did not score well according to the cosmin criteria . 
 several studies included unreported measures , designed specifically by the authors for the purpose of the smi study [ 44,4852 ] . with the exception of marsden et al . 
 an absence of psychometric data confounds the ability to draw reliable inferences from studies adopting those measures . 
 in addition , a lack of information regarding the development or modification of unreported measures limits the ability to make judgments upon the validity and appropriateness of the measure . 
 a further possible limitation on interpreting data from unreported measures may be a tendency for reporting positive results . without establishment of reliability and validity 
 the outcome measure is little more than a collection of items that have meaning to the developer alone . given the lack of consensus of how stroke self - management operates in the literature , and a lack of consensus upon the theoretical premises grounding stroke smis , the assumptions underpinning unreported measures remain speculative . 
 researchers and clinicians should exercise caution in considering findings from studies adopting unreported measures . of note 
 is that 11 studies ( 85% ) within this review adopted at least one outcome measure without reported validity and reliability with stroke populations . 
 the reporting of minimal , or non - significant , observed changes following stroke smis in those studies including measures without established psychometric properties in stroke populations may be indicative of a lack of relevance and meaningfulness of those measures to stroke populations . 
 problems exist in using unreported measures when determining whether change occurred as a result of an ineffective intervention or due to imprecise measures . 
 measures developed with intended user populations , facilitate the gaining of information about health , illness and the effects of health - care interventions from the perspective of the patient . as well as enhancing content validity , 
 this is of particular relevance to those involved in promoting self - management and increasing patient autonomy , such as nurses and therapists . 
 responsiveness is a necessary property of instruments intended for measuring clinically meaningful change , such as in stroke self - smis . 
 arguments exist that responsiveness should focus upon detecting change that is valued by the person rather than the clinician or researcher . 
 change attributed to an intervention is an important aspect of evaluating clinical effectiveness . in this review , 
 none of these measures scored  excellent  for this property , and only 15% scored  good  . aside from inadequate sample sizes , a common finding was that studies often were not clear about what happened to study populations between testing . 
 there is , therefore , a need for future measurement developers to specify these overlooked aspects of development more clearly in subsequent reporting . 
 the majority of smi study populations within this review experienced stroke < 24 months previously , with a number of studies using populations experiencing stroke no more than 6 months previously . 
 this may be a result of sampling to reduce the influence of additional factors upon study outcomes , such as the development of unhelpful coping behaviours , the likelihood of which might increase over time , and out of an assumption that more change may be observed in those early in their recovery . 
 however , in reality the number of people living in the community and recovering from stroke extends beyond those who are 624 months post - stroke . as engagement in self - management activities varies during recovery , particularly following adjustment to stroke as a long - term condition , there is a future need to consider outcome measures sensitive to change(s ) at different durations since stroke . 
 the role of proms , developed using rigorous investigation with the population to be measured extends beyond validating patient experience . 
 proms may improve the quality of interactions between health professionals and patients , assess levels of health and need , and provide evidence of outcomes of services , for the purposes of audit , quality assurance and comparative performance evaluation . 
 this is of particular importance when trying to capture the essence of self - management , since the experience of clients is vital in determining what is valued from their perspective . 
 there is a need to focus upon the development of measures of self - management developed with people recovering from stroke . 
 consensus between reviewers was used to determine eligibility and inclusion of smi articles . whilst we were in agreement 
 however , the use of a standardized critical appraisal tool may assist the selection of articles for future reviews . 
 where interpretation of the cosmin criteria differed , agreement was reached by discussion and consensus . 
 additional reviewers may have further validated this process however the criteria within cosmin are explicitly stated and differences were quickly resolved . 
 data extraction was facilitated by a standardized tool advocated by cosmin , with extraction and scoring checked in a random 10% of articles.we acknowledge that checking of 10% may be viewed as a limitation of this review , however , assert that a systematic process using a standard data extraction tool was followed throughout . that cosmin operates a 
 lowest score counts  scoring system may account for the lack of measures scoring well in the measurement property criteria . some studies used otherwise appropriate methodologies , but were rated as  poor  due to inadequate sample sizes for analyses . for example , for a measure to be rated as  good  for reliability , measurement error , criterion validity and responsiveness , a sample size of n = 5099 
 , the sample size required increased to five times the number of items within a measure ( and 100 or 57 * # items but < 100 ) . 
 therefore , if those studies were repeated with larger sample sizes , their ratings according to cosmin could change dramatically . 
 the tendency for measures to score poorly may be reflective of a floor effect of the cosmin checklist . 
 cosmin was developed following consensus of experts in health measurement , therefore if its stringent criteria is to be adopted this is indicative of a need to debate the rigorous methods for measure development required . 
 it is fair to comment that some of the measures in this review were developed before the focus upon involving potential users in measure development . 
 it may be that the measures examined in this review require further development and investigation to establish adequate measurement properties for use with stroke populations . 
 our recommendations for clinicians and researchers seeking to evaluate such interventions would be firstly to clarify the theoretical premise of the intervention in question , as advocated elsewhere . without this step , 
 it is difficult to identify the mechanisms by which the intervention may influence outcomes , and thus difficult to select an outcome measure which appropriately captures the potential outcome . 
 potential outcome measures should be selected on the basis that they appropriately reflect and capture the expected outcome change . 
 this review highlights that the reported theoretical drivers within stroke smis are unclear , not least because they are often not explicitly stated by researchers . 
 the heterogeneity of the outcome measures utilised by smis in this review may indicate a difficulty in determining the expected outcomes of stroke smis . 
 a systematic review demonstrated that interventions with specific aims , such as reduced systolic blood pressure in hypertension or glycosylated haemoglobin levels in diabetes , produced greater effect sizes than those without defined outcomes . 
 further work is therefore warranted to conceptualise stroke self - management and examine the theoretical premises supporting such interventions , and expected outcomes so that appropriate outcome measures which accurately reflect the concept can be selected and/or developed . until such clarification , researchers and clinicians should , where possible , select outcome measures with reliability and validity data in the population to be tested in the intervention . the selection of outcome measures developed with involvement from the target population is also advocated . 
 this ensures that what is meaningful to the patient is more likely to be captured appropriately , thus enhancing content validity . in the meantime 
 , researchers must support clinicians by conducting further work to examine the concept and theoretical premises of self - management and developing appropriate measures if required . 
 the range and number of different published outcome measures adopted by studies in this review   may suggest a current lack of consensus regarding the appropriate measures to assist evaluation of stroke smis . alternatively , the use of heterogeneous measures may be reflective of recognition by researchers that self - management embraces a range of differing concepts . 
 the current absence of consensus may in part reflect an underlying lack of consensus about the concept and operation of self - management in stroke . 
 in addition , most smis have been developed for generic audiences , which may partly explain the lack of specific measures developed for stroke self - management . 
 an argument exists for research to investigate the conceptual properties of stroke self - management , to examine which measurement concepts currently being used , if any , are appropriate . 
 some captured health outcomes , such as physical functioning , which the study authors anticipated may be affected by the smi ; others attempted to capture behaviours , such as resource utilisation or attitudes , such as changes in self - efficacy thought to be associated with self - management processes ( figure 2 ) . 
 however , how the concepts measured align with the patient experience of stroke self - management remain unknown . physical function ( pf ) was most often used as an indicator of effective self - management . of the 21 measures possessing at least one property of the cosmin checklist in this review , 11 related to pf ( 52% ) . 
 this is potentially suggestive of an assumption that effective self - management results in improved pf , or that improved pf is a desired outcome . 
 pf appears to remain a dominant concept within stroke rehabilitation , despite increasing evidence of the role of psychosocial factors in recovery [ 5355 ] . 
 for example , the measurement of pf is of limited value in studies that target speech disorder , depression , social participation or cognitive function , debatably all factors in effective stroke self - management . 
 questions regarding the differing priorities of rehabilitation between healthcare professionals and those affected by stroke have been raised before . 
 effective self - management extends beyond the ability to perform certain tasks , encompassing decision making and choices regarding health and behaviour . 
 the role of pf in stroke self - management requires further clarification before it can be adopted as a robust indicator of effective self - management . 
 . however , issues of potential greater importance to patients , for example a change in confidence or increased awareness about how to manage fatigue , may not be captured in measures focused upon management of health behaviours or resource utilisation . 
 there is a need to further conceptualise stroke self - management to ensure that self - management strategies pertinent to people recovering from stroke are captured in existing or new outcome measures . 
 eight of the studies in this review explicitly cited a theoretical basis to the intervention adopted in the study ( table i ) . 
 the most commonly cited theory was psychologist albert bandura s social cognition theory and the concept of self - efficacy ( n = 4 studies ) . 
 self - efficacy can be described as the belief in one s capabilities to organise and execute the course of action required , to produce given achievements . 
 the validity of outcome measures is contingent upon using them for the purpose they were intended for . 
 of the four studies citing self - efficacy as a theoretical premise underpinning the intervention , only two studies utilised outcome measures to reflect change attributed to this theoretical concept in the interventions . the measure adopted by kendall and colleagues , the self - efficacy scale , 
 has unknown psychometric properties in stroke populations , and therefore requires further examination to establish its validity for use in these populations . 
 the stroke self - efficacy scale adopted by jones and colleagues   was developed with stroke populations . 
 data were generated with people a relatively short time frame since stroke ( mean duration was 4.2 weeks and 16 days post - stroke for two of the development phases ) . 
 this may not represent sufficient time since stroke for individuals to adequately appraise their situation , especially as some were still in hospital . 
 the relationship to self - management of any of the measures in this review was not explicitly stated by any of the study authors . 
 this suggests that further clarification is required to determine the extent to which they reflect the process or outcomes of self - management . 
 whilst potential theoretical bases for the self - management of long - term conditions , such as self - efficacy , have gained increasing acknowledgement , the role in stroke self - management remains unclear . 
 this is in part due to a lack of robust outcome measures and , in addition , a lack of clarity regarding the purported theoretical foundations of stroke self - management . 
 a paucity of measures scored  excellent  or  good  for quality according to the criteria outlined by cosmin ( figure 2 ) . 
 the cosmin checklist does not advocate summarising the quality criteria into one overall quality score , as is often the case in other systematic reviews . 
 since measurement properties are in part affected by the context in which they have been determined , this approach would be misleading . 
 for example in our review , no measure was scored on cross - cultural validity , since the purpose of the review was not to assess how well a measure had been developed and validated in other languages or cultures . 
 outcome measures should be developed with involvement of the target population to identify what is meaningful from their perspective and hence enhance content validity and clinical utility . 
 three measures scored  excellent  in the content validity category ( sseq ; sipso ; sa - sip30 ) . 
 measures that did not include involvement of users in the development of the measure scored  poor  on the cosmin checklist , regardless of other aspects of the content validity process which may have been classed  fair 
 this is partly as a result of the cosmins scoring method in which the lowest score in any given category counts , but is also indicative of the importance of involving potential users in measurement development . 
 arguably , measures developed without user - involvement have questionable meaning and other types of validity , since without steps in the design to capture the experience of the population to be measured , the context of the measure remains largely that of the measure developers . 
 more recently techniques such as cognitive interviewing , have been used by researchers   to ensure the content validity of new measures is optimal . 
 difficulty exists in determining which measures used in the stroke smis in this review reflect self - management with validity since most measures did not score well according to the cosmin criteria . 
 several studies included unreported measures , designed specifically by the authors for the purpose of the smi study [ 44,4852 ] . with the exception of marsden et al . 
 an absence of psychometric data confounds the ability to draw reliable inferences from studies adopting those measures . 
 in addition , a lack of information regarding the development or modification of unreported measures limits the ability to make judgments upon the validity and appropriateness of the measure . 
 a further possible limitation on interpreting data from unreported measures may be a tendency for reporting positive results . without establishment of reliability and validity 
 the outcome measure is little more than a collection of items that have meaning to the developer alone . given the lack of consensus of how stroke self - management operates in the literature , and a lack of consensus upon the theoretical premises grounding stroke smis , the assumptions underpinning unreported measures remain speculative . researchers and clinicians should exercise caution in considering findings from studies adopting unreported measures . of note 
 is that 11 studies ( 85% ) within this review adopted at least one outcome measure without reported validity and reliability with stroke populations . 
 the reporting of minimal , or non - significant , observed changes following stroke smis in those studies including measures without established psychometric properties in stroke populations may be indicative of a lack of relevance and meaningfulness of those measures to stroke populations . 
 problems exist in using unreported measures when determining whether change occurred as a result of an ineffective intervention or due to imprecise measures . 
 measures developed with intended user populations , facilitate the gaining of information about health , illness and the effects of health - care interventions from the perspective of the patient . as well as enhancing content validity , 
 this is of particular relevance to those involved in promoting self - management and increasing patient autonomy , such as nurses and therapists . 
 responsiveness is a necessary property of instruments intended for measuring clinically meaningful change , such as in stroke self - smis . 
 arguments exist that responsiveness should focus upon detecting change that is valued by the person rather than the clinician or researcher . 
 change attributed to an intervention is an important aspect of evaluating clinical effectiveness . in this review , 
 none of these measures scored  excellent  for this property , and only 15% scored  good  . aside from inadequate sample sizes , a common finding was that studies often were not clear about what happened to study populations between testing . 
 there is , therefore , a need for future measurement developers to specify these overlooked aspects of development more clearly in subsequent reporting . 
 the majority of smi study populations within this review experienced stroke < 24 months previously , with a number of studies using populations experiencing stroke no more than 6 months previously . 
 this may be a result of sampling to reduce the influence of additional factors upon study outcomes , such as the development of unhelpful coping behaviours , the likelihood of which might increase over time , and out of an assumption that more change may be observed in those early in their recovery . 
 however , in reality the number of people living in the community and recovering from stroke extends beyond those who are 624 months post - stroke . as engagement in self - management activities varies during recovery , particularly following adjustment to stroke as a long - term condition , there is a future need to consider outcome measures sensitive to change(s ) at different durations since stroke . 
 the role of proms , developed using rigorous investigation with the population to be measured extends beyond validating patient experience . 
 proms may improve the quality of interactions between health professionals and patients , assess levels of health and need , and provide evidence of outcomes of services , for the purposes of audit , quality assurance and comparative performance evaluation . 
 this is of particular importance when trying to capture the essence of self - management , since the experience of clients is vital in determining what is valued from their perspective . 
 there is a need to focus upon the development of measures of self - management developed with people recovering from stroke . 
 consensus between reviewers was used to determine eligibility and inclusion of smi articles . whilst we were in agreement 
 however , the use of a standardized critical appraisal tool may assist the selection of articles for future reviews . where interpretation of the cosmin criteria differed , agreement was reached by discussion and consensus . 
 additional reviewers may have further validated this process however the criteria within cosmin are explicitly stated and differences were quickly resolved . 
 data extraction was facilitated by a standardized tool advocated by cosmin , with extraction and scoring checked in a random 10% of articles.we acknowledge that checking of 10% may be viewed as a limitation of this review , however , assert that a systematic process using a standard data extraction tool was followed throughout . that cosmin operates a 
 lowest score counts  scoring system may account for the lack of measures scoring well in the measurement property criteria . some studies used otherwise appropriate methodologies , but were rated as  poor  due to inadequate sample sizes for analyses . for example , for a measure to be rated as  good  for reliability , measurement error , criterion validity and responsiveness , a sample size of n = 5099 
 , the sample size required increased to five times the number of items within a measure ( and 100 or 57 * # items but < 100 ) . therefore , 
 if those studies were repeated with larger sample sizes , their ratings according to cosmin could change dramatically . 
 the tendency for measures to score poorly may be reflective of a floor effect of the cosmin checklist . 
 cosmin was developed following consensus of experts in health measurement , therefore if its stringent criteria is to be adopted this is indicative of a need to debate the rigorous methods for measure development required . 
 it is fair to comment that some of the measures in this review were developed before the focus upon involving potential users in measure development . 
 it may be that the measures examined in this review require further development and investigation to establish adequate measurement properties for use with stroke populations . 
 our recommendations for clinicians and researchers seeking to evaluate such interventions would be firstly to clarify the theoretical premise of the intervention in question , as advocated elsewhere . without this step , 
 it is difficult to identify the mechanisms by which the intervention may influence outcomes , and thus difficult to select an outcome measure which appropriately captures the potential outcome . 
 potential outcome measures should be selected on the basis that they appropriately reflect and capture the expected outcome change . 
 this review highlights that the reported theoretical drivers within stroke smis are unclear , not least because they are often not explicitly stated by researchers . 
 the heterogeneity of the outcome measures utilised by smis in this review may indicate a difficulty in determining the expected outcomes of stroke smis . 
 a systematic review demonstrated that interventions with specific aims , such as reduced systolic blood pressure in hypertension or glycosylated haemoglobin levels in diabetes , produced greater effect sizes than those without defined outcomes . 
 further work is therefore warranted to conceptualise stroke self - management and examine the theoretical premises supporting such interventions , and expected outcomes so that appropriate outcome measures which accurately reflect the concept can be selected and/or developed . until such clarification , researchers and clinicians should , where possible , select outcome measures with reliability and validity data in the population to be tested in the intervention . the selection of outcome measures developed with involvement from the target population is also advocated . 
 this ensures that what is meaningful to the patient is more likely to be captured appropriately , thus enhancing content validity . in the meantime 
 , researchers must support clinicians by conducting further work to examine the concept and theoretical premises of self - management and developing appropriate measures if required . 
 this is the first systematic review of international research on outcome measures used and selected in stroke self - smi studies . 
 we have identified important limitations in the measures used to evaluate the effectiveness of stroke self - smis , which has significant implications for the inferences we are currently able to draw about the evidence base . 
 none of the measures used in studies of stroke smis , purported to specifically measure self - management as a discrete concept . 
 this is indicative of the difficulty in conceptualisation and operation of this concept , a view expressed elsewhere . 
 further work is required to determine how the measures identified in this review , align with the concept of self - management . 
 the range of outcomes adopted , the lack of observed changes in outcomes following stroke smis and the lack of consensus surrounding which outcome measures to utilise , indicates that the causal mechanisms of stroke smis remain imprecise . 
 stroke smis have raced ahead of the evidence to support their theoretical basis , operation and effective evaluation . 
 work to conceptualise stroke self - management is required to help identify which outcomes are most appropriate for evaluating interventions , to further inform the theoretical basis for smis   and to assist the development of interventions . 
 there is a need for studies to explore the theoretical underpinnings of smi in stroke and for the development of robust outcome measures to enable evaluation of stroke smis .","purposeto systematically review the psychometric properties of outcome measures used in stroke self - management interventions ( smis ) to ( 1 ) inform researchers , clinicians and commissioners about the properties of the measures in use and ( 2 ) make recommendations for the future development of self - management measurement in stroke.methodselectronic databases , government websites , generic internet search engines and hand searches of reference lists . 
 abstracts were selected against inclusion criteria and retrieved for appraisal and systematically scored , using the cosmin checklist.resultsthirteen studies of stroke self - management originating from six countries were identified . 
 forty - three different measures ( mean 5.08/study , sd 2.19 ) were adopted to evaluate self - smis . 
 no studies measured self - management as a discreet concept . six ( 46% ) studies included untested measures . 
 eleven ( 85% ) studies included at least one measure without reported reliability and validity in stroke populations.conclusionsthe use of outcome measures which are related , indirect or proxy indicators of self - management and that have questionable reliability and validity , contributes to an inability to sensitively evaluate the effectiveness of stroke self - smis . 
 further enquiry into how the concept of self - management in stroke operates , would help to clarify the nature and range of specific self - management activities to be targeted and aid the selection of existing appropriate measures or the development of new measures.implications for rehabilitationthe evaluation of complex interventions such as self - management interventions is aided by clear outcome expectations and valid and reliable measurement.this review demonstrates a lack of outcome measures that specifically measure self - management of stroke . 
 a minority of outcome measures that were used as proxy indicators for sm fulfill some of the criteria for quality outlined in the cosmin checklist.clinicians should select measures which appropriately reflect expected outcomes , giving due consideration to the theoretical underpinnings of the intervention . 
 further work is required to establish which measures currently in use , if any , accurately reflect stoke self-management.in the meantime , researchers should seek to develop psychometrically sound measures of stroke self - management to assist effective evaluation of such interventions in stroke .",pubmed
"osteoarthritis ( oa ) in knee joint is the most common reason behind functional disability that leads to lowered quality of life in old age people of india . 
 some of the objective factors that may influence oa disease severity include but are not limited to age , height , weight , and bmi . in physiotherapy practice , lowering the pain , other symptoms , improving the activities of daily living ( adl ) , and quality of life ( qol ) are main outcome criteria for oa knee rehabilitation . 
 to achieve this rehabilitative goal , physiotherapists use both self - reported questionnaires as well as performance - based tests to monitor prognosis in the above said parameters , that is , pain , symptoms , adl , and qol . 
 self - reported questionnaires give subjective information about the disease process without examiner bias within short period of time , whereas , performance - based tests objectively measures patient 's ability of perform adl activities . 
 examples for self - reported questionnaire are womac , sf-36 , koos , and so forth . 
 examples for physical performance tests are timed up and go test ( tug ) , six - minute walk test ( 6-mwt ) , stair climbing test , and so forth . 
 physical performance tests are affected by motivation and not affected by psychogenic factors such as beliefs , expectations , cognitive impairments , and cultural , lingual , and educational level [ 3 , 4 ] . 
 possible difference between the two may be performance - based tests measure functional limitation , whereas self - reported questionnaire reveals disability , that is , combination of social and psychological side of the functional limitation . 
 functional limitation is sooner detected by performance - based tests than self - reported questionnaires [ 5 , 6 ] . 
 there is no consensus regarding the use of tests while some prefer to use self - reported questionnaires because they are easy to administer and have high internal consistency features ; others prefer performance - based tests as they consider that these tests are essential for adl evaluation of the patient . 
 ideally , both self - reported and functional tests should be used in knee rehabilitation setting [ 7 , 8 ] . in order to ascertain more valid results , performance - based tests should always be correlated with self - reported measures . in previous studies , womac , sf-36 , and koos 
 are compared with timed up and go ( tug ) test [ 7 , 911 ] 
 . however there is no study that compares self - reported questionnaire with 6-mwt which is more deals with adl as this measures both walking ability as well as endurance . 
 koos is a relatively newer self - reported questionnaire that was developed from womac by roos et al .   for oa knee patients . 
 thus , primary objective of present study was to see the correlation of subjectively measured koos questionnaire with objectively measured 6-minute walk test ( 6-mwt ) , age , height , weight , and bmi . 
 present study was a cross - sectional survey study in which data was extracted from individuals with clinical and nonradiographic idiopathic or primary knee 
 acr criteria for clinical diagnosis of idiopathic knee oa were based on knee pain in either knee on most days for at least 1 month in the previous year and at least two of the following signs / symptoms : stiffness , crepitus , bony tenderness , and bony enlargement . 
 exclusion criteria were ( 1 ) self - reports of currently doing lower - extremity exercise 2 times per week ; ( 2 ) self - reports of currently fitness walking 90 minutes per week ; ( 3 ) being unable to read english / urdu questionnaire ( koos ) ; ( 4 ) inflammatory arthritis ; ( 5 ) self - reports of having current knee conditions such as meniscus tears and knee ligament ruptures ; ( 6 ) fracture of spine or lower extremities ; ( 7 ) being scheduled to undergo a major surgical procedure in the next 6 months ; ( 8) those with neurological conditions ; ( 9 ) having contraindications for exercise testing based on american college of sports medicine   criteria . total of 251 patients ( 117 males , 134 females ) met the above said criteria and were included for the present study . 
 koos is a self - reported questionnaire consisted of 42 questions in total addressing five patient - related domains including pain ( 9 questions ) , other disease - specific symptoms ( 7 questions ) , activity of daily living ( adl ) ( 17 questions ) , sport and recreation function ( 5 questions ) , and knee - related quality of life ( 4 questions ) . 
 the walking track was a 30  m long empty corridor with marks on every 5 meters . 
 patients were asked to walk back and forth along the 30  m track and to cover the longest possible distance in 6 minutes . at the end of every minute patients 
 two weekly test - retest reliability of walking distance was reported to be 0.87 for patients with knee oa . 
 patients stood on a firm surface in their bare feet to have their height measured . 
 body mass was measured at the end of examination with a calibrated bathroom - type digital scale , on a firm surface . 
 patients were weighed in the standing position , wearing light indoor clothes , but no shoes , jewellery , or heavy clothing . 
 body mass index ( bmi ) was calculated with the formula ( weight/(height ) ) . 
 results are given in mean , standard deviation ( sd ) , and range ( minimum  maximum ) . 
 correlation was done using spearman rank correlation since self - reported data was ordinal variable . 
 the spearman correlation coefficient was interpreted as follows : < 0.3 : none ; 0.310.5 : weak ; 0.510.7 : strong ; 0.710.9 : very strong ; and > 0.9 : excellent . 
 table 2 shows mean and standard deviation along with range of all subscales of koos and 6-mwt in primary oa knee patients . 
 overall koos - sports and recreation had a minimum mean score followed by qol subscale . 
 6-mwt had a weak correlation with koos - adl ( rho 0.461 ) and strong correlation with koos - symptom , koos - pain , and koos - sports ( rho 0.578 , 0.619 , and 0.536 , resp . ) and very strong correlation with koos - qol ( rho 0.733 ) . 
 bmi had a strong correlation with koos - pain , koos - symptom , koos - adl , and koos - sports ( rho 0.683 , 0.641 , 0.523 , and 0.640 , resp . ) and very strong correlation with koos - qol ( rho 0.816 ) . 
 weight had a weak correlation with koos - symptom , koos - adl , and koos - sports ( rho 0.485 , 0.333 , and 0.413 , resp . ) and strong correlation with koos - pain and koos - qol ( rho 0.542 and 0.582 , resp . ) . 
 there was no correlation between age and any of koos subscales except age and koos - qol subscale ( rho 0.159 , p < 0.05 ) . 
 the aim of the present study was to see the correlation between koos five subscales and some of the objective measures , that is , 6-mwt , age , height , weight , and bmi . 
 the results show there is weak ( none ) correlation of koos subscales with height , progressively increase with weight and bmi . 
 mean score of koos all subscales were less than present study results but trend is similar between the two studies meaning sports and recreation subscale is the least followed by qol subscale and symptom subscale was the maximum in both studies . 
 the present study results are in agreement with sabirli et al .   who reported strong correlation between timed up and go ( tug ) and all subscales of koos . 
 they reported 0.66 , 0.521 , 0.531 , 0.694 , and 0.561 for pain , symptom , adl , sports , and qol whereas present results for them to 6-mwt was 0.619 , 0.578 , 0.461 , 0.536 , and 0.733 , respectively . 
 .   found strong correlation between womac and 5-mwt ( r  0.485 , 0.525 , and 0.577 for pain , stiffness , function subscales , respectively , p < 0.001 for all ) . 
 reported negative correlation between womac pain , function subscales , and 6-mwt ( r  0.205 for pain , p < 
 similarly , maly et al .   reported mild but significant correlation between 6-mwt , womac pain , and stiffness subscales ( r  0.39 for pain and r  0.48 for stiffness ) . 
 lin et al .   found mild correlation between womac and physical performance tests in oa knee patients ( r  0.330.54 ) . 
 .   observed mild but significant correlation between observed and self - reported physical performance ( r  0.200.26 ; p < 0.01 ) . 
 kennedy et al .   found mild to moderate correlation ( r  0.210.50 ) between self - report and actual physical performance on 1044 knee arthroplasty patients . 
 stratford et al .   reported moderate correlation between self - reported lower extremity function scale ( lefs ) and tug time ( r 0.42 ) on patients waiting for knee arthroplasty . 
 .   reported positive correlation between gait cycle and sf-36 pain domain ( r 0.6 - 0.7 , p < 0.001 ) as well as physical function domain ( r 0.5 , p < 0.05 ) . 
 recently , adegoke et al .   reported a positive correlation ( r = 0.56 ) between self - reported function and actual physical performance ( stair climbing and tug test ) which is similar to our result ( r = 0.461 ) . 
 reported that weight gain is associated with worsening pain , stiffness , and function in knee pain patients . 
 miller et al .   reported that 6 monthly changes in weight is correlated with womac pain ( r 0.346 , p < 0.01 ) , stiffness ( r 0.204 ) , and function ( r 0.310 , p < 0.05 ) , respectively . 
 penserga and tanque   reported a positive correlation between womac ( function ) and weight ( r = 0.260 ) . 
 correlation between weight and bmi is similar to that of marks   ( 0.764 in present study versus 0.83 and 0.896 in marks ' study ) and their reported r = 0.63 for self - determined walking pace with bmi is similar to our koos - adl with bmi ( r = 0.523 ) . 
 however contradictory to our finding , marks   reported lower values for vas pain scale with bmi ( r = 0.352 ) . 
 marks   concluded that bmi was significantly correlated with pain using vas ( r 0.270 , p < 0.01 ) and disability using aims ( r 0.357 , p < 0.01 ) . 
 reported bmi significantly correlated with all subscales of womac ( p < 0.001 ) and sf-36 but their correlation was < 0.30 . 
 previous studies also report similar correlation between bmi and womac ( sanghi et al . ; 
 r = 0.592 , 0.634 , 0.749 for pain , stiffness , and adl subscales in klc et al .   except cubukcu et al . 
 creamer et al .   reported positive correlation ( r = 0.42 ) between womac function subscale and bmi . 
 reported mild positive correlation between bmi and sit to stand performance ( r 0.47 , p < 0.01 ) . 
 marks   concluded that age was not significantly correlated with pain using vas ( r  0.122 ) and disability using aims ( r  0.175 ) . 
 penserga and tanque   reported lack of correlation between age and womac disability subscales ( r  0.077 ) . 
 lack of correlation between koos pain ( r 0.093 ) , symptom ( r 0.071 ) , and adl ( r 0.036 ) subscales with age and weak correlation between age and koos qol ( r 0.159 ) subscale in oa knee patients have already been published in our previous publication with larger sample size . 
 age found to be insignificant correlation with womac pain ( r 0.01 ) and function ( r 0.06 ) subscales in 257 knee arthroplasty patients . 
 elbaz et al .   also reported similar results in symptomatic oa knee patients using womac ( 0.028 , 0.023 , 0.09 for pain , stiffness , and function subscales , resp . ) . 
 reported similar result using womac ( correlations between age and womac pain , stiffness , and function were 0.081 , 0.49 , 0.114 , and resp . ; all were statistically insignificant ) . recently , 
 maly et al .   in their study found insignificant correlation between age and pain intensity ( r  0.03 ) and physical performance ( r 0.09 ) . 
 the present study results are in confirmation with previous studies and affirms the usage of koos in indian oa knee patients .","purpose . objective of the present study was to see the correlation of subjectively measured koos questionnaire with objectively measured 6-minute walk test ( 6-mwt ) , age , height , weight , and bmi . 
 participants . 
 251 subjects with oa knee based on american college of rheumatology criteria . methods . after passing inclusion and exclusion criteria , 
 the following parameters were recorded : age , height , weight , and bmi . then subjects were asked to fill koos questionnaire ; then all subjects were asked to do self - paced walk for 6 minutes . 
 analysis . 
 spearman rank test was done to see the correlation . 
 significant level was set at p < 0.05 . 
 results . 
 6-mwt had a weak correlation with koos - adl ( rho 0.461 ) and strong correlation with koos - symptom , koos - pain , and koos - sports and very strong correlation with koos - qol . 
 bmi had a strong correlation with koos - pain , koos - symptom , koos - adl , and koos - sports and very strong correlation with koos - qol . 
 weight had a weak correlation with koos - symptom , koos - adl , and koos - sports and strong correlation with koos - pain and koos - qol . 
 all the above values were significant with p < 0.001 . conclusion . 
 koos is strongly positively correlated with 6-mwt and negatively correlated with bmi . 
 its correlation strength has decreased with weight .",pubmed
"an increasingly sedentary lifestyle combined with rising consumption of caloric foods has transformed obesity into a global event . 
 obesity is a low - level , chronic inflammatory disease with a multifactorial etiology that includes eating habits , a sedentary lifestyle , and genetic predisposition that promotes the excessive accumulation of body fat [ 2 , 3 ] . 
 in addition to its roles as an energy reservoir and heat insulating material , adipose tissue also secretes cell - signaling molecules such as adipokines . 
 adipose tissue secretes proinflammatory factors like interleukin 6 ( il-6 ) , tumor necrosis factor - alpha ( tnf- ) , and leptin and anti - inflammatory factors as and interleukin 10 ( il-10 ) and adiponectin . 
 studies have demonstrated that the administration of a high - fat diet in rodents promotes metabolic changes , inducing the production of proinflammatory interleukins and a chronic inflammatory process [ 5 , 6 ] . 
 the presence of large concentrations of endotoxins and saturated fatty acids in the diet promotes the activation of toll - like receptor 4 ( tlr-4 ) , which triggers the production of proinflammatory cytokines [ 4 , 5 ] . through receptors associated with transmembrane proteins , 
 these cytokines interfere with the expression of other cytokines , thus affecting metabolic homeostasis [ 7 , 8 ] . among various implications that are closely correlated with obesity 
 , menopause deserves particular attention because of the increase in life expectancy of the population . 
 menopause is defined as the permanent cessation of menstruation due to loss of ovarian function [ 9 , 10 ] . 
 the loss of ovarian function , particularly the decline in estrogen production , promotes adverse changes in the profile of lipoproteins , metabolism of glucose and insulin , distribution of body fat , clotting , and vascular endothelium . in combination with weight gain , 
 these changes place women at a high risk of developing cardiovascular diseases , which are very common in this period of life [ 10 , 11 ] . 
 the physiological actions of estrogens in the body are mediated by two distinct estrogen receptors ( ers ) : er and er ; these receptors are nuclear transcription factors involved in the regulation of several complex physiological processes [ 12 , 13 ] . according to benedusi and colleagues , the inflammatory processes that occur following the menopause result from a reduction in circulating levels of estrogens and their receptors ( ers ) , which have anti - inflammatory properties 
 however , controversy persists as to whether the effects of estrogens are anti- or proinflammatory . 
 studies in premenopausal women suggest a beneficial role for estrogen in the prevention of vascular inflammation and consequent atherosclerosis . 
 estrogen exerts an anti - inflammatory effect on the vasculature , through antioxidant effects , generation of nitric oxide , prevention of apoptosis in vascular cells , suppression of proinflammatory cytokines , and modulation of the renin - angiotensin system . in vitro studies 
 have also demonstrated that estrogen has an anti - inflammatory effect in several cell lineages . 
 the hormonal changes that occur during the menopause are responsible for specific physical and metabolic remodeling that , when associated with weight gain and obesity , have a negative impact on women 's health . 
 therefore , it is essential that both obesity and menopause , in particular the effects of estrogen on the inflammatory state , are investigated to improve women 's quality of life . in this study 
 , we evaluated whether a high - fat diet with or without ovariectomy promoted the inflammatory process in rats by assessing the production of pro- and anti - inflammatory factors and their receptors and tlr-4 protein content . 
 the experimental research committee of the so paulo federal university approved all procedures for the care of the animals used in this study . 
 thirty - day - old female wistar rats were used in this study and were kept under controlled conditions of light ( 12  :  12  h light  dark cycle with lights on at 07:00 ) and temperature ( 22  1c ) . during the experimental period 
 the animals were maintained in collective cages and received water and the specific diet ad libitum . 
 thirty - day - old rats were divided into two groups and fed for 4 weeks with one of the following diets : control diet ( c ) or hyperlipidic diet ( h ) . 
 after this period the animals were divided into four groups : sham c : sham group treated with control diet;sham h : sham group treated with hyperlipidic diet;ovx c : ovariectomized group treated with control diet;ovx h : ovariectomized group treated with hyperlipidic diet . 
 sham c : sham group treated with control diet ; sham h : sham group treated with hyperlipidic diet ; ovx c : ovariectomized group treated with control diet ; ovx h : ovariectomized group treated with hyperlipidic diet . the food intake and body weight were measured weekly at 09:00 . the control diet was prepared according to the recommendations of the american institute of nutrition ( ain-93 m )   and the hyperlipidic diet was by ain-93 m modified . 
 the fat content in the control diet ( c ) was 4% containing 0.64  g of saturated fatty acids , and the hyperlipidic diet containing 20% of fat which 7.7  g was saturated fatty acids . 
 the energy content of c diet was 4114.71  kj and of hc diet 6438.58  kj/100  g ( table 1 ) . at 60 days of age , the rats were anesthetized and received intraperitoneal injection of ketamine ( 70  mg / kg ) and xylazine ( 10  mg / kg ) . 
 the ovx group was submitted to bilateral ovariectomy . for this , they were subjected to a muscular incision to open the peritoneal cavity for posterior connection of the uterine tubules and removal of the ovaries . 
 all the animals received antibiotic ( penicillin ) immediately after the surgery and 0.1  ml / kg body weight of ibuprofen 50  mg for 2 days . 
 the animals were euthanized by decapitation on the 90th day of life in the fasting state ( 10  h ) in the early morning to avoid chronobiological variations . 
 the sham groups were in estrus phase ( phase when estrogen secretions exert their biggest influence ) . 
 trunk blood was collected and immediately centrifuged at 4c and serum aliquots were taken and frozen at 80c to measure the concentrations of glucose , triacylglycerols , and total cholesterol using commercials kits from labtest diagnostic sa ( mg , brazil ) . 
 the concentrations of insulin , leptin , and adiponectin were determined by elisa ( linco research inc . 
 the retroperitoneal , parametrial ( par ) , and mesenteric ( mes ) adipose tissues , gastrocnemius muscle ( gast ) , and liver were dissected , weighed , frozen in liquid nitrogen , and stored at 80c until the protein extraction . for determination of carcass lipid and protein content 
 lipid content was measured as described by stansbie et al .   and standardized using the method described by oller do nascimento and williamson . 
 briefly , the eviscerated carcass was autoclaved at 120c for 90  min and homogenized with double the mass of water . 
 triplicate aliquots of this homogenate were weighed and digested in 3  ml of 30% koh and 3  ml of ethanol for at least 2  h at 70c in capped tubes . after cooling , 2  ml of 12  n h2so4 was added , and the sample was washed three times with petroleum ether for lipid extraction . 
 results are expressed as grams of lipid/100  g of carcass . for protein measurements , aliquots of the same homogenate ( approximately 1  g ) 
 were heated to 37c for 1  h in 0.6  n koh with constant shaking . 
 after clarification by centrifugation , protein content was measured according to bradford ( bio - rad , hercules , ca ) . 
 adipose tissue depots , gastrocnemius muscle , and liver ( 0.150.3  g ) were homogenized in ice - cold solubilization and total protein extraction buffer ( 100  mm tris , ph 7.5 , 100  mm sodium fluoride , 10  mm sodium orthovanadate , 2  mm phenylmethylsulfonyl fluoride , 10  mm sodium pyrophosphate , and 0.1  mg / ml aprotinin ) . 
 homogenates were centrifuged at 19283  g for 40  min at 4c . the supernatants were saved and the protein concentrations were determined using a bradford assay ( bio - rad , hercules , ca ) with bovine serum albumin as a reference . 
 fifty micrograms of the specific tissue total protein was loaded onto a sodium dodecyl sulfate - polyacrylamide gel ( 5% stacking gel ; 10% running gel ) , separated by electrophoresis , and then electroblotted onto nitrocellulose membranes ( hybond - c extra , amersham ) using a wet electroblotter ( bio - rad , ca , usa ) . 
 after blotting , the membranes were blocked in tris - buffered saline- ( tbs- ) tween buffer , ph 7.5 ( 20  mm tris/500  mm nacl/0.05% tween-20 ) , containing 1% bovine serum albumin and then exposed to specific antibodies diluted in tbs - tween buffer ( ph 7.5 ) containing 1% bsa for 2  h. the membranes then were washed and incubated with anti - rabbit ig or anti - mouse ig conjugated to horseradish peroxidase and diluted to 1/1000 in the same buffer for 1  h. after a series of washes in tbs - tween buffer , the bands were visualized with enhanced chemiluminescence scanned at uvitec ( cambridge ) after adding the ecl reagent ( ge healthcare bio - sciences ab , uk ) . 
 the antibodies against anti - tlr4 ( sc-99183 ) , anti - tnf - r1 ( sc-7895 ) , anti - il-6 r ( sc-660 ) , anti - myd88 ( sc-8197 ) , and anti--tubulin ( sc-58667 ) were obtained from santa cruz biotechnology ( santa cruz , ca , usa ) , and the anti - rabbit ig and anti - mouse ig conjugated to horseradish peroxidase were obtained from sigma ( usa ) . 
 the results were analyzed using two - way analysis of variance followed by tukey 's test . 
 the initial body , liver , gast , and par weights were similar in all groups . in the ovx h group , the ret weight was higher than in the sham c group and the mes weight was higher than in the sham h group . 
 administration of a hyperlipidic diet in the sham group did not modify these parameters compared with the administration of a control diet . 
 however , the association of a hyperlipidic diet with ovariectomy ( ovx h ) caused a significant increase in final body weight . 
 carcass protein content was greater in the ovx c group than both sham groups . however , carcass lipid content in the ovx h group was higher than that in the sham h group ( table 2 ) . 
 the initial 4 weeks of hyperlipidic diet administration did not modify body weight gain and food efficiency ( figures 1(a ) and 1(b ) ) . 
 however , the combination of ovariectomy and administration of a hyperlipidic diet ( ovx h ) increased body weight gain accompanied by high food efficiency ( figures 1(c ) and 1(d ) ) . 
 the serum concentrations of total cholesterol , hdl ( high - density lipoprotein ) cholesterol , glucose , adiponectin , and estradiol were similar in all groups . 
 serum levels of triacylglycerol ( tg ) were decreased in the sham h group compared with the sham c group . 
 serum insulin concentration was lower in the sham h , ovx c , and ovx h groups than in the sham c group . 
 ovariectomy combined with the administration of a hyperlipidic diet caused an increase in serum leptin levels compared with all other groups ( table 3 ) . 
 the cytokines content was similar in the sham c , sham h , ovx c , and ovx h groups and in the liver and gast ( figures 2 and 3 ) . 
 however , administration of a hyperlipidic diet caused a decrease in il-6 and il-10 levels in mes adipose tissues . 
 furthermore , administration of a hyperlipidic diet associated with ovariectomy decreased the tnf- content of mes ( figure 4 ) . 
 the il-6 content was decreased by the hyperlipidic diet ; however , in combination with ovariectomy , this effect was reversed in ret adipose tissues ( figure 5 ) . 
 the hyperlipidic diet caused a decrease in il-10 levels , but ovariectomy had the opposite effect , and the tnf- content of ret adipose tissues was high following ovariectomy ( figure 5 ) . 
 the il-6 receptor , tnf receptor , tlr-4 , and myd88 protein content did not differ between groups in the liver ( figure 6 ) , gast ( figure 7 ) , and mes adipose tissues ( figure 8) . 
 in this study , we demonstrated that administration of a hyperlipidic diet for 8 weeks combined with short - term ovariectomy promoted an increase in body weight gain and a decrease in metabolic efficiency , accompanied by hyperleptinemia . 
 the cytokines content of the liver and gast was unchanged ; however , levels of all cytokines analyzed were decreased in mes adipose tissues . 
 also , we demonstrated that 4 weeks of ovariectomy ( ovx c group ) per se did not modify these parameters compared with the sham c group . 
 however , ryou et al .   demonstrated that 8 weeks of ovariectomy in rats caused an increase in body weight and food efficiency with elevated plasma leptin . 
 these results suggest that the time period after ovariectomy may influence body weight gain and food efficiency . 
 previously , jen et al .   showed that high - fat diet fed male rats were heavier than chow - fed male rats by the sixth week of a high - fat diet , whereas female rats were heavier by the ninth week . 
 consistent with this , we demonstrated that the administration of a hyperlipidic diet for 8 weeks compared with a control diet did not cause an increase in body weight gain in female rats . 
 .   showed that ingestion of a high - fat diet for 8 weeks did not modify body or tissue weight . 
 however , the combination of a high - fat diet and ovariectomy caused an increase in body weight gain accompanied by an increase in ret and mes weight , similar to our findings . 
 this excessive weight gain is primarily due to fat deposition , reflected by increased serum leptin levels , which rise in direct proportion to fat mass . 
 carcass lipid content in the ovx h group was higher than in the sham h and sham c groups . 
 diets rich in saturated fatty acids decrease uncoupling protein-1 ( ucp-1 ) activity in brown adipose tissue 
 liu et al .   showed that low estrogens levels , characteristic of ovariectomy , increase ucp-1 expression in subcutaneous adipose tissue from 4 weeks until 12 weeks of ovariectomy . 
 in contrast , ko et al .   indicated that a high - fat diet combined with ovariectomy decreased ucp-1 levels relative to controls . 
 also , it has been reported that ovariectomy causes a decrease in the lipolytic responsiveness to norepinephrine of fat cells and an increase in adipose tissue lipoprotein lipase ( lpl ) . 
 lpl hydrolyzes tg from chylomicrons and very - low - density lipoproteins to free fatty acids and monoacylglycerols . 
 a hyperlipidic diet was shown to cause an increase in lpl activity in white adipose tissue . by these mechanisms , ovariectomy promotes increased adiposity , particularly in rats receiving a hyperlipidic diet . 
 taken together , these findings imply that a high - fat diet combined with ovariectomy may cause a decrease in energy expenditure and an increase in adipose tissue deposits , probably via enhanced uptake of tg from chylomicrons . also , in the present study , it was observed that serum tg was lower in sham h group compared with sham c group . 
 studies have reported that low - fat , high - carbohydrate diets increase the liver  de novo  lipogenesis and cause hypertriglyceridemia compared to high - fat diets [ 29 , 30 ] . 
 .   showed that the administration of estrogen promoted a slight decrease in body weight without entailing a significant reduction in adiposity index or in ret weight in obese rats . 
 these data suggest that this treatment is insufficient to restore these parameters , although there is a 77% reduction in the expression of lpl , which would reduce fat deposition by decreasing fatty acid uptake from the circulation . 
 however , this is inconsistent with studies in which estrogen replacement managed to recover both body weight and adiposity index in female control rats . 
 this discrepancy may be related to the structural form of estrogen used , in addition to the dosage and duration of estrogen replacement and the age at which the female rats were ovariectomized . 
 increase in serum leptin concentration was observed only in ovx h group , suggesting that the decrease in estrogen could potentiate the effect of high - fat diet on leptin secretion . 
 hyperleptinemia may arise as a result of the increased body weight of rats in the ovx h group . 
 showed that animals fed a high - fat diet were hyperleptinemic and overweight compared with animals receiving a control diet . 
 it is established that serum leptin levels rise with increasing adiposity and are directly proportional to fat mass . 
 loss of efficacy of endogenous or exogenous leptin in the therapeutic maintenance of body weight is attributable to the development of resistance to leptin , a common occurrence in obese individuals . 
 possible explanations for this include a lack of leptin at central sites resulting from defective transport , for instance , through the blood and the brain barrier , decreased production of leptin by the hypothalamus , or interruption of signal transduction between leptin and receptors in the hypothalamus . 
 saravanan et al .   showed that animals fed a high - fat diet had a state of hyperleptinemia accompanied by overweight when compared to animals that received control diet . 
 it is understood that ovariectomy promotes hyperphagia and weight gain , and although this hyperphagia is transitional , the gain in body weight is more durable . 
 estrogen replacement has been shown to decrease food intake and restore weight in ovariectomized rats . 
 these facts suggest that estrogen is involved in the modulation of energy homeostasis by direct action on the appetite and central pathways regulating energy and by controlling the secretion of hormonal signals peripherals such as leptin , which in turn regulates effector pathways central and essential to the integration of the hypothalamus in energy homeostasis . 
 content , administration of a hyperlipidic diet in this study caused a decrease in il-6 and il-10 content in both ret and mes adipose tissues . 
 previously , our group   showed that administration of a lard diet for 8 weeks promoted a decrease in il-10 in ret and mes adipose tissues of male mice , with no effect on il-6 or tnf-. cani et al 
 .   observed an increase in lps , accompanied by elevated mrna concentrations of pai-1 , il-1 , tnf- , and f4/80 in subcutaneous adipose depots and pai-1 , il-1 , and f4/80 in mes adipose tissues , of high - fat diet fed male mice relative to control diet fed mice . 
 these results suggest that the presence of female hormones could modify the response to a high - fat diet in terms of il-6 adipose tissue content , especially in ret adipose tissues . 
 previously , lafontan and berlan   reported that the physiology , metabolism , and function of white adipose tissue vary in a depot - specific manner . 
 several factors could contribute to this , such as difference in the amount of hormones receptors , blood flow , hormone , cytokines , and polypeptides production . 
 for instance , it has been demonstrated by yamashita et al .   that aerobic training was effective in reducing adipokines levels related to inflammation in mesenteric adipose tissue but not in retroperitoneal adipose tissue . in this study 
 , the rats were euthanized , between 9:00 and 12:00  h , in the estrus phase , a period when estrogen secretions exert their biggest influence . 
 in fact , in vitro studies demonstrated an inhibitory effect of estradiol on il-6 production in several cell types in humans and rodents , including macrophages , monocytes , osteoblasts , and bone stromal cells [ 3941 ] . 
 conversely , ovariectomy combined with a hyperlipidic diet promoted a decrease in the tnf- , il-6 , and il-10 content of mes adipose tissues . 
 riant et al .   demonstrated that estrogens , particularly chronic e2 administration to ovariectomized mice , enhance the expression of inflammatory factors , such as il-6 and tnf- , in mice fed with a high - fat diet for 4 or 12 weeks , indicative of a proinflammatory effect of e2 , at least in visceral adipose tissues . 
 accordingly , in this study , we observed a decrease in tnf- in mes adipose tissues in the ovx h group compared with the sham h group , supporting the idea of a possible in vivo proinflammatory effect of estrogen on white adipose tissue , in special visceral one . both hyperlipidic diet and estrogen 
 have been reported to influence the circadian rhythm modifying the expression of clock genes involved in the regulation of metabolism and protein expression [ 43 , 44 ] . in this sense , 
 cano et al .   demonstrated that high - fat diet disrupts the daily variations of circulating of several adipocytokines , particularly by a reduction in plasma concentration of tnf- from 9:00 to 13:00  h , compared to control diet treated rats . 
 it could be speculated that the results found in the present study , related to the effect of diet and ovariectomy on adipose tissues cytokines content , could be related to the disruption on the clock genes , since the presence of circadian clock genes has been reported in fat . 
 content in white adipose tissue by the administration of a hyperlipidic diet and/or ovariectomy , protein levels of il-6r , tnfr1 , tlr-4 , and myd88 were similar between groups in all tissues studied ( figures 6 , 7 , and 8) . 
 myd88 knockout mice showed no response to the tlr-4 ligand lps in terms of inflammatory mediator production by macrophages , b cell proliferation , or endotoxin shock [ 47 , 48 ] . 
 however , in the case of tlr-4 stimulation , lps - induced activation of nf-b and c - jun n - terminal kinases ( jnk ) was observed with delayed kinetics , even in myd88 knockout cells , although these cells did not produce any inflammatory cytokines in response to lps . 
 taken together , it is possible to speculate that the modification in cytokines content in white adipose tissue observed in this study may have been stimulated by another mechanism , such as mapk dependent pathway . in conclusion , 
 our results demonstrate that administration of a hyperlipidic diet for 8 weeks combined with short - term ovariectomy did not alter the cytokine content of the liver and gastrocnemius muscle but caused a decrease in all analyzed cytokines in mesenteric adipose tissue . 
 however , ovariectomy in rats fed a hyperlipidic diet caused hyperleptinemia and increased body weight gain , food efficiency , and carcass lipid content , which , in combination with long - term ovariectomy , may contribute to the induction of proinflammatory processes , particularly those associated with a high - fat diet . 
 these results emphasize that , after the menopause , women must decrease their consumption of fat .","four - week - old female wistar rats were divided into two groups and fed a control diet ( c ) or a hyperlipidic diet ( h ) for 4 weeks . 
 rats from each group underwent ovariectomy ( ovx ) or sham surgery ( sham ) . 
 they received c or h for the next four weeks . the body weight gain ( bw ) , food efficiency ( fe ) , and carcass lipid content were higher in the ovx h than in the sham h. the ovx h exhibited a higher serum leptin level than other groups . 
 il-6 , tnf- , and il-10 content of mesenteric ( mes ) adipose tissue was lower in the ovx h than in the ovx c. il-6 , tnf- , and il-10 content of retroperitoneal ( ret ) adipose tissue was lower in the sham h than in the sham c. the sham h showed decreased tg relative to the sham c. similar results were obtained in relation to il-6r , tnfr1 , tlr-4 , and myd88 contents in the mes and ret white adipose tissue among the groups . a hyperlipidic diet for 8 weeks combined with short - term ovariectomy decreases the cytokine content of mes adipose tissues but increases bw , enhancing fe and elevating serum leptin levels . 
 these suggest that the absence of estrogens promotes metabolic changes that may contribute to installation of a proinflammatory process induced by a hyperlipidic diet .",pubmed
"hemoptysis is one of the most common symptoms in patients with respiratory diseases , when massive , has a mortality rate of more than 50% 
 . therefore , significant or life - threatening hemoptysis calls for quick treatment to stop bleeding . in the past 
 , the control of hemoptysis had been obtained using surgical methods if conservative management failed to be effective . but 
 patients with massive hemoptysis may be associated with high morbidity and mortality using conservative or surgical management . 
 it is a safe and effective treatment , not only for massive hemoptysis , but also for chronic or recurrent hemoptysis that impairs the patient 's quality of life . 
 the first procedure for embolization therapy is a complete angiography of the pulmonary vascular tree when bleeding bronchial arteries are seen , embolization therapy is carried out . 
 this retrospective study was undertaken to determine the demographic characteristics , clinical features , results of imaging techniques , complications related to bae , and follow - up of 344 patients underwent bae . 
 this study was a retrospective analysis of 344 cases , who underwent bronchial arteriography at tianjin haihe hospital between 2006 and 2013 . 
 the patients were hospitalized due to hemoptysis , with the volume of blood ranging 502000 ml/24 h. each patient underwent standard medical management , but the hemoptysis still occurred , so they underwent bronchial arteriography at first and underwent bae when necessary . 
 there were 186 men and 158 women , with a mean age of 57 years old ( ranging 1783 years old ) . 
 two hundred and thirty - four patients had acute and massive hemoptysis , and 110 patients had chronic and recurrent hemoptysis ( recurrent rate at least 3 times in 1 year ) . 
 the data for outcome analysis were obtained from the clinical records , all of the radiological images were reviewed as possible . 
 we collected such data for analysis : clinical features ; chest computed tomography ( ct ) scan ; bronchial ateriography , bae , and results ; embolization material ; complications related to bae ; and follow - up . 
 about 317 patients ( 92.2% ) were suggestive of causes of hemoptysis , and 27 patients ( 7.8% ) showed focal hemorrhage . 
 blood lobe could be identified by chest ct scan in 105 cases ( 30.5% ) . 
 bleeding source could be localized in unilateral lung but could not be defined in an exact lobe in 90 cases ( 36.2% ) . 
 both of the lungs had unusual film but not focal hemorrhage in 149 patients ( 43.3% ) . 
 the findings of angiography suggested that few direct sign found during arteriography , most of the indirect appearances that support a site of bleeding include tortuosity , hypertrophy , hypervascularity , aneurysms , extravasation , and bronchial artery to pulmonary artery or vein shunting . in 131 
 patients ( 38.1% ) , only left - side bronchial arteries were abnormal ; in 69 patients , only right - side bronchial arteries were abnormal ; in the other 144 patients , bilateral bronchial arteries were abnormal . 
 as the statistics , 944 responsible bronchial arteries involves in according to the angiography : 275 right bronchial arteries ; 200 left bronchial arteries ; 170 combined right and left bronchial trunk ; 123 right intercostal arteries ; other 176 arteries [ figure 1 ] . 
 we analyzed the responsible arteries for tuberculosis , bronchiectasis , bronchogenic carcinomas and undefined reasons as follow [ table 1 ] . 
 a spinal artery was identified in three patients , one spinal artery originated from the left bronchial artery , one from the right intercostal artery , and one from the right subclavian artery . 
 the numbers of responsible arteries for different diseases despite of which kind of causations , the main responsible arteries were right bronchial artery , left bronchial artery and combined right and left bronchial trunk ; more right intercostal arteries were involved than left . 
 bronchial artery embolization was performed in 336 of 344 patients ( 97.7% ) , artery spasm and closure occurred in six patients , arterial subintimal dissection occurred in one patient , and the responsible vessel could nt be super - selective in one patient because of combining with the spinal artery . 
 overall , there were 1530 embolization coils for the 336 patients with a total of 920 arteries embolized ( including bronchial and nonbronchial systemic arteries ) . according to the chest ct scanning and bronchial arteriography , we concluded the etiologies of the hemoptysis in 344 cases : tuberculosis in 190 patients ( 55.2% ) , bronchiectasis in 99 patients ( 28.8% ) , malignancy in 20 patients ( 5.8% ) , undefined in 18 patients ( 5.2% ) , pneumonia in six patients ( 1.8% ) , deformity of bronchial artery in six patients ( 1.8% ) , bronchial artery aneurysm in one patients ( 0.2% ) , other pulmonary disease in four patients ( 1.2% ) . 
 bronchial artery embolization was successful in controlling hemoptysis immediately in 330 of 344 patients ( 96% ) . 
 61 patients ( 17.7% ) had recurrent hemoptysis within 1 month after undergoing bae , only three of them were performed a second bae , two patients had rebleeding in the previous site , one was found new bleeding area . 
 three of these patients died of massive hemoptysis in 1 month after undergoing embolization ( two patients with bronchogenic carcinoma and one with tuberculosis ) , one patient with severe chronic obstructive pulmonary disease died of hypercapnia in 3 days after undergoing embolization . 
 the two patients in whom an embolization procedure could not be performed ( one because of spinal artery combined with the hemorrhage vessel and one was under subintimal dissection ) , taken medical treatment for 1 week , the hemoptysis were gradually controlled . 
 each patient performed bae had a repeated chest ct scanning after 1 month , which showed that there was no coil dropping or shifting . 
 seventy four patients ( 21.5% ) had recurrent hemoptysis over 1 month after undergoing bae . 
 forty - four patients were performed a repeated embolization , all of the bronchial arteriograms bronchial arteriograms showed there was no dropping of the coil , in 17 patients developed recanalization of the previously embilzed vessels , the remaining 27 patients developed new blood vessels responsible for hemorrhage . 
 one hundred and seven patients ( 31.1% ) had a fever ( < 38.5c ) after bae . 
 chest pain postprocedure occurred in 65 patients ( 18.9% ) , back pain in 51 patients ( 14.8% ) , shoulder pain postprocedure in 29 patients ( 8.4% ) , and transient dysphagia or lower limb numbness in three patients ( 0.9% ) . 
 all of the patients had been followed up over 1 month after bae except the four patients had been died within 1 month . 
 two hundred and forty - eight patients had a telephone call follow - up recently ( follow - up periods ranging from 6 months to 8 years ) . 
 it revealed that 28 patients had been dead , six of them died of massive hemorrhage , seven died of deterioration of lung disease but not bleeding , the remainder died of other diseases or accident . 
 four patients died within 1 year after due to advanced lung cancer ( one had a massive bleeding out of controlling ) , 24 patients died during 24 years after bae . 
 twenty - one patients have recurrent hemoptysis after repeated bae , the volume of bleeding is < 5 ml / d . 
 one hundred and ninety - nine patients had follow - up periods ranging from 6 months to 8 years without evidence of recurrent hemoptysis . 
 about 317 patients ( 92.2% ) were suggestive of causes of hemoptysis , and 27 patients ( 7.8% ) showed focal hemorrhage . 
 blood lobe could be identified by chest ct scan in 105 cases ( 30.5% ) . 
 bleeding source could be localized in unilateral lung but could not be defined in an exact lobe in 90 cases ( 36.2% ) . 
 both of the lungs had unusual film but not focal hemorrhage in 149 patients ( 43.3% ) . 
 the findings of angiography suggested that few direct sign found during arteriography , most of the indirect appearances that support a site of bleeding include tortuosity , hypertrophy , hypervascularity , aneurysms , extravasation , and bronchial artery to pulmonary artery or vein shunting . in 131 
 patients ( 38.1% ) , only left - side bronchial arteries were abnormal ; in 69 patients , only right - side bronchial arteries were abnormal ; in the other 144 patients , bilateral bronchial arteries were abnormal . 
 as the statistics , 944 responsible bronchial arteries involves in according to the angiography : 275 right bronchial arteries ; 200 left bronchial arteries ; 170 combined right and left bronchial trunk ; 123 right intercostal arteries ; other 176 arteries [ figure 1 ] . 
 we analyzed the responsible arteries for tuberculosis , bronchiectasis , bronchogenic carcinomas and undefined reasons as follow [ table 1 ] . 
 a spinal artery was identified in three patients , one spinal artery originated from the left bronchial artery , one from the right intercostal artery , and one from the right subclavian artery . 
 the numbers of responsible arteries for different diseases despite of which kind of causations , the main responsible arteries were right bronchial artery , left bronchial artery and combined right and left bronchial trunk ; more right intercostal arteries were involved than left . 
 bronchial artery embolization was performed in 336 of 344 patients ( 97.7% ) , artery spasm and closure occurred in six patients , arterial subintimal dissection occurred in one patient , and the responsible vessel could nt be super - selective in one patient because of combining with the spinal artery . 
 overall , there were 1530 embolization coils for the 336 patients with a total of 920 arteries embolized ( including bronchial and nonbronchial systemic arteries ) . 
 according to the chest ct scanning and bronchial arteriography , we concluded the etiologies of the hemoptysis in 344 cases : tuberculosis in 190 patients ( 55.2% ) , bronchiectasis in 99 patients ( 28.8% ) , malignancy in 20 patients ( 5.8% ) , undefined in 18 patients ( 5.2% ) , pneumonia in six patients ( 1.8% ) , deformity of bronchial artery in six patients ( 1.8% ) , bronchial artery aneurysm in one patients ( 0.2% ) , other pulmonary disease in four patients ( 1.2% ) . 
 bronchial artery embolization was successful in controlling hemoptysis immediately in 330 of 344 patients ( 96% ) . 
 61 patients ( 17.7% ) had recurrent hemoptysis within 1 month after undergoing bae , only three of them were performed a second bae , two patients had rebleeding in the previous site , one was found new bleeding area . 
 three of these patients died of massive hemoptysis in 1 month after undergoing embolization ( two patients with bronchogenic carcinoma and one with tuberculosis ) , one patient with severe chronic obstructive pulmonary disease died of hypercapnia in 3 days after undergoing embolization . 
 the two patients in whom an embolization procedure could not be performed ( one because of spinal artery combined with the hemorrhage vessel and one was under subintimal dissection ) , taken medical treatment for 1 week , the hemoptysis were gradually controlled . 
 each patient performed bae had a repeated chest ct scanning after 1 month , which showed that there was no coil dropping or shifting . 
 seventy four patients ( 21.5% ) had recurrent hemoptysis over 1 month after undergoing bae . 
 forty - four patients were performed a repeated embolization , all of the bronchial arteriograms bronchial arteriograms showed there was no dropping of the coil , in 17 patients developed recanalization of the previously embilzed vessels , the remaining 27 patients developed new blood vessels responsible for hemorrhage . 
 one hundred and seven patients ( 31.1% ) had a fever ( < 38.5c ) after bae . 
 chest pain postprocedure occurred in 65 patients ( 18.9% ) , back pain in 51 patients ( 14.8% ) , shoulder pain postprocedure in 29 patients ( 8.4% ) , and transient dysphagia or lower limb numbness in three patients ( 0.9% ) . 
 all of the patients had been followed up over 1 month after bae except the four patients had been died within 1 month . 
 two hundred and forty - eight patients had a telephone call follow - up recently ( follow - up periods ranging from 6 months to 8 years ) . 
 it revealed that 28 patients had been dead , six of them died of massive hemorrhage , seven died of deterioration of lung disease but not bleeding , the remainder died of other diseases or accident . 
 four patients died within 1 year after due to advanced lung cancer ( one had a massive bleeding out of controlling ) , 24 patients died during 24 years after bae . 
 twenty - one patients have recurrent hemoptysis after repeated bae , the volume of bleeding is < 5 ml / d . 
 one hundred and ninety - nine patients had follow - up periods ranging from 6 months to 8 years without evidence of recurrent hemoptysis . 
 earlier , besides medical treatment , surgery was the fast and best method to control the bleeding . 
 but it required patients many preparations , such as chest ct , pulmonary function , even bronchoscopy to evaluate the patient physical condition and determine the range of surgery . 
 some preparations were impossible for the patients in emergency conditions , the impairment of surgery was so great for a lot of people that gravely lowered the quality of life . 
 bae is a well - established procedure used to control massive hemoptysis since it first performed in 1974 . 
 subsequently , bae was widely used , because non - operable patients could be treated and other patients could be stabilized prior to surgery . in clinical , 
 about 1 hour is needed to prepare before bae , and the physical requirements of patient are lower than for surgery . 
 the technology is mostly described the outcomes in control of hemoptysis due to varies causations . in some conditions , interventional embolization can be used before surgery . in our study , 
 of the 344 cases , an immediate control of bleeding was achieved with embolization in 330 patients ( 96% ) , similar to the immediate success rate was reported 7598% earlier . 
 recurrent bleeding despite apparently adequate embolotherapy remains a considerable problem , some reported with bleeding occurring in 942% of patients after embolization . in our series , rebleeding occurred within 30 days in 61 of 344 patients ( 17.7% ) . 
 a second embolization procedure was performed in three patients , two patients were found rebleeding in the previous points , and one was found new bleeding arteries . 
 overall the patients , 54 of whom rebleeding over 1 month in the follow - up , 44 of whom were performed a second bronchial arteriography . in 27 patients , 
 an additional bronchial or nonbronchial collateral artery was embolized resulting in the successful control of hemoptysis , 17 patients developed recanalization of the previously embilzed vessels and had been embolizied again . in the recurrent hemoptysis patients , 
 the etiologies were mainly due to cancer , tuberculosis , and severe bronchietasis ( at least two lobes were suffered ) . 
 the disease development contributed to the rebleeding . in our series , a patient with cancer was performed in bae for three times , and a patient with tuberculosis was performed for five times , both of whom were not suitable for surgery . 
 so it has shown that bae is an effective procedure with which to definitively treat some patients with hemoptysis and can be repeatedly utilized . 
 as we know , the source of massive hemoptysis in 90% of cases due to the brochial circulation , as well as the remaining 10% divided equally between the pulmonary circulation ( 5% ) and other systemic arterial supply to the lungs . 
 it is well - documented that the anatomy of bronchial arteries is variable form person to person , typically origin arises from the descending aorta between the levels of the t5 and t6 vertebrae . 
 four classic patterns of origin were described : type i ( 40% ) : two on the left and one on the right , presenting as an intercostobronchial trunk ( icbt ) ; type ii ( 21% ) : one on the left and one icbt on the right ; type iii ( 20% ) : two on the left and one on the right ( one icbt and one bronchial artery ) ; type iv ( 9.7% ) : one on the left and two on the right ( one icbt and one bronchial artery ) . according to the statistics , 
 the main responsible vessels for bleeding are right bronchial arteries , left bronchial arteries , combined right and left bronchial trunk , right intercostal arteries . in different etiologies , the rate of abnormal arteries is coincidence with the result . 
 the bronchial arteriography showed that more right intercostal arteries combining with bronchial artery than left . 
 the distribution and frequency of embolization of the arteries were agreed with kwon et al . usually 
 , a spinal artery can originate from a bronchial artery in up to 5% of patients , with right side being more common than the left side . as our observation , 
 a spinal artery was identified in three of our patients , with two occurring on the right side and one occurring on the left side . 
 our result showed that tuberculosis ( 55.2% ) , bronchiectasis ( 28.8% ) , malignancy ( 5.8% ) , undefined ( 5.2% ) , pneumonia ( 1.8% ) , deformity of bronchial artery ( 1.8% ) , bronchial artery aneurysm ( 0.2% ) , other pulmonary disease ( 1.2% ) . 
 , the etiologies of hemoptysis included tuberculosis ( 34% ) , bronchiectasis ( 26% ) , aspergilloma ( 18% ) , neumoconiosis in coal miners ( 13% ) , and bronchogenic carcinoma ( 3% ) . 
 knott - craig et al . retrospectively studied 120 patients with hemoptysis and found that the right lung was the source in 62% and the left lung in 38% , with the right upper lobe being the area that was most commonly affected . 
 our results were that the right lung was the source of bleeding in 131 patients ( 38.1% ) , the left lung was the source in 69 patients ( 20.0% ) , and both lungs were the source in 144 patients ( 41.8% ) . 
 the common complications of bae included subintimal dissection , arterial perforation by a guidewire , and reflux of embolic material into the artery without adverse sequelaed during the procedure , and fever , chest pain , dyspnea , transient dysphagia , groin hematoma , even headache or blindness etc . 
 several patients developed transverse myelitis as a result of inadvertent embolization of the spinal arteries . during the procedure of bae , when a spinal artery was founding , 
 but in contrast , mal et al . observed the brown - sequard 's syndrome , following three episodes of spinal cord complications , which regressed after 4 months without sequelae ; paraparesis with spontaneous regression after 2 weeks ; and complete paraplegia without regression . 
 if a spinal artery arises from a bronchial artery , we will only embolize the bronchial artery if we can achieve a stable distal position well beyond the spinal artery origin . 
 our patients showed transistent complications such as fever , chest pain , shoulder pain , groin hematoma , all the symptoms were disappeared in 48 hours , and fewer of our patients experienced any neurologic sequelae . 
 observed the outcome of bae : the hemoptysis control rate was 93.1% at the end of 2 weeks , 85.7% at the end of 1 month , 79.5% at 90 days , 63.2% at 180 days , 51% at 1 year and 38.7% at the end of 2 years . 
 the follow - up of our patients revealed that 28 patients had died , and six from massive hemorrhage and the remainder as a result of their disease process . 
 twenty - one patients have recurrent hemoptysis after repeated bae , the volume of bleeding is < 5 ml / d , taking medicine can control the bleeding . 
 one patient suffered recurrent hemoptysis wanted to take a surgical management , but the diffuse disease in lung limited the management . 
 there was no follow - up information available on 92 patients , because they did not return after treatment and the telephone numbers were null . 
 the remaining 199 patients had follow - up periods ranging from 6 months to 8 years without evidence of recurrent hemoptysis . in summary 
 , hemoptysis represents a significant clinical entity with high morbidity and potential mortality . medical management ( in terms of resuscitation and bronchoscopic interventions ) and surgery 
 bae procedures represent the first - line treatment for hemoptysis arising from bronchial arterial source . in our study 
 , it was very useful in obtaining immediate bleeding control which was slightly better than that in other report . 
 the technique was a relatively safe and effective method for control the hemoptysis , importantly , it can be used repeatedly . in our study , 
 more than 50% patients had massive hemoptysis , about 70% patients ca nt be identified bleeding point by chest ct , 41% patients hemorrhage source from two lungs according the bronchial arteriography . 
 although the long - term outcome in some patients is not good , bae may be the only life - saving treatment option in patients who are poor surgical candidates .","background : hemoptysis is a significant clinical entity with high morbidity and potential mortality . 
 both medical management ( in terms of resuscitation and bronchoscopic interventions ) and surgery have severe limitations in these patients population . 
 bronchial artery embolization ( bae ) represents the first - line treatment for hemoptysis . 
 this article discusses clinical analysis , embolization approach , outcomes and complications of bae for the treatment of hemoptysis.methods:a retrospective analysis of 344 cases , who underwent bronchial arteriography at tianjin haihe hospital between 2006 and 2013 . 
 several aspects of outcome were analyzed : demographics , clinical presentation , radiographic studies , results , complications and follow - up of bae.results:three hundred and forty - four consecutive patients underwent bronchial arteriography , 336 of 344 patients ( 97.7% ) performed bae ; there were 1530 coils for 920 arteries embolized ; the main responsible sources for bleeding were right bronchial artery ( 29.7% ) , left bronchial artery ( 21.6% ) , combined right and left bronchial trunk ( 18.4% ) , right intercostal arteries ( 13.3% ) ; 61 patients ( 17.7% ) had recurrent hemoptysis within 1 month after undergoing bae , 74 patients ( 21.5% ) had recurrent hemoptysis over 1 month after undergoing bae ; the common complications of bae included subintimal dissection , arterial perforation by a guide wire , fever , chest pain , dyspnea , etc . 
 the follow - up was completed in 248 patients , 28 patients had been dead , 21 patients still bleed , 92 patients had lost to follow-up.conclusions:the technique of bae is a relatively safe and effective method for controlling hemoptysis . 
 the complications of bae are rare . 
 although the long - term outcome in some patients is not good , bae may be the only life - saving treatment option in patients who are poor surgical candidates .",pubmed
"helicobacter pylori ( hp ) 
 infection may cause several clinical manifestations , ranging from asymptomatic 
 to significant gastroduodenal disease including ulcer , bleeding , perforation , 
 and adenocarcinoma . no pathogenic mechanism is known to explain the above 
 diversity . 
 celiac disease ( cd ) is a 
 t - cell - mediated disorder of the small bowel triggered by gluten in susceptible 
 subjects . 
 the damage is not confined only to the small bowel but may affect the 
 gastric mucosal structure and function . in 
 both conditions , hp and cd , a systemic humoral immune response is detected , but 
 much interest has been focused on the local immune inflammatory reaction . 
 it is 
 defined by the presence of > 25 intraepithelial lymphocytes ( iels ) ( surface 
 and upper foveolar epithelium ) per 100 epithelial cells , without taking into 
 account the mononuclear inflammatory 
 cell infiltration of the lp . 
 this condition may be recognized endoscopically as 
 varioliform gastritis , nodularity , hypertrophic gastropathy , and aphtous erosions 
 [ 5 , 6 ] . 
 a similar histological entity has been reported in association with a 
 variety of gastric infections , inflammatory diseases , and in autoimmune 
 disorders affecting the gastric mucosa including cd , in adults [ 79 ] , as well 
 as in pediatric patients [ 1012 ] . 
 additionally , hp is a major etiology for lg , 
 extensively documented in adults [ 13 , 14 ] and children [ 15 , 16 ] . 
 furthermore , 
 investigations of the relationship between hp infection and cd have yielded 
 conflicting results [ 9 , 17 ] , probably because of the different prevalence of hp 
 in the populations studied . 
 others have focused on the hp - related lg in cd 
 [ 6 , 8 ] and recently on the link between anemia , hp , and cd . 
 the pathogenetic 
 relationship between cd , hp and lg is even more complicated since gastric hp 
 existence attenuates duodenal lesions in cd patients . 
 it is generally accepted that the major cytokine 
 response to hp and cd has a t - helper 1 ( th1)-type profile . 
 the exact 
 nature of the cellular response contributing to this inflammatory profile has 
 not been determined , and less so in children . 
 conflicting results exist in the 
 literature on the lymphocytic subpopulations , quantity and quality , 
 infiltrating the gastric mucosa in cd and hp . 
 as lg is a morphologic endpoint of numerous etiologies , the literature is ripe with controversy   and since extensive characterization of the lymphocytic populations infiltrating the gastric mucosa in cd and hp infected children is lacking , the present study was undertaken . 
 the aims were to look for additional quantitative and qualitative histological features , to define the immunophenotype of the gastric mucosa of the two most prevalent etiologies of pediatric lg ( cd and hp infection ) . 
 our secondary aim was to demonstrate a unique histopathology profile of lg by immunohistocemistry staining , trying to clarify the interrelationship between hp and cd in the pediatric population , thus shedding new light on the two entity 's enigma . 
 forty patients referred 
 for endoscopy due to upper gastrointestinal symptoms ( mostly recurrent 
 abdominal pain , or iron deficiency anemia ) were included in the study . 
 patients 
 were divided into 4 groups group1 is eight normal controls ( without cd nor hp 
 infection ) ; group 2 is ten patients with new active cd without hp infection ; 
 group 3 is twelve celiac negative patients infected by hp ; and group 4 is ten new 
 active cd patients infected by hp ( table 1 ) . 
 none of these patients had any 
 other gastrointestinal underlying disease including evidence for food allergy , 
 giardiasis , or inflammatory bowel disease . 
 the diagnosis of cd was based on the 
 accepted histological findings   supported by positive serology for cd 
 ( antiendomysial and antitissue transglutaminase antibodies ) . 
 all cd 
 patients were under normal gluten containing diet at the time of diagnosis . 
 hp 
 status was assessed according to conventional biopsy - based criteria plus 
 positive urease test . 
 all patients underwent esophago - gastro - duodenoscopy using 
 gif - xp 20 endoscope ; pentax , tokyo , japan . 
 at least 
 6 biopsies were obtained : 3 from the second part of the duodenum for diagnosing 
 or exclusion of cd , and 3 from the antrum ; one for quick urease test and two 
 for histologic examination . 
 sections obtained from 
 gastric biopsies were immediately fixed in buffered formalin and embedded on 
 edge in paraffin . 
 the diagnosis of lg was established if 25 lymphocytes per 100 
 gastric epithelial cells infiltrated the surface epithelium . 
 furthermore , we 
 estimated the extent to which the inflammatory infiltrate involved the lp and 
 the mucosal glands . in addition , the number of 
 mucosal and submucosal lymphoid aggregates were counted . to clarify the 
 nature of these inflammatory cells 
 three  m sections were prepared from formalin - fixed , paraffin - embedded 
 tissue blocks , air dried , and subjected to deparaffinization with xylene and 
 absolute alcohol . 
 the immunoperoxidase stains were performed using a panel of 
 antibodies that included cd20 , cd3 , cd4 , cd8 , cd57 , cna42 , and ki67 ( table 2 ) , 
 with appropriate dilutions as recommended by the manufactures , using the 
 ventana es autoimmunostainer and the iviewdab detection kit from ventana . 
 the gastric iels were counted on the 
 hematoxylin - eosin stains per 100 consecutive gastric surface mucosal cells with 
 a x400 magnification ( objectivex10 ) . 
 the density of subepithelial lymphocytes was 
 determined semiquantitatively using a 4 tier grading system , according to the 
 percentage of the area in the lp infiltrated by the inflammatory cells . 
 grade 0 is if less than 5% of the area was infiltrated by inflammatory cells , grade 1 is 
 between 530% , grade 2 is between 3060% , and grade 3 is if > 60% of the area was 
 infiltrated . in order to evaluate the inflammatory glandular involvement , 
 glands containing inflammatory cells 
 were counted out of a fixed total number 
 of superficial glands ; calculated as the percent of involved glands . 
 continuous variables were compared across groups using 
 analysis of variance ( anova ) with bonferroni 's correction for multiple 
 comparisons . 
 discrete ( semi quantitative ) variables were compared using 
 kruskal - wallis nonparametric one - way analysis of variance , with multiple 
 comparisons . a p - value less or equal than 0.05 was considered significant . 
 demographic data of the four examined groups 
 of patients are summarized in table 1 . 
 the mean age of all groups and gender 's ratio 
 were not statistically significant different . in cd as well as in hp 
 gastritis patients , increased numbers of iel 
 compatible with the diagnosis of lg ( 45.518.5 per 100 gastric epithelial 
 cells ) compared to patients without lg ( 10.84.5 per 100 gastric epithelial 
 cells ) , were observed . 
 the highest proportion of lg ( 50% ) was found in cd patients without hp 
 infection , followed by patients with cd infected by hp ( 20% ) , hp positive 
 patients without cd ( 8.3% ) and 
 none in the control group . 
 the distribution and 
 total number of lymphoid aggregates in the antrum among the four different 
 groups are shown in table 3 . 
 patients infected with hp had the highest number 
 of lymphoid aggregates irrespective of the cd status . 
 three cd patients with hp 
 and nine hp infected patients without cd had more than 2 lymphoid aggregates . the mean number of 
 intraepithelial lymphocytes ( iels ) positive for cd3 was increased significantly 
 in cd patients with or without hp infection compared to controls [ p0.01,p0.05 
 respectively ] , ( table 4 ) . 
 similarly the mean number of cd8 + iels was increased 
 significantly in cd patients with or without hp infection compared to controls 
 ( p<0.05 ) ( figure 1 ) . 
 hp infection did not contribute to the number of cd8 + 
 iels . in the hp infected group without cd , the number of cd8 + iels was not 
 significantly different from the normal controls [ table 4 ] . 
 a slight increase 
 in cd8 + lymphocytes was noted in the lp and intra mucosal glands in the cd 
 group patients compared with all the other groups , although these results did 
 not reach statistical significance . 
 cd4 + lymphocytes were 
 slightly increased in the lp and in the mucosal glands of the hp+ patients 
 compared with controls and cd cases . 
 no statistical 
 significant differences were found for the number and distribution of b 
 lymphocytes ( cd20 + ) , natural killer ( nk ) lymphocytes ( cd57 ) and follicular 
 dendritic cells ( cna42 ) within all different examined compartments ( iels , lp 
 and glands ) . 
 in order to investigate the differential contribution 
 of hp infection to lg in an inflammatory condition like cd , the gastric tissue 
 inflammatory subpopulation profile was investigated , by immunohistochemistry , 
 and compared between the 4 groups of patients . 
 past studies focused mainly on 
 the surface epithelial infiltrate as well as on the superficial pit gastric 
 epithelium invasion by lymphocytes , and to a lesser extent on the infiltrate in 
 the lp and in the gastric glands . 
 the main finding of the 
 present study was the highest prevalence lg in pediatric cdhp followed by cdhp . 
 forty five percent of cd 
 patients are estimated to have lg like in our study [ 8 , 12 ] 
 . however , lg appears 
 to be similarly frequent in hp positive children with and without cd . 
 previous studies demonstrated that lg was found to be more common in hp 
 positive children without cd than in hp negative children without cd . 
 there are still controversial results on 
 the contribution of cd and hp infection to lg . 
 moreover , the association 
 between these pathologies to lg is not well established [ 1315 ] . 
 lymphocytic 
 gastritis was reported in 3645% of children with cd [ 6 , 10 , 13 ] , and disappears 
 after a gluten free diet . 
 hp infection is less frequently found in patients 
 with lg [ 13% ] than with the usual chronic antral gastritis [ 6590% ] . 
 cd is 
 considered to be a population at greatest risk for lg compared to hp affected 
 patients . 
 our study confirmed the 
 published results of an increased number of iels in both cd and hp infected 
 patients . 
 surprisingly , cd patients with associated hp infection showed a lower rate of 
 lg . 
 this evidence might be explained by the well known limited roll of 
 bacterial infection in cytotoxicity . 
 hp convergent the immune response towards 
 th2 response and suppress the th1 immune response . 
 previous investigators 
 have published several sets of data concerning the composition of the 
 lymphocytic infiltrates in the different diseases [ 1620 ] . 
 drut et al found 
 that lg in pediatric cd patients contains a peculiar cd3 , cd7 and cd8 
 intraepithelial lymphocyte population , that is not associated with the presence 
 of cd4 , cd20 , cd56 and cd57 iels . in hp gastritis there is an increased 
 number of mononuclear cells in the gastric lp , including b and t lymphocytes , plasma cells , macrophages and mast 
 cells . 
 it has been shown that hp stimulates b lymphocytes and causes an increase 
 in their numbers predominantly in the lp . 
 although immunity against hp 
 infection appears not to be dependent on b cells , the role of t cells still 
 remains to be clarified . 
 it appears however that the b cell proliferation 
 might be driven by activated lymphocytes ( cd4 + cells ) that might recall and 
 activate mononuclear phagocytes . 
 demonstrated 
 that the cellular response includes an innate nonspecific response represented 
 mainly by polymorphonuclear cells and macrophages , as well as a t cell response 
 with abundant positive staining with anti - cd8 antibodies , was observed 
 indicative of a predominance of suppressor / cytotoxic t lymphocytes both in the 
 lp and in the epithelium . 
 similarly we also showed 
 the presence of cd3 + iels in both diseases ( cd ; hp infection ) . despite a 
 potential additive effect between these two pathological processes , it is 
 impossible to differentiate between them , based only on the number of the cd3 + 
 iels in the antral mucosa . 
 cd3 staining is a pan t lymphocyte marker and does 
 not differentiate between cd4 helper and cd8 suppressor / cytotoxic cells , 
 therefore we aimed to characterize the t cell subsets immunophenotype ( cd4 + , 
 cd8 + , cd57 ) . in our study the cd8 + 
 iels were significantly higher in cd patients ( 20/100 epithelial cells ) , 
 compared to controls ( 1/100 epithelial cells ) or hp infected patients without 
 cd ( 2/100 epithelial cells ) . 
 we assume that in undiagnosed patients with 
 histological features compatible with lg , higher counts of cd8 + iels may imply 
 that the diagnosis is cd rather than hp gastritis . 
 our findings support the 
 published data that cd8+/cd4 iels are involved significantly in the 
 pathogenesis of cd . 
 in our study , the low expression of cd57 in 
 the lymphoid cells , both in the epithelium , in the lp as well as in the mucosal 
 glands indicate that nk cells may play a negligible role in these two pathologies . 
 we also tried to differentiate between 
 these two causative agents of lg by looking at the proliferation marker , ki67 . 
 although both conditions were associated with a prominent adaptive immune 
 activity , no increase in the proliferation index of the surface epithelium was 
 demonstrated in these pathologies . 
 dendritic cells ( cna42 + ) which serve as 
 professional antigen presenting cells did not show an increased expression in 
 both pathologies . 
 this may indicate that the process of antigen presentation 
 occurred in the lymph nodes . 
 this notion is supported by the absence of cd4 ( t 
 helper ) cells in the immune cell infiltrates found in the affected tissues . 
 in addition to all the 
 above , we confirmed the published results that the highest number of lymphoid 
 follicles was observed in a similar proportion of children with or without cd , 
 who were hp positive independent of the presence of lg . 
 the inflammatory 
 infiltrate in the lp and in the mucosal glands does not contribute to the 
 differentiation between these diseases . in summary , our study 
 aimed to explore the contribution of cd and hp infection to lg and to 
 characterize the different immunoprofiles of the gastric inflammatory cells 
 involved in these diseases . 
 we were looking for an applicable histological tool 
 that might differentiate between cases of cd and hp infection with overlapping 
 clinical and histological features . 
 we suggest that in very 
 young patients infected by hp and suspicion for having atypical cd ( negative 
 serology with increased iels with normal villous architecture - marsh i 
 classification ) with a debatable diagnosis of cd versus hp infection , the 
 number of cd8 + iels in the antrum might hint toward the diagnosis of cd rather 
 than hp infection , and the number of lymphoid follicles directs toward the 
 diagnosis of hp infection . 
 thus , it is important to include immunohistochemical 
 analysis of cd8 lymphocytes in the antrum in undefined cases of cd .","lymphocytic gastritis ( lg ) is associated with helicobacter pylori ( hp ) and celiac disease ( cd ) . 
 we aimed to clarify 
     the relationship between hp infection and cd by defining a unique histopathology profile of lg in these two diseases . 
 forty patients who underwent upper endoscopy were divided into four groups : eight controls , ten active cd patients 
     without hp , twelve cd negative with hp , and ten active cd with hp infection . 
 antral samples were assessed by 
     immunohistochemical staining for cd20 , cd3 , cd4 , cd8 , cd57 , cna42 , and ki67 for lymphoid aggregates , 
     intraepithelial lymphocytes ( iels ) number , density of lamina propria ( lp ) lymphocytes , and inflammatory glandular 
     involvement . only iels positive for cd3 and cd8 were increased significantly in cd patients with or without hp infection . 
 hp did not contribute to the number of cd8 iels . 
 in complicated cases with hp and suspicious for cd , the number of 
     cd8 + iels hints toward a cd rather than hp infection .",pubmed
"angiogenesis , the process by which the existing vascular network expands to form new blood vessels , is required for the growth of solid tumors . for this reason , 
 vascular endothelial growth factor ( vegf ) , a primary stimulant of angiogenesis , binds and activates vegf receptor 1 ( vegfr1 ) and vegfr2 . vegf is an important and powerful factor increasing vascular permeability and promoting metastasis . without blood vessels , 
 the tumors can not be larger than a few millimeters , so the inhibition of angiogenesis with the use of several drugs could represent an important tool in cancer treatment for several reasons . 
 ( 1 ) angiogenesis occurs at high levels during fetal development , the menstrual cycle , and in wound healing . 
 therefore , the treatments should have low toxicity ; in fact , they might be expected to interfere with this process and should not harm most normal dividing cells . 
 ( 2 ) the antiangiogenic treatments should not be designed to attack directly the cancer cells . 
 the targets of several of these treatments are normal processes controlled by normal cells and not by the tumor cells themselves . 
 the high mutation rates of cancer cells that often render chemotherapy ineffective will not interfere with these drugs .   in this paper 
 , we underline the importance of inhibition of vegf as attractive therapeutic target in the treatment of cancer . 
 vegf is a primary stimulant for tumor angiogenesis , making it a critical target for cancer therapy [ 3 , 4 ] . in breast cancer , elevated levels of vegf 
 actually , bevacizumab , a humanized monoclonal antibody that binds human vegf and prevents vegf from binding vegfr1 and vegfr2 , is approved for the treatment of metastatic her2/neu - negative breast cancer . 
 anti - vegf therapy with bevacizumab , the phenethylamine of the 2c family 2c3 or the fully human antibody that inhibits vegf binding to vegfr2 r84 inhibits the growth of established orthotopic mda - mb-231 breast cancer cell line in severe combined immunodeficiency ( scid ) mice , reduces tumor microvessel density , and limits the infiltration of tumor - associated macrophages , but it is associated with elevated numbers of tumor - associated neutrophils [ 9 , 10 ] . 
 selective inhibition of vegfr2 with an anti - vegf antibody is sufficient for effective blockade of the protumorigenic activity of vegf in breast cancer xenografts . 
 these findings further define the complex molecular interactions in the tumor microenvironment and provide a translational tool that may be relevant to the treatment of breast cancer . 
 inhibition of vegf binding to vegfr2 by 2c3 has been shown to reduce tumor size both in pancreatic [ 1113 ] and breast tumors . 
 also the effect on tumor growth after the treatment with r84 in an orthotopic breast cancer model , similar to 2c3 , has been evaluated . 
 in fact , mda - mb-231 cells ( 5  10 ) were injected into the mammary fat pad of nonobese diabetic nod / scid mice , and the therapy was initiated on day 26 after tumoral cell injection , when tumor volume reached 150 mm . in this orthotopic human breast cancer xenograft model , the chronic treatment with r84 , 2c3 , or bevacizumab significantly reduced ( p < 
 .001 ; days 44 and 48 versus control ) the tumoral growth , such that there was a 55% , 62% , and 58% decrease , respectively , in tumor volume compared with control - treated animals . 
 thus , these data show that inhibition of the vegf factor is sufficient to reduce the mass volume of mda - mb-231-derived tumors . to determine if the effect of r84 , 2c3 , and bevacizumab on mda - mb-231 tumor growth in vivo could be due directly to the block of vegf activation of tumor cells , the tumor cell proliferation and migration were also evaluated in vitro , demonstrating that mda - mb-231 cells migrated strongly toward vegf and , consequently , this migration was blocked by the addition of 2c3 , r84 , or bevacizumab . 
 vegf is an abundant cytokine in the tumor microenvironment and is known to stimulate immune cell chemotaxis ; however , few studies have looked directly at the effect of inhibitors of vegf on immune cell infiltration into tumors . 
 it has been shown previously that 2c3 inhibits macrophage infiltration in an orthotopic pancreatic cancer model [ 16 , 17 ] . in a mouse model of thyroid cancer , 
 in nonobese diabetic ( nod)/scid mice , inoculated with mda - mb-231 cells , anti - vegf therapy with 2c3 , r84 , or bevacizumab reduced tumor microvessel density by 50% , 45% , and 58% , respectively . cancer and inflammation are connected by two pathways : the intrinsic and the extrinsic pathways . 
 these two pathways converge , resulting in the activation of transcription factors , such as nuclear factor-b ( nf-b ) , signal transducer and activator of transcription 3 ( stat3 ) , and hypoxia - inducible factor 1 ( hif1 ) , in tumour cells . 
 these transcription factors modulate the production of inflammatory mediators , including cytokines and chemokines . these factors recruit and activate various leukocytes . 
 the cytokines activate the same key transcription factors in inflammatory cells , stromal cells , and tumor cells , resulting in more inflammatory mediators being produced and a cancer - related inflammatory microenvironment being generated . 
 the mediators and cellular effectors of inflammation are important constituents of the local environment of tumours ; in some cancers , inflammatory conditions are present before a malignant change occurs . to examine the importance of inflammatory microenvironment on tumor site , 
 where a massive macrophage infiltration is associated , breast tumors from bevacizumab , 2c3 , or r84 treatment groups were analyzed using three different macrophage markers : the mouse f4/80 antigen , a 160  kd glycoprotein expressed by murine macrophages , and cd16 + cells , a cluster of differentiation found on the surface of natural killer cells , neutrophil polymorphonuclear leukocytes , monocytes , and macrophages . 
 immunofluorescence staining of tumor sections revealed that anti - vegf therapy with 2c3 , r84 , and bevacizumab reduced cluster of differentiation the infiltration of f4/80 +   and of cd16 + natural killer cells , neutrophil polymorphonuclear leukocytes , monocytes and macrophages . 
 the reduction of macrophage infiltration in the phase of treatment with 2c3 and r84 is presumably due to expression of vegfr2 on tumor - associated macrophages . 
 the major findings of this study were that 2c3 , r84 , and bevacizumab effectively decrease tumor size , microvessel density , and macrophage infiltration in an orthotopic model of breast cancer . 
 these data are consistent with previous findings [ 1618 ] and support the concept that reducing macrophage infiltration is an important aspect of anti - vegf therapy . in addition , these studies showed changes in other immune cell infiltrates : myeloid - derived suppressor cells ( mdscs ) , dendritic cells ( dcs ) , and neutrophils , following anti - vegf therapy . 
 in fact , infiltrating inflammatory cells constitute a large component of the overall tumor mass . 
 vegf is a major chemoattractant for inflammatory cells , including macrophages , neutrophils , dendritic cells , myeloid - derived suppressor cells , and t - cells [ 20 , 21 ] . to evaluate 
 if inhibition of vegf reduces angiogenesis and modulates immune cell infiltration of orthotopic breast cancer xenografts , roland et al . 
 use preclinical models of breast cancer to compare the effect of different anti - vegf therapies on breast cancer growth , vascular parameters , immune cell infiltration , and intratumoral cytokine levels . 
 they confirmed that the inhibition of vegf receptor activation resulted in changes in intratumoral levels of il-1 and cxcl1 that correlated with changes in immune cell infiltration . 
 in fact ,   ma et al . constructed a plasmid encoding vegf shrna to knock down vegf both in vitro and in vivo . in vitro , specificity and potency of the targeting sequence 
 were first validated in a549 lung adenocarcinoma cells by rtpcr and elisa assays . in vivo 
 the vegf shrna expressing plasmids were administered systemically in combination with low dose of cis - diclorodiamminoplatino ( ddp ) that is an antineoplastic chemotherapy agent that interferes with all phases of the cell cycle by binding to dna through the formation of crosslinks between complementary strands . 
 the combinated treatment of the two agents had a significantly enhanced antitumoral effect compared with the treatment with pshvegf or ddp alone , resulting in reduction of the tumor weight by 83.13% . to demonstrate that the therapeutic effects were related to downregulation of vegf expression instead of other nonspecific reactions , 
 it was analyzed the distribution of immunoreactive vegf in the tumors and observed a general decrease of vegf staining in the tumors belonging to the mice treated with pshvegf , whereas the tumors belonging to the mice treated with pshhk exhibited significantly more vegf staining . 
 consistently , the elisa assay showed that pshvegf caused significant reduction in intratumoral vegf expression compared with pshhk . 
 these data reinforce the hypothesis that the effect of anti - vegf agents extends beyond the inhibition of angiogenesis because many immune cells express vegfrs , including macrophages , neutrophils , mdscs , dcs , and t - cells [ 23 , 24 ] . 
 in fact , it was shown a reduction in macrophage number in tumors from animals treated with anti - vegf therapy with respect to the control mice . 
 vegf is a potent stimulator of endothelial cell survival , mitogenesis , migration , and differentiation [ 1 , 2 ] . 
 several vegf inhibitors have been approved by the us food and drug administration for the treatment of tumors or age - related macular degeneration [ 3 , 4 ] ( figure 1 ) . 
 recently , in many clinical trails , angioinhibitors were also being used in combination with conventional chemotherapy . 
 clinical trials generally combine very low dose of chemotherapy followed by angioinhibitor therapy . combination of angioinhibitors will need to be tested vigorously in the future , as single angioinhibitors are approved for use of cancer 
 .   preventive angioinhibitory therapy may also be possible in the future , because angioinhibitory therapy is generally less toxic and less susceptible to induction of acquired drug resistance .","cancer is the second leading cause of death in the world after cardiovascular diseases . 
 some types of cancer cells often travel to other parts of the body through blood circulation or lymph vessels , where they begin to grow . 
 this process is recognized as metastasis . 
 angiogenesis is the formation of new blood vessels from existing vessel . 
 normally angiogenesis is a healthy process , that helps the body to heal wounds and repair damaged body tissues , whereas in cancerous condition this process supports new blood vessels formation that provide a tumor with its own blood supply , nutrients and allow it to grow . 
 the most important proximal factor for angiogenesis is the vascular endothelial growth factor vegf . 
 angioinhibition is a form of targeted therapy that uses drugs to stop tumors from making new blood vessels . 
 therefore , in this paper we analyse the importance of vegf as target of cancer therapy , analysing murine models .",pubmed
"vulvar and vaginal atrophy ( vva ) , resulting from the loss of estrogen stimulation on vaginal and vulvar tissue , is a common medical condition in postmenopausal women 
 one that will occur in most postmenopausal women at some point in their lives.1,2 there are an estimated 64 million postmenopausal women in the united states ( us ) , and as many as 32 million women may suffer from vva symptoms including dyspareunia ( pain during sexual intercourse ) , vaginal dryness , and vaginal irritation.1,3,4 vva is chronic , progressive , and , unlike vasomotor symptoms , will not resolve with time and without treatment . 
 left untreated , vva symptoms can not only cause discomfort but can also negatively impact women s quality of life , including sexual relationships and emotional well - being.1,5 severe vva may affect other quality - of - life aspects , including clothing choices , exercise options , and general pelvic floor comfort . 
 the recently published revive ( real women s views of treatment options for menopausal vaginal changes ) survey , administered to a large cohort of postmenopausal women in the us , offers many insights into the impact of vva symptoms on women s lives . 
 findings of generally poor understanding of vva among women , coupled with concerns about efficacy , convenience , and safety of vaginal over - the - counter ( otc ) products and prescription therapies for vva , emphasize the need for better communication between women and their healthcare providers ( hcps ) about vva and its treatment options.6 this article reviews findings from the revive survey , discusses implications of these findings for hcps who care for postmenopausal women , and provides practical treatment strategies for the care of women with vva . 
 the revive survey was an online evaluation of postmenopausal women in the us , conducted from may 31 , 2012 through june 14 , 2012 , and published online on may 16 , 2013.6 a total of 15,576 women aged 4575 years were contacted through knowledgepanel ( gfk custom research , princeton , nj ) , a demographically representative panel of us citizens , making the revive survey the largest study cohort of postmenopausal women in recent years 
 . of 10,486 women who responded , 8081 ( 77% ) identified themselves as postmenopausal ( ie , having no menstrual period for the previous 12 months for natural or surgical reasons ) . among 8081 postmenopausal women , 3046 ( 38% ) reported 1 symptom consistent with vva ( dyspareunia , vaginal dryness , local irritation , tenderness , bleeding with sexual activity , or pain with exercise ) . 
 the common medical terms vva and vulvar and vaginal atrophy were unfamiliar to most women reporting vva symptoms . 
 most women were unaware that their vaginal symptoms could caused by menopause or hormonal changes ; only approximately one - quarter of women specifically identified menopause as the cause of their symptoms.6 thus , the revive survey demonstrates that many postmenopausal women have low awareness and poor understanding of vva and its associated symptoms . 
 these findings show the contrast between perceptions of vva symptoms and other symptoms that are more readily associated with menopause ( eg , hot flushes ) . 
 women who associate vva with menopause may assume that it will abate over time , similar to vasomotor symptoms . 
 almost half of the study population had never discussed their vva symptoms with an hcp . 
 forty percent of women with vva symptoms said they expected hcps to initiate this conversation ; however , among those who had discussed vva symptoms , the hcp was the initiator only 13% of the time . 
 similarly , among participants who had an hcp for gynecologic needs , only 19% reported being asked about sexual health during routine examination . 
 the most common reasons for not mentioning symptoms to hcps were the assumption that their symptoms were a natural part of aging or were not bothersome enough at that time.6 among women who initiated discussions about vva , 73% waited until a scheduled physical examination and ~50% waited greater than 7 months to do so.6 the most common symptoms prompting a visit specifically to discuss vva were vaginal irritation ( 50% ) , dyspareunia ( 27% ) , and vaginal dryness ( 24% ) . among women who discussed vva symptoms with an hcp , 
 ~50% felt neutral or negative about the information and recommended treatment options they received.6 this inadequate understanding of vva , along with poor communication between women and their hcps regarding vva , may contribute to delayed diagnosis and treatment . 
 data from the revive survey demonstrate the considerable impact of vva on women s lives . 
 the most commonly reported vva symptoms among postmenopausal women were vaginal dryness ( 55% ) , dyspareunia ( 44% ) , and local irritation ( 37% ) ( table 1).6 more than half of participants reported that vva had the greatest impact on enjoyment of sex ( table 2 ) . in all , 12% of women without a sexual partner noted they were not seeking one because of discomfort caused by their vva symptoms . 
 approximately one in four women reported that other areas of their life were negatively affected by vva , including sleep , general enjoyment of life , and temperament . 
 although some participants in the revive survey stated that the earliest onset of vva symptoms was premenopause ( 13% ) , or during the first year after their last menstrual period ( 20% ) , most ( 67% ) reported that their earliest symptoms began during postmenopause ( greater than one year after their last menstrual period).6 the onset of individual symptoms occurred at variable times , with irritation being the most likely symptom occurring premenopause , and dryness or tenderness more likely to begin during the first year after cessation of menstrual periods . 
 use of vva - specific treatments ( vaginal otc products [ eg , astroglide and replens ] or vaginal prescription therapies [ eg , estrace , vagifem , and estring ] ) was reported by 40% of participants.6 otc products were used by 67% of those who ever used a treatment , and vaginal estrogen therapies were used by 27% . even among women who had discussed symptoms with an hcp , the use of otc products as monotherapy was common ( 62% ) ; 23% of women who had spoken with an hcp were on prescription therapy and 15% of women were using vaginal prescription therapy + otc vaginal therapy . 
 reported limitations of participants current otc or prescription treatments included inadequate symptom relief , inconvenience , and dislike of the accommodations needed for vaginal administration ( ie , privacy and nighttime administration ) . 
 previous population - based surveys710 have identified that many women who are using vaginal therapies to treat their vva symptoms are dissatisfied and discontinue treatment because they had concerns about side effects , or had found the treatment messy and inconvenient or not to have an effect on their symptoms . 
 an additional analysis of data collected in the revive survey6 looked at women s perspectives on their satisfaction / dissatisfaction with their current treatments for vva including otc lubricants and moisturizers as well as prescription vaginal estrogen therapies . 
 of the 3046 postmenopausal women in the revive survey who reported vva symptoms , 41% were currently using some form of treatment for vva . 
 however , of those women who actually had been given a clinical diagnosis of vva ( 9% recalled being given a diagnosis ) , 27% were not using any treatment for their symptoms  neither a prescription nor otc treatment . 
 only 35% of postmenopausal women currently on any treatment for vva ( otc , prescription vaginal estrogens , or both ) were satisfied with their current vva treatment , with women using vaginal estrogen generally more satisfied than women using otc lubricants or moisturizers ( 42% vs. 32% , respectively ) . in the assessment of dislikes among current treatment options , long - term safety ( 41% ) 
 messiness ( 43% ) was the major concern identified by users of otc lubricants or moisturizers ( table 3 ) . 
 many women recognized that otc lubricants or moisturizers were able to neither restore the vagina to its natural state ( 39% ) nor provide adequate relief of vva symptoms ( 28% ) . 
 for the women who remained nave to treatment despite a diagnosis of vva , major concerns for not initiating treatment included safety concerns ( 28% ) and concerns about hormone exposure ( 16% ) . 
 however , many of these women ( 44% ) felt their symptoms were not bothersome enough to warrant treatment . with regard to satisfaction with current treatment(s ) , 40% of those using vaginal estrogen therapy felt they had no other treatment option , with an additional 22% feeling inconvenienced with the mode of administration . 
 in addition , 38% of all women who had ever used prescription products to treat vva chose not to refill their vaginal estrogen therapy because of a variety of concerns related to safety or side effects ( 45% ) including long - term safety concerns ( 28% ) , administration ( 8% ) , messiness ( 15% ) , or overall treatment efficacy ( 47% ) , with 16% citing  not enough relief from vva symptoms . 
 similar concerns were raised by those women who discontinued the use of otc lubricants and moisturizers , including issues related to administration ( 10% ) , messiness ( 14% ) , and overall treatment efficacy ( 38% ) . 
 some women also stated the symptoms not being bothersome enough as a reason for discontinuing their current therapy ( otc , 9% ; vaginal estrogen , 14% ) . when asked about preferences regarding the method of treatment for their vva symptoms 
 an oral treatment was highly preferred among younger patients as well as those patients who had never used any vva treatment , especially among those who indicated a direct preference . 
 for those women currently using any type of treatment for their vva symptoms , there was no difference in preference between an oral treatment ( 35% ) or a vaginal treatment ( 41% ) ( table 4 ) . 
 the common medical terms vva and vulvar and vaginal atrophy were unfamiliar to most women reporting vva symptoms . 
 most women were unaware that their vaginal symptoms could caused by menopause or hormonal changes ; only approximately one - quarter of women specifically identified menopause as the cause of their symptoms.6 thus , the revive survey demonstrates that many postmenopausal women have low awareness and poor understanding of vva and its associated symptoms . 
 these findings show the contrast between perceptions of vva symptoms and other symptoms that are more readily associated with menopause ( eg , hot flushes ) . 
 women who associate vva with menopause may assume that it will abate over time , similar to vasomotor symptoms . 
 almost half of the study population had never discussed their vva symptoms with an hcp . 
 forty percent of women with vva symptoms said they expected hcps to initiate this conversation ; however , among those who had discussed vva symptoms , the hcp was the initiator only 13% of the time . 
 similarly , among participants who had an hcp for gynecologic needs , only 19% reported being asked about sexual health during routine examination . 
 the most common reasons for not mentioning symptoms to hcps were the assumption that their symptoms were a natural part of aging or were not bothersome enough at that time.6 among women who initiated discussions about vva , 73% waited until a scheduled physical examination and ~50% waited greater than 7 months to do so.6 the most common symptoms prompting a visit specifically to discuss vva were vaginal irritation ( 50% ) , dyspareunia ( 27% ) , and vaginal dryness ( 24% ) . among women who discussed vva symptoms with an hcp , ~50% felt neutral or negative about the information and recommended treatment options they received.6 this inadequate understanding of vva , along with poor communication between women and their hcps regarding vva , may contribute to delayed diagnosis and treatment . 
 data from the revive survey demonstrate the considerable impact of vva on women s lives . 
 the most commonly reported vva symptoms among postmenopausal women were vaginal dryness ( 55% ) , dyspareunia ( 44% ) , and local irritation ( 37% ) ( table 1).6 more than half of participants reported that vva had the greatest impact on enjoyment of sex ( table 2 ) . in all , 12% of women without a sexual partner noted they were not seeking one because of discomfort caused by their vva symptoms . 
 approximately one in four women reported that other areas of their life were negatively affected by vva , including sleep , general enjoyment of life , and temperament . 
 although some participants in the revive survey stated that the earliest onset of vva symptoms was premenopause ( 13% ) , or during the first year after their last menstrual period ( 20% ) , most ( 67% ) reported that their earliest symptoms began during postmenopause ( greater than one year after their last menstrual period).6 the onset of individual symptoms occurred at variable times , with irritation being the most likely symptom occurring premenopause , and dryness or tenderness more likely to begin during the first year after cessation of menstrual periods . 
 use of vva - specific treatments ( vaginal otc products [ eg , astroglide and replens ] or vaginal prescription therapies [ eg , estrace , vagifem , and estring ] ) was reported by 40% of participants.6 otc products were used by 67% of those who ever used a treatment , and vaginal estrogen therapies were used by 27% . even among women who had discussed symptoms with an hcp , 
 the use of otc products as monotherapy was common ( 62% ) ; 23% of women who had spoken with an hcp were on prescription therapy and 15% of women were using vaginal prescription therapy + otc vaginal therapy . reported limitations of participants current otc or prescription treatments included inadequate symptom relief , inconvenience , and dislike of the accommodations needed for vaginal administration ( ie , privacy and nighttime administration ) . 
 previous population - based surveys710 have identified that many women who are using vaginal therapies to treat their vva symptoms are dissatisfied and discontinue treatment because they had concerns about side effects , or had found the treatment messy and inconvenient or not to have an effect on their symptoms . 
 an additional analysis of data collected in the revive survey6 looked at women s perspectives on their satisfaction / dissatisfaction with their current treatments for vva including otc lubricants and moisturizers as well as prescription vaginal estrogen therapies . 
 of the 3046 postmenopausal women in the revive survey who reported vva symptoms , 41% were currently using some form of treatment for vva . 
 however , of those women who actually had been given a clinical diagnosis of vva ( 9% recalled being given a diagnosis ) , 27% were not using any treatment for their symptoms  neither a prescription nor otc treatment . 
 only 35% of postmenopausal women currently on any treatment for vva ( otc , prescription vaginal estrogens , or both ) were satisfied with their current vva treatment , with women using vaginal estrogen generally more satisfied than women using otc lubricants or moisturizers ( 42% vs. 32% , respectively ) . in the assessment of dislikes among current treatment options , long - term safety ( 41% ) 
 messiness ( 43% ) was the major concern identified by users of otc lubricants or moisturizers ( table 3 ) . 
 many women recognized that otc lubricants or moisturizers were able to neither restore the vagina to its natural state ( 39% ) nor provide adequate relief of vva symptoms ( 28% ) . 
 for the women who remained nave to treatment despite a diagnosis of vva , major concerns for not initiating treatment included safety concerns ( 28% ) and concerns about hormone exposure ( 16% ) . however , many of these women ( 44% ) felt their symptoms were not bothersome enough to warrant treatment . 
 with regard to satisfaction with current treatment(s ) , 40% of those using vaginal estrogen therapy felt they had no other treatment option , with an additional 22% feeling inconvenienced with the mode of administration . 
 in addition , 38% of all women who had ever used prescription products to treat vva chose not to refill their vaginal estrogen therapy because of a variety of concerns related to safety or side effects ( 45% ) including long - term safety concerns ( 28% ) , administration ( 8% ) , messiness ( 15% ) , or overall treatment efficacy ( 47% ) , with 16% citing  not enough relief from vva symptoms . 
 similar concerns were raised by those women who discontinued the use of otc lubricants and moisturizers , including issues related to administration ( 10% ) , messiness ( 14% ) , and overall treatment efficacy ( 38% ) . 
 some women also stated the symptoms not being bothersome enough as a reason for discontinuing their current therapy ( otc , 9% ; vaginal estrogen , 14% ) . when asked about preferences regarding the method of treatment for their vva symptoms 
 an oral treatment was highly preferred among younger patients as well as those patients who had never used any vva treatment , especially among those who indicated a direct preference . 
 for those women currently using any type of treatment for their vva symptoms , there was no difference in preference between an oral treatment ( 35% ) or a vaginal treatment ( 41% ) ( table 4 ) . 
 results of the revive survey verify that many women suffering from vva symptoms do not discuss them with their hcp 
 . they may find it easier to discuss hot flushes rather than vaginal symptoms such as painful sex , because of the sensitivity of the latter topic.11 however , even women who have discussed vva with their hcp may not have had an optimal experience , as illustrated by the high number of women who continue using otc products despite inadequate symptom relief . 
 bridging communication gaps in the difficult topics of sexual health and menopause can result in increased patient adherence and satisfaction with vva treatment . 
 this can be accomplished not only by physicians but also by hcps in gyn and primary care practices such as nurse practitioners and physician assistants who may have more opportunity to engage in in - depth discussions and provide individualized care.12,13 when a potentially uncomfortable topic arises , a key to fostering open communication is to first put the woman at ease by  normalizing  the conversation , commenting that vva is a common medical condition that most postmenopausal women experience . 
 this can often be accomplished by prefacing questions about vva with general broad statements such as  many women have vaginal changes after menopause , so i ask all of my patients about vaginal and sexual health ,  or 
 tell me if you have experienced symptoms of vaginal changes , such as dryness .  opening statements such as 
 these can be followed by more specific questions about bothersome vaginal symptoms.11,14,15 willingness to discuss these issues can even be facilitated in the waiting room by providing a comfortable environment and pamphlets or other materials addressing the topic . 
 if signs of vva are present , although the patients did not complain of any symptoms , a statement can be made such as  i notice some changes that i ve seen in many postmenopausal women that may cause symptoms such as vaginal dryness or pain with intercourse ,  followed by  tell me about any symptoms you may have experienced . 
 simple screening questions can be incorporated into routine visits for women in their mid-40s to facilitate an open and honest dialogue about vva and the potential negative impact of vva symptoms . 
 the goal is to identify women who are experiencing vva symptoms but have not mentioned them because of assumptions and misconceptions such as those revealed in the revive survey . before the publication of the women s health initiative study , hormone therapy was prevalent and 
 however , the utilization and acceptance of systemic hormone replacement therapy is currently low,16 resulting in more women presenting with vva symptoms . with the trend toward using the lowest possible hormone dose for the shortest possible time , vva can even occur among postmenopausal women who are taking systemic hormone therapy.17 furthermore , as shown by the revive survey , women often do not understand that vva is related to menopause . 
 effective patient education might begin with the explanation that vva is a common medical condition associated with reduced estrogen levels after menopause , though less well known than vasomotor symptoms ( hot flushes ) . at the same time , hcps should point out that , unlike hot flushes , vva will worsen over time if left untreated.1 findings from the revive survey indicate that , for some women , symptom onset may occur at early stages of menopause and postmenopause ; these women may experience vva symptoms for a prolonged period if treatment is not initiated as soon as symptoms appear . 
 additionally , it may be helpful to describe the physical changes occurring in the vaginal tissues that result in vva and how these changes lead to the symptoms that women experience . 
 changes in the number of superficial and parabasal cells in the vagina lead to reduced moisture and elasticity , which contribute to symptoms such as vaginal dryness and dyspareunia . 
 increases in vaginal ph lead to greater susceptibility to infection , which in turn can produce symptoms such as itching and irritation . 
 visual examination findings , such as vaginal dryness , petechiae , pallor , friability , and redness of the mucosa , may also prompt discussion of menopausal vaginal changes ( fig . 
 1 ) . an effective way to describe these changes to patients may be as follows :  before menopause , due to the presence of estrogen , the vagina is moist and has ridges and folds , like pleats , that allow the vagina to be more flexible and adaptable for intercourse and childbirth . without estrogen , 
 physical changes occur in the vagina , leading to symptoms such as painful sex and dryness , and the chemistry of the vagina changes , resulting in greater likelihood of infection , itching , and irritation . 
 a suggested approach to screening and managing vva and associated symptoms is presented in figure 2 . 
 another important point to emphasize during patient education is that otc products do not effectively treat the underlying pathological causes of vva and therefore do not halt or reverse the progression of this condition . 
 however , certain prescription therapies ( ie , estrogens and estrogen agonist / antagonist therapies ) directly improve the physical changes underlying vva by increasing superficial cells and reducing parabasal cells and vaginal ph . although otc products may be the reasonable first - line option for women with mild symptoms 
 thus , if the otc product is tried and found to be ineffective , as was the case for ~40% of the revive survey participants who used them , the patient would already have a prescription in hand , and thus treatment would not have to be delayed . 
 women should be counseled on the many treatment options available for vva - related symptoms,18 and they can make choices based on their personal preferences and needs . treatment adherence is improved when women participate in the decision - making process . 
 table 5 provides an overview of currently available options for vva , including otc products ( lubricants and moisturizers ) , vaginal estrogen therapies ( conjugated equine estrogen and estradiol creams , estradiol tablet and ring ) , and a new non - estrogen oral therapy , ospemifene , which is an estrogen agonist / antagonist recently approved by the us food and drug administration for the treatment of moderate to severe dyspareunia associated with postmenopausal vva . 
 the revive survey offers important insights on vva from postmenopausal women experiencing undesirable vaginal symptoms . 
 this survey illustrates that women s awareness / understanding of vva is low , and almost half of the revive survey participants had not discussed their vaginal symptoms with their hcp . 
 moreover , 40% of women expected their hcp to initiate a conversation about menopausal symptoms . 
 these findings underscore the need for increased vigilance by hcps caring for postmenopausal women . for women approaching menopause , simple screening questions about vaginal symptoms 
 can be asked during routine visits , and the responses can help identify those in need of treatment . 
 in addition , proactive patient education can be provided on the physical changes underlying vva and how the condition can progress if left untreated . 
 only 41% of respondents were current treatment users , whereas 27% had never been treated and 33% had stopped using treatment . 
 the use of otc products that do not treat the physiological changes underlying vva was prevalent , even among women who had discussed their symptoms with an hcp . 
 nurse practitioners ( nps ) should engage their patients in a candid discussion of symptom severity , preferences , and concerns regarding vva treatments . 
 this approach can help guide treatment selection and patient education , which ultimately may improve patient adherence and outcomes . 
 hcps are in a position to proactively identify women suffering from vva , provide essential patient education regarding the condition , and guide their patients to appropriate treatment .","vulvar and vaginal atrophy ( vva ) is a chronic and progressive medical condition common in postmenopausal women 
 . symptoms of vva such as dyspareunia , vaginal dryness , irritation , and itching can negatively impact sexual function and quality of life . 
 the revive ( real women s views of treatment options for menopausal vaginal changes ) survey assessed knowledge about vva and recorded attitudes about interactions with healthcare providers ( hcps ) and available treatment options for vva . 
 the revive survey identified unmet needs of women with vva symptoms such as poor understanding of the condition , poor communication with hcps despite the presence of vaginal symptoms , and concerns about the safety , convenience , and efficacy of available vva treatments . 
 hcps can address these unmet needs by proactively identifying patients with vva and educating them about the condition as well as discussing treatment preferences and available therapies for vva .",pubmed
"in denmark , women planning to conceive are advised to take a daily supplement of 400 g folic acid ( fa ) until the 12th week of gestation to reduce the risk of neural tube defects ( ntds).1 starting fa supplementation before conception is an important component of effective prevention of ntds , since the neural tube closes at gestational week 6 , only a few weeks after the pregnancy may be recognized.2 fa is primarily taken as single supplements or as a component of multivitamins ( mvs ) and other vitamin preparations made for pregnancy planners or pregnant women . despite campaigns promoting the fa recommendation in denmark in 1999 and 2001 , women in the preconceptional period appear to have neither adequate dietary intake of folate nor adequate supplementation from fa tablets or mv supplements . according to the national survey of dietary habits from 2003 to 2008 , only 510% of danish women of fertile age have a sufficient intake of dietary folate.3 the most recent data on periconceptional use of fa supplements in denmark are from the danish national birth cohort ( dnbc ) . between 2000 and 2002 
 , the proportion of fa compliers among dnbc participants who planned their pregnancy increased from 14% to only 22% , and thus remained low even after the campaigns.4,5 several retrospective studies of pregnant women in other countries also reported poor compliance , with 12% of women in the uk,6 17% in norway,7 2330% in australia8,9 and 44% in the us10 reporting fa supplement use in the preconception period . 
 in addition , a prospective cohort study reported that only 6% of the women who became pregnant within 3 months of being interviewed in a general woman s survey in the uk followed the fa recommendation.11 in previous studies , young age , low education and income , smoking , overweight , and parity were reported to be predictors of noncompliance.58,10 to date , no study has examined other health - related predictors of compliance during the preconceptional period , and we lack recent estimates of compliance with the fa recommendation reported from pregnancy planners during the preconceptional period . in the current study , we estimated the prevalence of fa and mv supplement use and identified sociodemographic , lifestyle , reproductive , and medical predictors of such use among danish pregnancy planners . 
 the present study was a cross - sectional analysis of baseline data from an internet - based prospective cohort study of danish pregnancy planners  snart - gravid ( soon pregnant).the study design has been described in detail elsewhere.12,13 briefly , the study was initiated in june 2007 . 
 participants were enrolled via the study website , and data were collected by email and self - administered questionnaires . 
 recruitment was achieved by a pop - up advertisement placed on a well - known health - related website ( http://www.netdoktor.dk ) and two press releases . before enrolment 
 , potential participants entering the study website were required to read a consent form and fill in a screening questionnaire in order to confirm eligibility . 
 further , participants were required to provide a valid email address and their personal civil registry ( cpr ) number , which is a unique ten - digit personal identification number allowing linkage to a number of nationwide registries . 
 eligible women were invited to complete a baseline questionnaire and bimonthly follow - up questionnaires for 12 months or until conception occurred , after which active follow - up ended . 
 participants were initially randomized to receive either a short- or a long - form baseline questionnaire . 
 completion rates and missing data were similar for both questionnaire versions.13 eligible women were danish residents aged 1840 years , living in a stable relationship with a male partner , not using birth control , not receiving any type of fertility treatment , and attempting to conceive for no more than 12 months . from june 1 , 2007 to august 3 , 2011 
 of these , one woman did not provide a valid cpr number in the screener , one woman was excluded because she was already pregnant ( 14 weeks ) , and 533 women were excluded because they had been attempting to conceive for more than 12 months . in total , 5383 eligible women enrolled in the study during the 4-year study period . 
 data on preconceptional use of fa and mv supplements and sociodemographic , lifestyle , reproductive , and medical variables were assessed at baseline . in the baseline questionnaire , women were asked  do you take vitamins on a regular basis  daily or almost every day ?  ,  how long have you been taking vitamins on a regular basis 
 less than one year , 15 years , more than 5 years , or do nt know ?  , and  which of the following vitamins or minerals do you take on a regular basis 
 mvs , vitamin a , beta - carotene , vitamin b , vitamin c , vitamin d , vitamin e , fa , calcium , magnesium , selenium , or other ?  participants who reported  multivitamin  or wrote the name of an mv product were defined 
 similarly , participants who reported  folic acid  or wrote  folate  were defined 
 users  were defined as women who used either fa supplements , mv supplements , or both . 
 women who used single - vitamin or mineral supplements other than fa and women who did not take any dietary supplements were defined  nonusers . 
 a total of 197 ( 3.7% ) women did not answer the initial vitamin question and were also defined as nonusers . 
 participants reported weight , height , physical activity , and smoking history in the baseline questionnaire , allowing the calculation of body mass index ( bmi ) , total metabolic equivalents ( mets ) , and pack - years of smoking . 
 bmi was calculated by dividing weight ( kg ) with height squared ( m ) . 
 mets were estimated by summing the mets from moderate and vigorous physical activity ( hours per week multiplied by 3.5 and hours per week multiplied by 7.0 , respectively).14 data on smoking were categorized as pack - years of ever - smoking where 1 pack - year was defined as smoking 20 cigarettes per day in 1 year . to assess sociodemographic predictors of compliance among pregnancy planners , we examined the association between preconceptional fa or mv supplement use and the following variables : age ( categorized as < 25 , 2529 , 3034 , and 35 years ) , schooling ( primary and lower secondary school , high school , and other ) , vocational training ( none , short [ <3 years ] , medium [ 34 years ] , and long [ > 4 years ] ) and total monthly household income ( < 12,500 , 12,50024,999 , 25,00039,999 , 40,00064,999 , and 65,000 dkk / month ) . 
 we also examined lifestyle factors that could indicate intense efforts to conceive , such as intercourse frequency ( categorized as < 1 , 13 , and 4 times per week ) and months attempting pregnancy at study entrance ( 01 , 24 , 56 , and 712 months ) . 
 furthermore , compliance with other health recommendations , such as attending the national screening program for cervical cancer ( pap smear ) during the last 3 years ( none vs one or more ) , smoking history ( never smoked , < 5 , 59 , and 10 pack - years ) , current alcohol intake ( none , 13 , 47 , 814 , and 15 
 drinks / week ) , bmi ( < 18.5 , 18.524.9 , 2529.9 , 3034.9 , and 35 kg / m ) , and engagement in physical activity ( < 10 , 1019 , 2039 , and 40 mets / week ) , were also of interest . finally , we studied the associations with previous spontaneous abortion ( yes vs no ) , parity ( nulliparous vs parous ) , and medical conditions that may increase health awareness in relation to pregnancy , which included hypertension ( yes vs no ) , diabetes ( yes vs no ) , thyroid disease ( yes vs no ) , pelvic inflammatory disease ( yes vs no ) , and infection with chlamydia ( yes vs no ) . 
 we estimated the prevalence proportion ( pp ) of fa or mv users in each level of study predictors . 
 multivariate binomial regression was used to obtain prevalence proportion differences ( ppds ) with 95% confidence intervals ( cis ) for each predictor level in relation to the reference group . 
 the multivariate analyses for the pp and ppd for each study predictor were adjusted for all other covariates . 
 stata statistical software ( version 11.2 ; college station , texas ) was used for all analyses . 
 missing values ranged between 0.1% ( schooling ) and 8.7% ( total monthly household income ) . 
 because 1540 ( 28.6% ) participants were initially randomized to receive the short - form baseline questionnaire , they did not receive the questions about hypertension , diabetes , thyroid disease , and pelvic inflammatory disease . 
 the present study was a cross - sectional analysis of baseline data from an internet - based prospective cohort study of danish pregnancy planners  snart - gravid ( soon pregnant).the study design has been described in detail elsewhere.12,13 briefly , the study was initiated in june 2007 . 
 participants were enrolled via the study website , and data were collected by email and self - administered questionnaires . 
 recruitment was achieved by a pop - up advertisement placed on a well - known health - related website ( http://www.netdoktor.dk ) and two press releases . before enrolment 
 , potential participants entering the study website were required to read a consent form and fill in a screening questionnaire in order to confirm eligibility . 
 further , participants were required to provide a valid email address and their personal civil registry ( cpr ) number , which is a unique ten - digit personal identification number allowing linkage to a number of nationwide registries . 
 eligible women were invited to complete a baseline questionnaire and bimonthly follow - up questionnaires for 12 months or until conception occurred , after which active follow - up ended . 
 participants were initially randomized to receive either a short- or a long - form baseline questionnaire . 
 eligible women were danish residents aged 1840 years , living in a stable relationship with a male partner , not using birth control , not receiving any type of fertility treatment , and attempting to conceive for no more than 12 months . from june 1 , 2007 to august 3 , 2011 
 of these , one woman did not provide a valid cpr number in the screener , one woman was excluded because she was already pregnant ( 14 weeks ) , and 533 women were excluded because they had been attempting to conceive for more than 12 months . in total , 5383 eligible women enrolled in the study during the 4-year study period . 
 data on preconceptional use of fa and mv supplements and sociodemographic , lifestyle , reproductive , and medical variables were assessed at baseline . 
 in the baseline questionnaire , women were asked  do you take vitamins on a regular basis  daily or almost every day ?  ,  how long have you been taking vitamins on a regular basis 
 less than one year , 15 years , more than 5 years , or do nt know ?  , and  which of the following vitamins or minerals do you take on a regular basis 
 mvs , vitamin a , beta - carotene , vitamin b , vitamin c , vitamin d , vitamin e , fa , calcium , magnesium , selenium , or other ?  participants who reported  multivitamin  or wrote the name of an mv product were defined 
 similarly , participants who reported  folic acid  or wrote  folate  were defined 
 users  were defined as women who used either fa supplements , mv supplements , or both . 
 women who used single - vitamin or mineral supplements other than fa and women who did not take any dietary supplements were defined  nonusers . 
 a total of 197 ( 3.7% ) women did not answer the initial vitamin question and were also defined as nonusers . 
 participants reported weight , height , physical activity , and smoking history in the baseline questionnaire , allowing the calculation of body mass index ( bmi ) , total metabolic equivalents ( mets ) , and pack - years of smoking . 
 bmi was calculated by dividing weight ( kg ) with height squared ( m ) . 
 mets were estimated by summing the mets from moderate and vigorous physical activity ( hours per week multiplied by 3.5 and hours per week multiplied by 7.0 , respectively).14 data on smoking were categorized as pack - years of ever - smoking where 1 pack - year was defined as smoking 20 cigarettes per day in 1 year . to assess sociodemographic predictors of compliance among pregnancy planners , we examined the association between preconceptional fa or mv supplement use and the following variables : age ( categorized as < 25 , 2529 , 3034 , and 35 years ) , schooling ( primary and lower secondary school , high school , and other ) , vocational training ( none , short [ <3 years ] , medium [ 34 years ] , and long [ > 4 years ] ) and total monthly household income ( < 12,500 , 12,50024,999 , 25,00039,999 , 40,00064,999 , and 65,000 dkk / month ) . 
 we also examined lifestyle factors that could indicate intense efforts to conceive , such as intercourse frequency ( categorized as < 1 , 13 , and 4 times per week ) and months attempting pregnancy at study entrance ( 01 , 24 , 56 , and 712 months ) . 
 furthermore , compliance with other health recommendations , such as attending the national screening program for cervical cancer ( pap smear ) during the last 3 years ( none vs one or more ) , smoking history ( never smoked , < 5 , 59 , and 10 pack - years ) , current alcohol intake ( none , 13 , 47 , 814 , and 15 drinks / week ) , bmi ( < 18.5 , 18.524.9 , 2529.9 , 3034.9 , and 35 kg / m ) , and engagement in physical activity ( < 10 , 1019 , 2039 , and 40 mets / week ) , were also of interest . finally , we studied the associations with previous spontaneous abortion ( yes vs no ) , parity ( nulliparous vs parous ) , and medical conditions that may increase health awareness in relation to pregnancy , which included hypertension ( yes vs no ) , diabetes ( yes vs no ) , thyroid disease ( yes vs no ) , pelvic inflammatory disease ( yes vs no ) , and infection with chlamydia ( yes vs no ) . 
 we estimated the prevalence proportion ( pp ) of fa or mv users in each level of study predictors . 
 multivariate binomial regression was used to obtain prevalence proportion differences ( ppds ) with 95% confidence intervals ( cis ) for each predictor level in relation to the reference group . 
 the multivariate analyses for the pp and ppd for each study predictor were adjusted for all other covariates . 
 stata statistical software ( version 11.2 ; college station , texas ) was used for all analyses . missing values ranged between 0.1% ( schooling ) and 8.7% ( total monthly household income ) . because 1540 ( 28.6% ) participants were initially randomized to receive the short - form baseline questionnaire 
 , they did not receive the questions about hypertension , diabetes , thyroid disease , and pelvic inflammatory disease . 
 overall , 412 ( 7.7% ) women used fa supplements exclusively , 1100 ( 20.4% ) used mv supplements exclusively , and 1831 ( 34.0% ) used both , yielding 62.1% users . 
 a total of 82 ( 1.5% ) used single vitamins or minerals other than fa , and 1958 ( 36.4% ) did not use any dietary supplements , yielding 2040 ( 37.9% ) nonusers of fa or mv supplements ( table 1 ) . among users , 
 after mutual adjustment for covariates , higher age was associated with increased prevalence of fa or mv use ( table 2 ) . 
 our data also indicate that women who finished high school were more likely to use fa or mvs compared with women with less schooling . 
 similarly , compared with women with a long vocational training ( > 4 years ) , ppds for having no vocational training and a short ( <3 years ) vocational training were 5.7% and 7.1% , respectively , and compared with total monthly household of 65,000 dkk / month , ppd for < 12,500 dkk / month was 8.7% . while intercourse frequency  1 times / week was associated with an increased prevalence of fa or mv use , there was no clear association between months attempting to conceive at study entrance and fa or mv use . 
 furthermore , women who had pap smears once or more during the last 3 years were more likely to use fa or mv supplements compared with women who did not have pap smears ( ppd was 9.7% for none versus one or more ) . 
 kg / m ) were associated with a decreased prevalence of fa or mv use . compared with women who never smoked , 
 ppd for 10 pack - years of smoking was 11.3% . compared with no alcohol intake ppd for 15 drinks / week was 18.4% . compared with normal weight ( bmi = 18.524.9 kg / m ) , 
 ppds of fa or mv use for bmis of 3034.9 and 35 kg / m were 5.1% and 7.1% , respectively . 
 furthermore , women who engaged in physical activity were more likely to use fa or mvs than sedentary women ( < 10 mets / week ) . among women who had a previous spontaneous abortion , 
 the ppd of fa or mv use was 7.7% higher than women who did not report having a previous abortion . 
 finally , there was no clear association between being diagnosed with either hypertension , diabetes , thyroid disease , pelvic inflammatory disease , or infections with chlamydia and preconceptional fa or mv supplement use . 
 our data indicate that 62% of the 5383 pregnancy planners in this study adhere to the danish fa recommendation . by interpreting 
 users  as compliers , we may overestimate the proportion of compliers following the fa recommendation . the questionnaire provided information on whether the women took an fa or mv supplement or not , but there was no information on the exact dose of fa ingested or the brand name of the fa or mv product . in addition , 
 mv supplements have no standard definitions,1720 it can not be assumed that all mvs contained fa . however , most mvs on the danish market contain 400 g fa , especially those made for use during pregnancy . 
 the prevalence among pregnancy planners in our study is higher than the prevalence of preconception fa ( 12%44%)510 and mv use ( 11%)8 reported by pregnant women in previous studies . however , these studies collected data during the second and third trimesters , which may decrease the recall of fa and mv use during the preconceptional period . in addition , the studies were relatively small , with 588 women attending an antenatal clinic in melbourne in gestational weeks 3638,8 1541 women attending an antenatal ultrasound screening in gestational weeks 1719 in oslo,7 and 327 pregnant women calling the california teratogen information service in gestational week 13 in the us.10 consequently , prevalence estimates reported in these studies may have been imprecise . 
 the definition of compliance with the fa recommendation differs among studies . in the study by forster 
 et al,8 29% took prepregnancy fa supplements , but only 23% reported taking fa for at least 4 weeks before pregnancy . in the study by braekke and staff,7 17% of 
 this proportion may be more comparable with our findings , since some  users  in our study may have been in gestational weeks 04 at baseline . 
 women with unplanned pregnancies are less likely to use fa supplements before pregnancy . in the studies by forster 
 et al8 and goldberg et al,10 the prevalence of unplanned pregnancy was 19% and 36% , respectively . 
 these studies estimated fa use among all pregnancies ( planned and unplanned ) , making their results difficult to compare with ours . in the study by braekke and staff,7 there was no information about unplanned pregnancies or contributions of fa from mv supplements , both increasing the possibility of underestimation of the proportions of fa users in this study . in denmark , the largest and most recent study of lifestyle factors in relation to compliance with the fa recommendation was conducted among 18,294 pregnant women ( gestational weeks 1012 ) enrolled in the dnbc during 20002002.5 in this study , fa supplements were derived from single fa tablets , mv tablets , and vitamins made for pregnant or lactating women . 
 compliance was defined as taking 320 g fa per day , from 4 weeks before the date of the last menstrual period until gestation week 6 . 
 a total of 76% of the pregnancies were planned , and the overall proportion of women who complied with the recommendation was only 16% among planners . during the 10-year time period to the present study , there may have been an increase in the awareness as well as more available information about prepregnant recommendations , which may partly explain the observed increase in the prevalence of compliers with the fa recommendation found in this study . 
 in contrast to the dnbc , our data on supplement use and potential predictors were reported during the preconceptional period , and information was recorded prospectively , which increases the validity of the study . on the other hand , snart - gravid study participants may be more health - conscious than the general population of pregnancy planners , possibly overestimating the prevalence of fa or mv use . 
 even so , it is unlikely that an overestimation of supplement use would meaningfully distort the associations between predictors and fa or mv use . 
 in addition , differences in sociodemographic factors among snart - gravid study participants compared with the general population may also contribute to an overestimation of fa or mv use . 
 our study agrees with previous studies that found that maternal age,5,7,10 education,5,6,10 income,6,8 smoking,5,8 and bmi10 were associated with preconception fa use . 
 although a history of childbirth may increase awareness of preconceptional fa use , previous studies found that a previous childbirth either decreased the likelihood of fa use in subsequent pregnancies or was not related to fa use.5,7,8 we found only little association between parity and fa use in our study . 
 the positive associations between previous spontaneous abortion and intercourse frequency and fa or mv use found in our study have not been reported previously . 
 these findings may indicate a relation between a high desire to conceive and increased knowledge about the preconceptional fa guidelines . while lifestyle recommendations during pregnancy are widely available , less advice is available for women trying to conceive . 
 in addition to the fa recommendation , danish women planning a pregnancy are advised to avoid alcohol altogether.1 a total of 32% reported no alcohol intake in our study , which was slightly more than the 12% reported from the dnbc previously.21 in addition , we found the lowest proportion of fa or mv users among women with the highest alcohol intake . 
 together with the concern that excessive alcohol intake impedes the normal bioavailability and metabolism of folate,22 these results underscore the need to target women at high risk of not following the fa recommendation , as well as those not following other preconceptional guidelines . 
 being diagnosed with a chronic disease has previously been associated with increased dietary supplement use in general.23 pregnancy planners with a chronic illness might be more prone to use fa or mvs because of increased health awareness , but we found little association between a diagnosis of hypertension , diabetes , thyroid disease , pelvic inflammatory disease or infections with chlamydia and preconceptional use of fa or mvs . 
 our data indicate that 62% of the 5383 pregnancy planners in this study adhere to the danish fa recommendation . by interpreting 
 users  as compliers , we may overestimate the proportion of compliers following the fa recommendation . the questionnaire provided information on whether the women took an fa or mv supplement or not , but there was no information on the exact dose of fa ingested or the brand name of the fa or mv product . in addition , 
 mv supplements have no standard definitions,1720 it can not be assumed that all mvs contained fa . however , most mvs on the danish market contain 400 g fa , especially those made for use during pregnancy . 
 the prevalence among pregnancy planners in our study is higher than the prevalence of preconception fa ( 12%44%)510 and mv use ( 11%)8 reported by pregnant women in previous studies . however , these studies collected data during the second and third trimesters , which may decrease the recall of fa and mv use during the preconceptional period . in addition , the studies were relatively small , with 588 women attending an antenatal clinic in melbourne in gestational weeks 3638,8 1541 women attending an antenatal ultrasound screening in gestational weeks 1719 in oslo,7 and 327 pregnant women calling the california teratogen information service in gestational week 13 in the us.10 consequently , prevalence estimates reported in these studies may have been imprecise . 
 the definition of compliance with the fa recommendation differs among studies . in the study by forster 
 et al,8 29% took prepregnancy fa supplements , but only 23% reported taking fa for at least 4 weeks before pregnancy . in the study by braekke and staff,7 17% of 
 this proportion may be more comparable with our findings , since some  users  in our study may have been in gestational weeks 04 at baseline . 
 women with unplanned pregnancies are less likely to use fa supplements before pregnancy . in the studies by forster 
 et al8 and goldberg et al,10 the prevalence of unplanned pregnancy was 19% and 36% , respectively . 
 these studies estimated fa use among all pregnancies ( planned and unplanned ) , making their results difficult to compare with ours . in the study by braekke and staff,7 there was no information about unplanned pregnancies or contributions of fa from mv supplements , both increasing the possibility of underestimation of the proportions of fa users in this study . in denmark , the largest and most recent study of lifestyle factors in relation to compliance with the fa recommendation was conducted among 18,294 pregnant women ( gestational weeks 1012 ) enrolled in the dnbc during 20002002.5 in this study , fa supplements were derived from single fa tablets , mv tablets , and vitamins made for pregnant or lactating women . 
 compliance was defined as taking 320 g fa per day , from 4 weeks before the date of the last menstrual period until gestation week 6 . 
 a total of 76% of the pregnancies were planned , and the overall proportion of women who complied with the recommendation was only 16% among planners . during the 10-year time period to the present study , there may have been an increase in the awareness as well as more available information about prepregnant recommendations , which may partly explain the observed increase in the prevalence of compliers with the fa recommendation found in this study . 
 in contrast to the dnbc , our data on supplement use and potential predictors were reported during the preconceptional period , and information was recorded prospectively , which increases the validity of the study . on the other hand , snart - gravid study participants may be more health - conscious than the general population of pregnancy planners , possibly overestimating the prevalence of fa or mv use . 
 even so , it is unlikely that an overestimation of supplement use would meaningfully distort the associations between predictors and fa or mv use . 
 in addition , differences in sociodemographic factors among snart - gravid study participants compared with the general population may also contribute to an overestimation of fa or mv use . 
 our study agrees with previous studies that found that maternal age,5,7,10 education,5,6,10 income,6,8 smoking,5,8 and bmi10 were associated with preconception fa use . 
 although a history of childbirth may increase awareness of preconceptional fa use , previous studies found that a previous childbirth either decreased the likelihood of fa use in subsequent pregnancies or was not related to fa use.5,7,8 we found only little association between parity and fa use in our study . 
 the positive associations between previous spontaneous abortion and intercourse frequency and fa or mv use found in our study have not been reported previously . 
 these findings may indicate a relation between a high desire to conceive and increased knowledge about the preconceptional fa guidelines . while lifestyle recommendations during pregnancy are widely available , less advice is available for women trying to conceive . 
 in addition to the fa recommendation , danish women planning a pregnancy are advised to avoid alcohol altogether.1 a total of 32% reported no alcohol intake in our study , which was slightly more than the 12% reported from the dnbc previously.21 in addition , we found the lowest proportion of fa or mv users among women with the highest alcohol intake . together with the concern that excessive alcohol intake impedes the normal bioavailability and metabolism of folate,22 these results 
 underscore the need to target women at high risk of not following the fa recommendation , as well as those not following other preconceptional guidelines . 
 being diagnosed with a chronic disease has previously been associated with increased dietary supplement use in general.23 pregnancy planners with a chronic illness might be more prone to use fa or mvs because of increased health awareness , but we found little association between a diagnosis of hypertension , diabetes , thyroid disease , pelvic inflammatory disease or infections with chlamydia and preconceptional use of fa or mvs . 
 our findings indicate that more than one - third of the pregnancy planners in this study do not follow the preconceptional fa recommendation . 
 we found that pregnancy planners with other risky behaviors , such as smoking and alcohol use , obesity , and being physically inactive , are less likely to comply with the fa recommendation or use mvs .","purposecompliance with the danish preconceptional folic acid ( fa ) recommendation  a daily supplement of 400 g  is reported to be poor . 
 uncertainty remains , however , about the prevalence of compliers and health - related predictors of compliance in the preconceptional period.methodswe used self - reported baseline data from 5383 women , aged 1840 years , enrolled in an internet - based prospective cohort study of danish pregnancy planners during 20072011 . 
 we estimated the prevalence proportions of fa or multivitamin ( mv ) use in relation to selected sociodemographic , lifestyle , reproductive , and medical characteristics . 
 multivariate binomial regression was used to obtain prevalence proportion differences with 95% confidence intervals for each level of study predictors , adjusted for all other predictors.resultsoverall , 7.7% of women used fa supplements , 20.4% used mv supplements , 34.0% used both , 1.5% used other single vitamins or minerals , and 36.4% did not use any dietary supplements . 
 the prevalence of fa or mv supplement use was higher among older women , women with higher education and income , and women with healthy lifestyle factors such as being a nonsmoker , nondrinker , physically active , maintaining a normal body mass index and having regular pap smears . 
 greater intercourse frequency and a history of spontaneous abortion were also positively associated with fa or mv supplement use . 
 we found no clear association between use of fa or mv supplements and a diagnosis of hypertension , diabetes , thyroid disease , pelvic inflammatory disease , or chlamydia.conclusiona large proportion of pregnancy planners do not use fa or mv supplements . 
 pregnancy planners with generally risky lifestyle behaviors are less likely to comply with the fa recommendation .",pubmed
"peripheral arterial disease ( pad ) commonly results from progressive narrowing of arteries in the lower extremities due to atherosclerosis . 
 it has become evident that pad is an important predictor of substantial coronary and cerebral vascular risk [ 2 , 3 ] . 
 the most common symptomatic manifestation of mild to moderate atherosclerotic pad is intermittent claudication , observed in ~5% of individuals older than 60 years [ 5 , 6 ] . 
 several studies , using the ankle - brachial index , have revealed that the presence of pad , even when being asymptomatic and in patients with no history of other cardiovascular diseases , is a marker of greatly increased cardiovascular morbidity and mortality [ 7 , 8 ] . 
 oxidative stress , through the extra generation of reactive oxygen species ( ros ) , is also involved in the development and progression of pad . 
 moreover , risk factors associated with pad , similar to those for coronary heart disease , include high blood concentrations of total cholesterol and ldl - cholesterol . 
 inhibitors of 3-hydroxy-3-methylglutaryl coenzyme a ( hmg - coa ) reductase , called statins , are well - known lipid - lowering drugs . 
 statins not only reduce lipid levels , but they also have important pleiotropic effects , improving endothelial function , reducing oxidative stress , affecting the lipid metabolism enzyme activities , and so forth [ 1214 ] . 
 three main reasons have been suggested for initiating statin therapy for patients with pad of the lower limbs : ( 1 ) prevention of coronary heart disease ; ( 2 ) treatment of lower extremity peripheral arterial disease ; and ( 3 ) perioperative treatment of patients with peripheral arterial disease . 
 although several trials have shown that treatment with statins is associated with a reduction in coronary heart disease events , such as stroke [ 18 , 19 ] and mortality [ 2023 ] , most of these trials did not specifically address the treatment of patients with pad . 
 the aim of the present study , therefore , was to evaluate lipid peroxidation parameters and total antioxidant capacity in the serum of patients with pad before surgery as well as 35 days and 710 days after surgery . 
 we also compared these parameters with those in a group of patients receiving simvastatin therapy . 
 the study group consisted of pad patients , treated either surgically ( group i , n = 35 males , aged 61  8 years ) or pharmacologically ( group ii , n = 18 males , aged 62    7 years ) in the department of general and vascular surgery at the poznan university of medical sciences . patients included in group i were admitted for surgical treatment ( implantation of an aortobifemoral bypass graft ) . 
 they were diagnosed by measurement of the ankle brachial index ( abi ) and arteriography of the lower limbs , and were clinically stable without any accompanying diseases . 
 the patients treated surgically had shown critical ischemia ( abi less than 0.90 , rest pain , ulcer or necrosis of the lower limbs , and ankle pressure  50  mmhg ) . 
 blood samples were collected without anticoagulant , in the fasting state , before surgery as well as 35 days and 710 days after surgical treatment . 
 group ii had been receiving statin therapy , with 20  mg of simvastatin daily , for at least 3 months . 
 the study protocol was approved by the ethics committee of poznan university of medical sciences , and the subjects were fully informed and gave their written consent . 
 all the reagents were of analytical grade of purity ( sigma - aldrich , st . 
 spectrophotometric measurements were carried out on sp-8001 uv / visible spectrophotometer ( metertech inc . , taiwan ) . 
 the subjects ' lipid profile ( total cholesterol , hdl cholesterol , ldl cholesterol , and triglycerides ) , glucose , uric acid , apolipoproteins ( apo a - i , apo b ) and fibrinogen concentrations in serum were measured by standard laboratory procedures , using the roche cobas 6000 analyzer system , dade behring bn ii system , and the sysmex ca-500 series system . the total antioxidant potential ( capacity ) of serum 
 was determined by ferric reducing antioxidant power ( frap ) assay according to the benzie and strain method , based on the reduction of ferric ion fe(iii ) to ferrous ion fe(ii ) at a low ph . 
 this method measures the change in absorbance at 593  nm owing to the formation of a blue - colored fe(ii)-tripyridyltriazine complex from the colorless oxidized fe(iii ) form , by the action of electron - donating antioxidants . 
 lipid peroxidation was measured by estimation of both lipid hydroperoxides ( loohs ) and malondialdehyde ( mda ) concentrations by the sodergren method   and ohkawa et al . 
 the concentration of thiol ( -sh ) groups was determined according to the hu method . 
 ceruloplasmin , in the presence of o - dianisidine hydrochloride in an acidic solution ( ph 5.0 ) , forms a stable yellow - brownish product . 
 absorbance was measured at 540  nm and ceruloplasmin activity was expressed in u / l ( mol of oxidized substrate in 1  ml of serum in 1  min ) . 
 the paired student 's t - test ( to compare the values before and after surgery ) , the two - tailed unpaired t - test ( to compare group i and group ii ) , and the f - test were used for statistical analysis of the data .   the mean changes (  ) in lipid peroxidation parameters and antioxidant capacity , that is , 35 days ( 1 ) and 710 days ( 2 ) after surgery , in relation to the values obtained before surgery , were calculated . 
 pearson 's correlation coefficients r1 and r2 were computed for the results found before surgery and the changes observed during treatment ( 1 , 2 ) . 
 as shown in table 1 , generally , the clinical parameters of the two groups of patients included in this study did not differ significantly . 
 however , the concentrations of total cholesterol and ldl cholesterol were found to be significantly higher in group i before surgery than those in group ii on simvastatin therapy . 
 group i patients before surgery had enhanced oxidative stress , as demonstrated by elevated serum levels of mda , in comparison to those treated pharmacologically . 
 we did not observe any significant decrease in looh and mda concentrations in group i patients after surgery ( figures 1 and 2 ) .   the total antioxidant capacity ( expressed as frap ) and -sh group concentrations in the serum of patients on statin therapy and in those before surgery were similar ( figures 3 and 4 ) . 
 however , a decrease was observed in frap level 35 days after surgery , and 710 days after surgery , it was significantly lower in comparison to the values before surgery and on simvastatin therapy . 
 moreover , thiol group concentrations 35 days postoperatively were lower than those before surgery and 710 days after surgery and were lower than those in patients treated with simvastatin ( figure 4 ) . a significant difference in the oxidase activity of ceruloplasmin between group ii and group i before surgery was observed . 
 its oxidase activity in serum was higher in group ii and 710 days after surgery in group i , in comparison to the values before and 35 days after surgery ( figure 5 ) . 
 in addition , all the studied parameters ( looh , mda , frap , -sh groups , and ceruloplasmin ) in group i patients before surgery were significantly correlated with mean changes at 35 days ( 1 ) and/or 710 days ( 2 ) after surgery ( table 2 ) . 
 oxidized ldl is a biochemical marker of oxidative damage and has atherogenic effects , including the stimulation of foam cell formation and activation of the inflammatory process in the vascular wall . 
 the findings of the present study are broadly consistent with the hypothesis that lipid peroxidation may be one mechanism through which several risk factors may promote pad . 
 , we observed an increased mda level in the patients before surgery , which suggests that they were in a state of high oxidative stress . at neutral ph , mda exists as a low reactive enolate anion , but it is still toxic and may damage many biologically important molecules . 
 determination of the lipid peroxidation product mda in pad is important because , as was shown earlier , mda damages collagen by forming transverse intramolecular bonds that cause rigidity of blood vessels . 
 in addition , modification of native ldl by mda causes the accumulation of cholesteryl esters within the cell in which the atherosclerotic reaction takes place . 
 several trials have shown that statin therapy relieves symptoms and improves function in patients with lower extremity pad [ 32 , 33 ] . 
 mda concentrations in the serum of the patients treated with simvastatin were lower than those in group i patients before surgery . since pad patients are usually undertreated with regard to the use of lipid - lowering agents , compared to patients with coronary heart disease , our results provide strong support for the efficacy of statin therapy in pad . 
 human cells and tissues contain many antioxidant enzyme systems and nonenzymatic antioxidants , which protect against the action of toxic free radicals . 
 the frap assay used here is a useful indicator of the body 's antioxidant status , which determines the level of non - enzymatic antioxidants , such as uric acid , ascorbic acid , -tocopherol , and some protein - containing -sh groups . 
 an increase in frap level in the patients before surgical treatment is consistent with similar results obtained by gawron et al .   in a group of patients with coronary heart disease and by lantos et al .   in patients with hypertension . 
 an increased antioxidant capacity of serum seems to be response to an increase in the concentration of lipid peroxidation products . 
 the reduction in frap level observed in our group i patients 35 and 710 days after surgery can be partly explained by a decrease in their serum antioxidant status and an increase in serum oxidant levels following surgery , as indicated by the negative correlations found ( table 2 ) . in their study , girona et al . 
 they showed that simvastatin therapy increases the resistance of both ldl and hdl to oxidation . 
 in addition , simvastatin inhibits the ability of activated macrophages to oxidize ldl in vitro in a dose - dependent manner . 
 a similar effect has been shown for other statins , such as atorvastatin , which reduces ldl levels and hdl oxidation and protects paraoxonase  an enzyme bound to hdl .   with regard to frap in our study , simvastatin therapy ( 20  mg daily ) did not influence the total antioxidant potential 
 . however , shin et al .   found a significant increase in antioxidant ability in their group of hypercholesterolemic patients treated with 40  mg simvastatin daily . 
 they suggested the possibility of a dose - dependent effect of simvastatin on plasma antioxidant status . 
 this could be the reason why we did not detect any effect of simvastatin on frap in our study . 
 glutathione and other proteins , such as albumin , are an important part of serum antioxidant activity against free radicals . 
 the thiol groups react with peroxide radicals in the first stage of the oxidation reaction and also act in the lag phase , which is an accepted marker of serum oxidizability . in our group of patients with pad , 
 the decrease in thiol group concentrations , observed 35 days after surgery , followed by an increase 710 days after surgery , may be explained by oxidative stress intensification in reperfusion and by the actions of -sh groups , leading to their reduction . 
 furthermore , a decrease in both lag phase and albumin level has also been reported in the serum of patients with aneurysmal or arterial occlusive disease . 
 some studies have confirmed that ceruloplasmin is an independent risk factor for cardiovascular disease [ 43 , 44 ] . 
 authors of that study suggest that increased ceruloplasmin activity is related to the acute phase response to inflammation and necrosis in atherosclerosis obliterans and results from increased ros production , mostly of superoxide radicals . 
 therefore , the preferential oxidative destruction of thiol groups can explain the greater susceptibility of ceruloplasmin to oxidative stress . 
 recent data show that statins may reduce oxidative stress   by decreasing the generation of ros , thereby synergizing with the biological effects of antioxidants . 
 the results of our study show that ceruloplasmin activity was influenced by both simvastatin therapy and surgical treatment , but only 710 days postoperatively . in our opinion , simvastatin may be implicated in antioxidant properties of ceruloplasmin , as reflected by the increase in its activity in men on simvastatin therapy . 
 several in vitro studies have indicated that ceruloplasmin is a potent catalyst of ldl oxidation and that has been attributed to the presence of a reduced copper atom on its surface . 
 ros dissociate copper from ceruloplasmin and therefore can markedly alter its structure and function , which could explain the initial lack of differences in its activity 35 days after surgery . in view of the escalating prevalence of pad in our aging population , 
 further studies are needed to clarify precisely not only the mechanisms involved but the relative importance of the lipid - related and nonlipid - related effects of statins as well . 
 results of this study show diverse oxidative state responses to surgical treatment and confirm the beneficial effects of statin therapy in pad .","the various risk factors for peripheral arterial disease ( pad ) are almost identical to those for atherosclerosis and include abnormal levels of lipids or lipoproteins . lipid peroxidation parameters and total antioxidant capacity in the serum of male patients with pad before surgery as well as 
 35 days and 710 days after surgery were measured . 
 we also compared these parameters with those in a group of patients receiving simvastatin therapy . 
 concentrations of lipid hydroperoxides ( loohs ) and malondialdehyde , the total antioxidant capacity ( assessed by ferric reducing antioxidant power assay ) , concentration of thiol ( -sh ) groups , and ceruloplasmin activity were determined spectrophotometrically in pad patients treated surgically ( group i ) or pharmacologically ( group ii ) . 
 the patients before surgical treatment had significantly higher concentrations of malondialdehyde but lower ceruloplasmin activity than those observed in group ii , treated with simvastatin . 
 no significant differences before surgery in ferric reducing antioxidant power or thiol concentrations were found between the two groups . 
 however , in group i , both ferric reducing antioxidant power and thiol group concentrations decreased 35 days postoperatively , and ceruloplasmin activity increased 710 days after surgical treatment . 
 the presented results demonstrate diverse oxidative stress responses to surgical treatment and confirm the beneficial effects of statin therapy in pad .",pubmed
"in recent years , the demand for herbal remedies in nigeria has been on the rise . 
 yet there are little or no scientific data to support medicinal claims of these herbal formulations . 
 herbals are usually sought after for their health benefits and these have become common medicines in many nigerian homes . 
 it is recognized that herbal remedies prepared from medicinal plants may have several traditional applications especially in developing nations like nigeria where access to formal healthcare is limited 
 . however , the increasing commercial promotion of herbals necessitate the need for assessment of safety and validity of medicinal claims . in recent past 
 , several studies have demonstrated the toxic effects of indiscriminate use of packaged herbals [ 1 , 4 , 5 ] . 
 furthermore , the general belief that herbal medicines are natural , effective , and without adverse effects has immensely contributed to the upsurge in the patronage of herbal formulations [ 68 ] . the popular belief that herbal remedies are without toxic or undesirable effects 
 these and other factors form part of several concerns associated with the use of herbal remedies in developing nations . in nigeria , studies have revealed that the number of nigerian medicinal plants screened for the validation of biological activity far outweighs the assessment for potential toxic compounds and contaminants [ 2 , 10 , 11 ] . also , many herbal formulations being promoted and marketed in nigeria lack scientific data to support acclaimed medicinal benefits and may pose great health risk to the numerous unsuspecting consumers . 
 according to the fijk leaflet , the herbal is prepared from a mixture of 13 medicinal plants including cassia alata , citrus medica var acida , aloe vera , cassia augustifolia , cassia siamea , kyaha sengalensis , xylopia aethiopica , gongronema latifolium , khaya grandifoliola , morinda lucida , anthocelesta djalonensis , citrullus lanatus , and aloe barbaris . 
 this must have contributed to the wide patronage and consumption among the nigerian populace . according to the fijk label 
 , the herbal remedy is indicated for treatments of several ailments which include obesity , diabetes , sexual improvement , menstruation pains , infertility , high blood pressure , crawling sensation , and malaria , amongst others . 
 there are scientific reports on the medicinal relevance of the individual plant constituents of the fijk mixture 
 . however , there are no empirical data to support claims of medicinal benefits or otherwise of fijk . a search through online research libraries including the cochrane , pubmed , and google - scholar revealed the absence of scientific data on fijk herbal remedy . 
 the scenario makes likely toxic potential from repeated exposure to the fijk mixture largely unknown . 
 for the safety of public health , it is essential that herbal remedies have available scientific data on safety / toxicity profiles in order to aid informed choices by consumers . 
 this study aimed to determine the effect of oral and repeated exposure to fijk on rat plasma biochemical indices and liver morphology . 
 commercial reagent kits for the assay of alkaline phosphatase ( alp ) , aspartate aminotransferase ( ast ) , alanine aminotransferases ( alt ) , total cholesterol ( tc ) , total protein ( tp ) , triglyceride ( tag ) , and high density lipoprotein cholesterol ( hdl - c ) were as supplied by randox diagnostics , crumlin , uk . 
 twenty - four male wistar rats with weight between 190 and 200  g were obtained from the experimental animal farm at the university of ilorin , ilorin , nigeria . 
 the wistar rats were housed in animal cages in a well - ventilated experimental room . 
 the rats were allowed to acclimatize for a period of 14 days before the commencement of treatments . 
 handling of animals was in accordance with relevant institutional and ethical guidelines as approved for scientific study . 
 mg / kg : received fijk at a dosage of 15  mg / kg body weight . 
 fijk 30  mg / kg : received fijk at a dosage of 30  mg / kg body weight . 
 fijk 45  mg / kg : received fijk at a dosage of 45  mg / kg body weight . control : received distilled water . 
 15  mg / kg : received fijk at a dosage of 15  mg / kg body weight . 
 fijk 30  mg / kg : received fijk at a dosage of 30  mg / kg body weight . 
 fijk 45  mg / kg : received fijk at a dosage of 45  mg / kg body weight . the dosages were selected based on corresponding therapeutic doses for human consumption as recommended by the herbal producer . 
 the treatment was daily and lasted for 21 days . at the end of treatments , 
 the blood samples were collected into edta bottles , spun at 5000  g for 10 minutes using a refrigerated centrifuge ( anke tdl-5000b , shanghai , china ) to obtain the plasma , which was subsequently used for the biochemical analysis . 
 the liver tissues were excised , weighed , and fixed in buffered neutral formalin and used for histopathological examinations . 
 the biochemical indices were determined in rat plasma using a uv / vis spectrophotometer ( jenway , staffordshire , united kingdom ) where applicable . 
 the levels of rat plasma total protein ( tp ) , aspartate aminotransferase ( ast - ec : 2.6.1.1 ) , alanine aminotransferase ( alt - ec : 2.6.1.2 ) , alkaline phosphatase ( alp - ec : 3.1.3.1 ) , and lipid profile including total cholesterol ( tc ) , triglyceride ( tag ) , high - density lipoprotein - cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) were determined using randox assay kits . reduced glutathione level ( gsh ) was determined by the procedure described by ellman   with slight modification . 
 lipid peroxidation was ascertained by monitoring the degrees of lipid peroxides in the supernatant fraction of plasma using the method described by niehaus jr . and samuelsson . 
 the rat liver was fixed in 10% buffered neutral formalin ( bnf ) immediately following excision from animals . 
 capture and scoring for morphological changes were done by a pathologist blind to the treatments , at the pathology unit , university of ilorin teaching hospital , ilorin , nigeria . 
 data were subjected to statistical analysis using the one - way anova ( graphpad software inc . , san diego , ca ) . 
 the oral administration of fijk to rats did not produce significant change to average body weights of animals , even though gradual decreases in rat body weights were observed for groups that received fijk herbal mixture ( figure 1 ) . 
 likewise , the relative organ to body weight ratio showed no significant difference among the various treatment groups ( figure 2 ) . 
 however , the oral administration of fijk herbal mixture at double and triple doses caused significant reduction in rat plasma protein level compared to the control group . 
 the determination of lipid profile showed that the oral exposure of rats to fijk at the various doses did not produce significant change in the plasma level of total cholesterol when compared to the control . 
 however , the exposure to fijk at the highest dosage caused significant elevation in rat plasma level of tryglyceride . 
 further assessment of lipid profile revealed that the daily administration of fijk mixture depleted rat plasma level of hdl - c ( figure 3 ) . 
 meanwhile , the level of rat plasma ldl - c rose with increasing doses of fijk herbal . 
 furthermore , the atherogenic index was elevated significantly in the groups exposed to fijk mixture ( figure 4 ) . 
 on the other hand , rat plasma alp level was lowered ( p < 0.05 ) following oral exposure to fijk herbal mixture but the reduction was only significant in the group that received the highest dose of fijk . 
 in contrast , treatment of rats with fijk herbal mixture at all doses significantly increased the levels of plasma alt and ast relative to the control group ( figure 5 ) . to assess whether oral exposure of fijk in rats would predispose to lipid peroxidation , the plasma level of mda was determined . 
 there was significant elevation in rat plasma level of mda in the fijk treated groups relative to the control ( figure 6 ) . 
 conversely , oral administration of fijk to rats significantly depleted the plasma levels of gsh compared to the control . the light microscopic examination of rat liver sections for morphological changes revealed lesions caused by fijk herbal mixture ( figure 7 ) . 
 the fijk herbal mixture caused hepatic alterations including hypochromic , haemorrahage , ruptured vascular channels , and hyperemia . 
 however , the rat liver in the control group had no incidence of this cellular lession . 
 unfortunately the use of herbal products is not strictly regulated in nigeria thus making them freely available , a scenario which predisposes to possible abuse by consumers . 
 although herbal mixtures enjoy wide patronage in nigeria , little is known about likely toxicity that may be associated with repeated consumption . 
 herbal remedies may have recognizable therapeutic effects ; they also may have toxic side effect . 
 more so , many herbal preparations lack scientific facts to back up acclaimed medicinal benefits . currently , there are no available empirical data on fijk herbal mixture . 
 the present study sought to determine the influence of oral and repeated adminstration of fijk herbal mixture on rat biochemical and morphological parameters . 
 this is coming against the background that previous studies have demonstrated toxic potentials of packaged herbals [ 4 , 10 , 11 ] . in the present study , 
 although rat liver weights were reduced in the treatment groups , the relative organ weights were not significantly affected by exposure fijk mixture . however , 
 the evaluation of rat lipid profile revealed that oral administration of fijk herbal mixture did not significantly alter the total cholesterol in all the treatment groups . 
 however , there were dose - dependent increases ( p < 0.05 ) to the plasma levels of tag and ldl - c in rats orally exposed to fijk herbal mixture . 
 conversely , levels of plasma hdl - c were significantly lowered by repeated administration of fijk mixture and the effect was dose dependent . 
 the lipid profile indices are useful in monitoring health status of the cardiovascular system . although the rat total cholesterol level was not affected by fijk herbal mixture administration , elevated levels of tag and ldl - c may predispose to cardiovascular related disorders . increased level of ldl - c has been associated with higher risk of atherosclerosis while elevated level of hdl - c is linked to reduced occurences of cardiovascular disorder [ 19 , 20 ] . 
 the level of rat plasma hdl - c decreased with increasing doses of fijk leading to elevated atherogenic index . 
 the atherogenic index can be used to predict the risk for development of cardiovascular disorders . 
 therefore , the low ratio of hdl - c to ldl - c , caused by repeated oral exposure to fijk , may implicate increased tendency for the development of atherosclerosis . 
 the rat plasma levels of alt and ast were elevated by exposure to repeated administration of fijk mixture relative to control . 
 the alt and ast are normally found in the red blood cells , liver , heart , and kidney tissues . 
 the levels of alt and ast have long been used to assess the functions of liver . increased plasma levels of both alt and ast have been linked to tissue toxicity [ 21 , 22 ] . normally , a basal level of the enzymes is found in the plasma ; however , when there is cellular damage , the enzymes extrude into the extracellular fluid thus raising the concentrations in the plasma . in the present study , the significant alterations to levels of rat plasma alt and ast 
 this is further supported by the fact that the ratio of ast to alt in plasma was less than one ( < 1 ) , which may indicate ensuing acute or chronic liver injury . on the other hand , 
 the rat plasma alp levels were significantly reduced in the group which received the highest dosage of fijk herbal mixture . the decreased level may be as a result of inactivation or decreased protein synthesis . 
 previous studies have shown that herbal mixtures have capability to alter the levels of alt , ast , and alp in rats [ 1 , 9 ] . furthermore , in order to evaluate whether the oral and repeated exposure to fijk mixture was capable of predisposing to oxidative stress , we determined the levels of rat plasma mda and gsh . 
 the mda is a by - product of lipid peroxidation while the gsh level could be used to assess the antioxidant status of a cellular system . 
 the increased level of rat plasma mda suggested the presence of lipid peroxidation caused by oral exposure to fijk herbal mixture . 
 the increased mda level in rats may be due to the generation of free radical species potentiated by exposure to fijk mixture . 
 on the other hand , ensuing oxidative stress caused by the oral administration of fijk was further demonstrated by the depleted level of rat plasma gsh . 
 the gsh molecule is a nonenzymatic antioxidant capable of scavenging free radicals and it could also serve as substrate to other antioxidant enzymes like the glutathione peroxidase . 
 the low level of gsh may be attributed to increased usage in order to scavenge free radical species or consumption as substrate by antioxidant enzymes which function to protect against oxidative damage . 
 recent studies have associated oral and repeated exposure to herbal mixtures with lipid peroxidation and oxidative stress [ 1 , 5 ] . 
 the light microscopic examinations of rat liver sections showed intact and normal cellular architecture in the control group . 
 however , the examination of rat liver sections in groups exposed to various dosages of fijk mixture revealed incidences of morphological lesions . 
 the rat hepatic alterations caused by exposure to fijk herbal mixture included haemorrahage , ruptured vascular channels , hyperemia , and inflammation . 
 the histopathological findings support the biochemical alterations caused by oral and repeated exposure to fijk herbal mixture and are reminiscent of rat hepatic injury . 
 this is consistent with a previous report   which demonstrated organ toxicity caused by herbal formulations . 
 the study revealed elevated atherogenic index caused by oral and repeated exposure to fijk mixture in rats . 
 furthermore , oral and repeated exposure to fijk herbal mixture in rats caused lipid peroxidation and potentiated hepatic lesion .","this study determined the effect of the oral and repeated administration of fijk herbal mixture on rat biochemical and morphological parameters . 
 twenty - four wistar rats were distributed into four groups of 6 . 
 group a served as control and received oral administration of distilled water daily . 
 the experimental groups b , c , and d were daily and orally exposed to fijk herbal mixture at 15 , 30 , and 45  mg / kg , respectively . 
 treatments lasted for 21 days . 
 the rats were sacrificed under mild diethyl ether anesthesia 24  hr after cessation of treatment . 
 the blood and liver samples were collected and used for the biochemical and morphological analyses . oral exposure to fijk caused elevated levels of rat plasma alt , ast , triglycerides , ldl , and mda . 
 in contrast , rat plasma hdl , gsh , and alp levels were lowered by fijk oral exposure . 
 also , the herbal remedy caused a dose - dependent elevation in the plasma atherogenic index . 
 the histopathology examinations of rat liver sections revealed inimical cellular alterations caused by repeated exposure to fijk . 
 study provides evidence that oral and repeated exposure to fijk in rats raised the atherogenic index and potentiated oxidative stress as well as hepatic injury .",pubmed
"identifying carriers of autosomal recessive or x - linked disorders before pregnancy has the potential to benefit prospective parents . 
 couples can become aware of the possible genetic risks to future offspring and of the reproductive options available . 
 these options include not only prenatal diagnosis followed ( or not ) by termination of the pregnancy in case of an affected fetus or by coming to terms with the risk , but also the choices of using preimplantation genetic diagnosis , using donor sperm or oocytes , seeking adoption or refraining from having children . in some culturally related marriage practices 
 there are two approaches to the identification of carriers : carrier screening and carrier testing . 
 carrier screening is defined as the detection of carrier status in persons who do not have an a priori increased risk for having a child with a certain disease , whereas with carrier testing , the persons do have a higher a priori risk based on their or their partners personal or family history ( castellani et al . , 2010 ) . 
 carrier screening can be offered on an individual basis , but also as an organized screening programme , either during or before pregnancy . 
 while most screening programmes in health care aim to prevent , treat and alleviate disease , above and beyond these aims , the particular goal of preconceptional carrier screening is to strengthen reproductive autonomy and informed decision - making . 
 if offered preconceptionally , this can mean less time constraints , less pressure and less emotional stress than when a test is performed during pregnancy ( bombard et al . , 
 2010 ) internationally , the potential of preconceptional carrier screening has been studied extensively for cystic fibrosis ( cf ) ( bekker et al . , 1993 ; tambor et al . , 1994 ; axworthy et al . 
 these studies have repeatedly revealed positive attitudes towards cf carrier screening from health care providers ( poppelaars et al . 
 , 2004a ; mcclaren et al . , 2008 ) , patients and their relatives ( poppelaars et al . , 2003 ) and from the general public ( poppelaars et al 
 , 2004c ) . furthermore , preconceptional carrier screening for cf for couples with no family history of cf was recommended a decade ago by the national institutes of health ( nih ) ( national institutes of health , 1999 ) , the american college of medical genetics ( acmg ) and the american college of obstetricians and gynaecologists ( acog ) ( grody et al . , 2001 ) . despite this , screening is not currently offered in most countries . 
 carrier testing for disorders such as cf is still usually restricted to families and partners of cf patients and carriers . apart from some specific regions ( e.g. thalassemia carrier screening in cyprus and sardinia ) and specific communities ( e.g. carrier screening for ashkenazi jews ) 
 , traditional healthcare systems have hesitated to implement carrier screening programmes for the entire population of couples planning a pregnancy . 
 various concerns underlie this stance , including the psychological impact ( axworthy et al . , 1996 ) and risk of stigmatization / discrimination of being a carrier ( mcqueen , 2002 ) , impairment of the freedom of choice because of the undue pressure on individual choice ( godard et al . , 2003 ) , medicalization ( poppelaars et al . , 2004c ) , the fact that couples are difficult to reach before conception ( lakeman et al . , 2009 ) , and provider - related barriers such as a lack of familiarity with genetics and selection criteria for offering screening ( morgan et al . , 2004 ) . 
 recently , commercial companies have been offering preconceptional carrier tests directly - to - consumers . 
 these direct - to - consumer ( dtc ) tests provide information about monogenic diseases and the risk of having affected offspring . 
 dtc genetic testing is often defined as the offer and/or marketing of genetic tests directly to the public without the intermediary of a health care professional from the traditional health care system . 
 as has been suggested by the human genetics commission , however , we also include in this discussion  tests that are commissioned by the consumer from a commercial company outside the traditional health care system  but where a medical practitioner or a health professional is involved in the provision of the service ' ( human genetics commission , 2010 ) . 
 the availability of dtc genetic testing services in general has created a huge debate about the desirability of these activities ( howard and borry , 2008 ; hunter et al . , 2008 ; kaye , 2008 ) . 
 however , until now , these discussions have mainly focused on the commercial dtc offer of genome - wide - testing ; a service which offers individual risk assessments for common disorders ( janssens et al . , 2008 
 ; melzer et al . , 2008 ) with limited or no medical or health benefits to the individual user . 
 various concerns were raised with regard to this dtc offer of genetic tests , including , among others , concerns related to the validity and utility of tests offered , the absence of individualized medical supervision , the lack of an adequate consent procedure , the absence and/or quality of pre- and post - test genetic counselling , the inappropriate testing of minors , the respect for privacy and the potential burden on public health resources . because of the great heterogeneity in types of dtc genetic tests , and the fact that the consequences of their results vary widely , a one - size - fits - all approach is not appropriate for all dtc genetic tests . since preconception carrier screening has been accepted by various medical associations , dtc testing for these tests may be regarded differently when compared with the dtc tests for common disorders that have been criticized because of , for example , the lack of clinical utility ( i.e. how likely the test is to significantly improve patient outcomes ) and analytical validity ( i.e. how accurately and reliably the test measures the genotype of interest ) . therefore , the aim of this article is to discuss various ethical issues raised by the dtc offer of preconceptional carrier tests for autosomal recessive disorders from commercial companies that are not embedded in regular health care . in the following order , 
 this article will present and discuss : ( i ) the current offer of preconceptional carrier tests through online providers ; ( ii ) the implications for the informed consent procedure and the possible consequences for the provision of information ; ( iii ) the need for medical supervision and follow - up and ( iv ) the current offer in relation to the appropriate use of existing resources . 
 the details of the offer from each company , however , vary greatly . in may 2010 , 
 the company dna direct ( www.dnadirect.com ) advertised one individual carrier test ( for cf ) and one carrier testing panel ( for ashkenazi jews ) . on its website 
 professional medical groups including the nih (  ) and acog recommend that cf carrier screening be offered to all couples who are planning a pregnancy or are currently pregnant ( https://www.dnadirect.com/web/article/testing-for-genetic-disorders/cystic-fibrosis/30/who-should-consider-testing accessed 19/01/2011 ) . 
 the company dnatraits says that they are  committed to making all medically validated tests available to consumers rapidly , inexpensively and understandably ( www.dnatraits.com/compare accessed 03/05/2010 ) and is selling a limited number of individual tests ( e.g. for alpha-1-antitrypsin deficiency ) as well as panels of disorders ( e.g. an ashkenazi jewish genetic disease panel or a sickle cell / beta - thalassaemia panel ) . along with risk assessment information for other disorders , pathway genomics ( www.pathway.com ) and 
 23andme ( www.23andme.com ) include in their full genome testing report the carrier status for 37 and 24 different single - gene conditions , respectively . 
 finally , in february 2010 the company counsyl ( www.counsyl.com ) launched their offer of a prepregnancy 
 universal carrier test which tests an individual or couple for over 100 , mostly autosomal recessive , genetic diseases . 
 counsyl considers these activities as  a cause , a campaign to finally end the needless suffering of preventable genetic disease ( https://www.counsyl.com/about/counsyl/ accessed 19/01/2011 ) . 
 these abovementioned dtc genetic tests providers do not make a distinction between screening individuals with an a priori low population risk or testing individuals with a high a priori probability of having an affected child . 
 because of this increasing possibility for determining an individuals risk of transmitting a condition to his or her offspring and the potential opportunities and threats generated by these results to individual citizens and society as a whole , the health council of the netherlands ( 2008 ) , for example , emphasized the need for criteria for a responsible offer of genetic dtc screening as well . in this specific report , the original genetic screening criteria , as outlined by the who ( wilson and jungner , 1968 ) , which relate to the prerequisites for starting community - wide screening , are extended with a much larger view on screening . 
 specific individuals being invited to undergo a medical test included in the definition of a genetic carrier screening programme , but also a wider population is included by  highlighting the opportunity to be tested in brochures or in the press , via advertising or promotion by commercial providers. screening can take the form of large - scale programmes for which all pregnant women , all newborn babies or all men or women in a particular age group are eligible . 
 but it can also entail  people responding to an advertisement or a website offer for a health check with a clinic or a health organization ( health council of the netherlands , 2008 ) . 
 the large number of monogenic disorders included in the dtc genetic testing companies services is in clear contrast with the selected number of disorders that is usually considered for inclusion in preconception carrier screening programmes in the traditional health care system . on the basis of 
 generally accepted genetic screening guidelines ( health council of the netherlands , 2008 ; european society of human genetics , 2003 ; uk national screening committee , 2003 ; nuffield council on bioethics , 2006 ) , carrier testing for a larger number of disorders challenges at least two established specifications that have been followed in this field until now : ( i ) the condition should be an important health problem , and ( ii ) there should be a suitable test with known predictive value . moreover , testing should lead to a favourable ratio of advantages to disadvantages , in which the voluntary character of consent , the information provision , the embedding in medical supervision and the appropriate use of existing resources are guaranteed ( see below ) . 
 first , an important criterion that should be fulfilled before a screening programme is initiated is that the condition should be an  important health problem ( european society of human genetics , 2003 ; uk national screening committee , 2003 ) or a  significant health problem ( health council of the netherlands , 2008 ) . 
 significance does not necessarily relate to the number of people affected ; it also addresses the severity of a health problem even if a condition is rare ( european society of human genetics , 2003 ; health council of the netherlands , 2008 ) . the universal carrier test offered by counsyl , 
 therefore , is whether dtc carrier testing should be seen as an activity for which these screening criteria are applicable . 
 a disorder such as gaucher disease , for example , was already a matter of debate with regard to its integration in a panel for ashkenazi jews , since the most common gaucher disease mutation in this population leads to a highly variable but usually milder phenotype ( zimran et al . , 1997 ; zuckerman et al . , 2007 ) . 
 this then raises the question of whether it is acceptable to systematically identify carrier couples for disorders that are less severe ( borry et al . , 2008 ) . 
 severity judgments are complex and a particularly challenging base on which to make reproductive choices . 
 providing severe and possibly less severe disorders within the same panel undermines the consistency of that panel . 
 secondly , it has been emphasized that screening should be based on a  simple , safe , precise and validated test ( uk national screening committee , 2003 )  with known predictive value ( european society of human genetics , 2003 ) . although the analytical validity of the test for each mutation included in panels offered by dtc genetic testing companies has likely been validated , the clinical validity ( i.e. how consistently and accurately the test detects or predicts the intermediate or final outcomes of interest ) of the panel of mutations may be far from 100% . 
 moreover , the clinical utility of testing for some disorders is debatable as some homozygotes , because of low penetrance , may never develop overt disease and/or the expression may be variable ( levenson , 2010 ) . 
 for example , this is the case for hereditary haemochromatosis , which is also included in the counsyl test panel . 
 in addition , companies offering large panels of disorders may base their inclusion criteria on technical and economic aspects rather than on policy considerations , which take into account , among other things , carrier frequency , severity of the disorder and feasibility of testing in a particular population . 
 it also raises doubts about the individual disorders included in these large testing panels , and questions whether  more is really better ( leib et al . , 2005 ) . 
 moreover , the question is raised of whether good information and informed decision - making is still possible when the test panel contains such a heterogeneous group of disorders , for which test sensitivity and specificity are variable . 
 to ensure informed decision - making , it is crucial that individuals receive the necessary information about the purpose of the test , the reproductive choices resulting from the test , the reliability and limitations of the test , the possible psychological impact and the potential consequences of the test for the individual and his / her family members ( mcqueen , 2002 ) . 
 privacy and confidentiality of the results , as well as possible consequences related to their disclosure to third parties , such as insurance companies and employers , should also be discussed . 
 moreover , the nature of the reproductive options makes it imperative to adopt a non - directive approach with potential users . 
 although some dtc genetic testing companies do include a lot of this information on their websites , the question is whether , while being commercially driven , the information presented is balanced enough to enable informed choice . 
 moreover , according to a recent study ( lachance et al . , 2010 ) , many users would struggle to find and understand the important information on companies websites needed to make an informed decision . finally , 
 although all companies selling dtc genetic tests require some form of consent from the consumer when ordering a test , the process of informed decision - making can not be reduced to clicking a box to accept the terms of service or signing a document . educating people about carrier testing 
 2009 ) , and the fact that carrier tests have a test sensitivity of < 100% ( causing a residual risk to people who are not found to be carriers ) make the goal of transmitting information about these tests a non - trivial matter . 
 studies have shown that a significant proportion of participants who tested negative when undergoing preconceptional screening for cf or hbps wrongly believed that they were definitively not carriers , while some carriers falsely believed that they were only likely to be carriers ( axworthy et al . , 1996 ; honnor et al . , 2000 ; 
 an offer of dtc testing is likely to face the same problems in educating people as has been demonstrated in these screening programmes . 
 multiple studies have also demonstrated that both carriers and non - carriers may experience negative feelings , such as anxiety and worry , when participating in genetic screening , although anxiety levels often decrease after a few months ( bekker et al . , 1994 ; henneman et al . , 
 the dtc offer of genetic tests is worrisome in the light of these findings , where the practices of existing dtc genetic testing companies mainly only offer written information found on their website . as dna direct puts it : 
 pretest education is provided at no additional charge through in - depth , online materials for all tests ( www.dnadirect.com/web/consumers accessed 4/05/2010 ) . pre- or post - test counselling is often only possible at an additional cost ( $ 250/hour for dna direct ; $ 99/hour for pathway genomics ) , which may be a barrier for consumers to ask for it , although some insurance companies may reimburse it . 
 in contrast , dnatraits offers a free optional consultation with their genetic counsellors before testing and a free mandatory consultation after to discuss the results ( http://www.dnatraits.com/services accessed 04/05/2010 ) . 
 23andme does not offer pre- or post - test counselling and urges consumers  to seek the advice of health professionals if questions or concerns arise ( https://www.23andme.com/about/consent/?version=1.3 accessed 19/01/2011 ) . 
 similarly , counsyl initially considered their test as an  at - home carrier test , as a successor to the at - home pregnancy test and did not consider offering any pre- or post - test counselling ( http://precedings.nature.com/documents/4192/version/1 accessed 19/01/2011 ) . 
 it has been suggested that general practitioners or midwives may be the most appropriate health care providers to offer carrier screening , since they can provide preconception care as part of ongoing primary care ( poppelaars et al . , 2004b ) . 
 in addition , some carrier screening programmes are offered by gynaecologists or midwives in antenatal care . 
 embedding in a healthcare setting can ensure adequate information provision to increase informed choice , a more optimal informed consent procedure , a medical follow - up if necessary and psychosocial counselling . 
 an offer of carrier testing through the internet by commercial companies runs the risk of disconnecting the service completely from its usual embedding in a medically supervised context . when these companies started to offer carrier tests , they referred consumers to seek medical supervision in the established health care system at the consumer 's own discretion . 
 pathway genomics suggested  you should consult with a physician or other appropriate health care professional regarding the diagnosis , treatment and prevention of any disease or health condition ( http://www.pathway.com/more_info/terms_of_service accessed 04/05/2010 ) . at the start of offering their service , 
 counsyl also sent out this message by underlying that people could order the test directly from their website to receive your kit immediately . 
 everyone has a prescription : the acmg recommends that adults of reproductive age be offered carrier testing for cf and spinal muscular atrophy , two of the many conditions assayed by the universal genetic test . 
 alternatively , you may get the test through your doctor ( https://www.counsyl.com/learn/easy/ accessed 04/05/2010 ) . 
 counsyl recently changed its stance and since the beginning of may 2010 , testing can only be requested through a physician , who can be found , among others , via the list offered on counsyl 's website . 
 the company also sends the results directly to the physician for interpretation , thereby , technically no longer selling ( but still advertising ) tests directly to consumers . in october 2010 , 
 dna direct also changed its policy ; one can find on their website that they 
 are no longer offering testing services directly to consumers ; but are now focusing their 
 efforts on providing comprehensive yet easy - to - understand information and tools to consumers , physicians , hospitals , employers and health plans ( http://www.dnadirect.com/web/consumers accessed 04/11/2010 ) . 
 pathway genomics has also , in the last few months , changed the way they offer tests ; consumers must now obtain a physician prescription and the company offers consumers the opportunity to send an email to the treating physician of the consumer directly from the company site in order to request the prescription . the offer through physicians may eliminate some of the concerns that were raised about information provision , but does not solve the issue about the appropriateness of the test provided . the absence of medical supervision for some dtc tests may compromise or fail to foster patient health especially in the case of carrier couples who may need intensive counselling regarding their reproductive choices , as well as concerning the risks for other family members . 
 as preconceptional carrier screening programmes have an impact on healthcare budgets , careful consideration should be given to whether the expenses for such a programme are justified within the realm of the healthcare system ( modell and kuliev , 1991 ) . 
 namely the option for and the cost of a specific screening programme might mean that other forms of screening can not be carried out . usually a balance should be made between the health gain obtained and the costs incurred . in the context of preconceptional carrier screening this has to be analysed carefully , as the primary goal of preconceptional carrier screening is enabling informed reproductive choice . 
 furthermore , the identification of a high number of carriers or false positives may create a higher number of visits to medical doctors for more information , and thus leading to higher costs in follow - up activities . 
 commercial companies underline that most users will pay for this test  out of pocket and therefore , the health care system will not be burdened . 
 since the need for follow - up information or tests is likely to happen in the traditional health care system , this is not completely true . 
 it is foreseeable that companies testing for a whole list of disorders will create a higher number of patients visiting health services . 
 for example , for the test panel offered by counsyl , the company foresees that 35% of the participating individuals would be carriers of at least one disease . 
 furthermore , every mentioned company underlines the value of privacy and the possibility to perform the test outside the control of insurers or the healthcare system . 
 they often play on people 's fear that if testing is conducted within the traditional health care system , third parties could access their genetic information and that this might lead to discrimination . as expressed by counsyl , 
 no one will ever have access to your data without your express consent ( https://www.counsyl.com/about/privacy/ accessed 04/05/2010 ) . 
 pathway genomics states that  your dna and results belong to you and no one else . 
 nobody should see your data or results unless you want them to ( http://www.pathway.com/more_info/dna_security accessed 04/05/2010 ) . 
 in contrast , dna direct and counsyl want health insurers to be involved in their services . 
 dna direct underlines that their services will be reimbursed by most insurers , although insurance companies may still ask for a letter of medical necessity which people with no a priori high risk may have . 
 counsyl advances that  for many people the test will be entirely free [ paid by the insurer ] ; for many others insurance will cover 70% or more of the cost ' . 
 in contrast , dnatraits refuses any reimbursement :  dnatraits does not accept reimbursement from insurance companies as this might limit our ability to ensure your privacy ( http://www.dnatraits.com/philosophy accessed 04/05/2010 ) . 
 as it is to be expected that more companies will want their services to be reimbursed by insurance companies for their customers , it becomes crucial that carrier tests involve a required degree of usefulness , have a scientific basis and are voluntary in nature . 
 the implementation of a preconceptional carrier test should result in the optimization of the advantages whilst reducing as much as possible the risks or adverse effects of the offer . 
 therefore , it is crucial that the tests offered for important health problems are suitable and have a known predictive value . furthermore , it is important to ensure quality pre- and post - test information and counselling that can lead to informed decision - making for the participants . 
 all potential negative psychosocial impacts resulting from participation in a carrier test should be avoided or minimized and the testing process should lead to a correct understanding of the test result . in our opinion , a commercial offer of dtc genetic carrier testing through the internet makes it difficult to meet all these criteria . 
 the purpose of carrier screening is to enlarge people 's reproductive autonomy by enabling carrier couples to make informed reproductive decisions . in this paper , we have criticized dtc genetic testing companies that have included large numbers of disorders in the test panel , including less severe ones , as well as disorders for which only suboptimal tests are available according to the traditional screening criteria . 
 it is , however , questionable whether commercial ( preconceptional ) carrier test providers can continue to exist using this business model as long as such genetic carrier tests are not integrated in larger , more comprehensive prenatal , or preferably in preconceptional , care settings ( which are , admittedly , not really established in general healthcare yet ) . 
 the company university diagnostics ltd ( www.udlgenetics.com , only to be consulted on http://www.archive.org/ ) , for example , started to offer a service to discover cf carrier status , but has ceased to exist ( advisory committee on genetic testing , 1999 ) . 
 furthermore , an analysis of the companies activities in this field shows that various genetic tests that were marketed online in recent years are no longer available for purchase . without the support of the healthcare system 
 , it may be that only a relatively small percentage of the population will become aware of these services and will use them ( kolor et al . , 2009 ) . 
 that being said , involving a physician in the dtc offer of genetic tests may not solve many of our concerns . 
 if physicians are not well educated about which tests should be given based on specific criteria , they may simply become a pawn in the commercial companies attempt at increasing their market size . 
 various recent events have created the expectation that regulatory oversight will increase in the near future ( borry et al . , 2010 ) . 
 for example , the european society of human genetics recently endorsed a statement in which it recommended to ensure , among other issues , the quality of the testing services , the provision of pretest information and genetic counselling , a face - to - face consultation and oversight of this industry ( european society of human genetics , 2010 ) . 
 as the government has a duty to protect citizens against the risks of unsound testing , the government can be expected to ensure that specific standards are being held and to promote the responsible provision and responsible use of specific genetic testing services to the public . 
 if we agree that reproductive autonomy should be respected , carrier screening for various serious disorders should be available , but careful implementation is necessary in order to ensure optimization of the screening offer , counselling and follow - up . 
 all other authors were involved in redrafting and revising of the paper and approved the final version of the manuscript for submission . 
 is funded by the european commission fp7 marie curie initiative . l.h . and m.c.c . 
 are financially supported by the centre for medical systems biology and the centre for society and genomics in the framework of the netherlands genomics initiative . 
 funding to pay the open access publication charges for this article was provided by the section community genetics , clinical genetics & emgo institute for health and care research , vu university medical center , amsterdam .","recently , a number of commercial companies are offering preconceptional carrier tests directly - to - consumers . 
 this offer raises a number of concerns and issues above and beyond those encountered with preconceptional tests offered within the traditional health care setting . in order to bring some of these issues to light and to initiate dialogue on this topic 
 , this article discusses the following issues : the current offer of preconceptional carrier tests ( until the end of 2010 ) through online commercial companies ; the implications for the informed consent procedure and the need for good information ; the need for medical supervision and follow - up ; and the appropriate use of existing resources . 
 the article concludes with some reflections about the potential sustainability of the offer of preconceptional carrier tests directly - to - consumers .",pubmed
"substantial growth has occurred in the options for operative fracture treatment in the last few decades.1 this , in proportion to newer and better internal fixation devices or implants . 
 specially designed intramedullary ( i m ) nails , and plates and screws for every bone and even parts of bones have helped increase treatment efficacy and union rates . 
 titanium alloys and stainless steel are generally employed for the manufacture of orthopedic implants.1 after fracture union , the implant loses its purpose and continues to exist only as a foreign object inside the patient s body . 
 removal of asymptomatic hardware is still debated , and there is a lack of clear guidelines.2 although there are several presumed benefits of implant removal , like functional improvement and pain relief , the surgical procedure can be challenging and may lead to complications such as neurovascular injury and refractures.2 there is wide country to country and surgeon to surgeon variations in the viewpoint on removal of hardware , with some advocating and many opposing routine removal.3 add to this the patients demands owing to their own perceptions and fears about the  foreign device  inside their body . in children , 
 though , routine implant removal after fracture union is still standard procedure.4 implants may disturb function , and some theoretical long - term risks such as growth disturbance , foreign body reaction , chronic infection , and corrosion are used as arguments for removal . 
 however , benefits should outweigh risks and removal should not require a more extensive operation than insertion.4 the consensus dilemmas over routine implant removal in adults notwithstanding , there are circumstances when removal of hardware does become necessary . 
 these include pain and discomfort to the patient , infection and implant failure . in our center 
 we designed this short study to investigate the commonest indications of implant removal , and to detect which implants were most likely to require removal . 
 the study was conducted prospectively on patients admitted for removal of implants in the orthopedics department of a teaching hospital . 
 adult patients aged 18 years or more who presented in the outpatient department ( opd ) with hardware related problems that necessitated removal was admitted . 
 patients admitted over a period of 7-month starting february 2013 were included in the study . 
 patients who had fixation devices intended to be removed after a definite interval to begin with , like percutaneous k - wires , external fixators and tarsal screws , were not included in the study . 
 patients requiring removal of joint prostheses were also excluded from the study . at the time of admission , the potential risks of the operation and the possibility of non - favorable outcomes were explained to all patients . after admission 
 , routine inpatient investigations were performed on all patients to evaluate their fitness for surgery . 
 postoperatively , the patients were retained in the hospital for variable periods depending on the indication of removal and the condition of the wound . 
 antibiotics were continued for longer duration in patients with infected hardware . at discharge , all the patients were strictly advised to protect the extremity for a variable length of time as demanded by the bone and the implant removed . 
 they were followed in the opd for another 4 months and evaluated for symptom relief / persistence / new problems , and the data were collected . 
 the data were analyzed by the authors and also by statistician using spss software and applying t - test , and results were compiled . 
 eighty - seven patients fulfilled the inclusion criteria , but four did not return for follow - up . that left us with eighty - three patients on whom to conduct the study ( n = 83 ) . 
 seventy - one patients were males ( 85.55% ) and 12 were females ( 14.45% ) . 
 their ages ranged from 20 years to 78 years , and the mean age was 38 years . 
 the reasons for removal of implants were found to lie in five categories : pain / discomfort / prominent hardware , infected hardware , implant failure , elective ( patient s insistence ) , and other reasons ( table 1 ) . 
 distribution of cases thirty - three patients out of eighty - three had hardware pain or discomfort or prominence ( 39.75% ) . 
 they ranged in age from 20 to 60 years ( mean age 31.6 years ) . 
 the time since fracture fixation ranged from 4 months to 96 months ( average 38 months ) . 
 the implants most commonly responsible in order of frequency were patella tension band wiring ( tbw ) ( n = 8) , olecranon tbw / plates ( n = 7 ) , distal humeral plates ( n = 6 ) , and femoral i m nails ( n = 4 ) . 
 the mean duration of hospital stay in these patients was 5 days . at 4 months follow - up , 15 patients out of 34 reported complete relief of pain ( 44.11% ) . 
 the average pain visual analog scale ( vas ) score decreased from 5.2 preoperatively to 2.1 postoperatively , which was statistically significant ( p < 0.05 ) . 
 one had an ulnar nerve palsy postoperatively , which recovered ( chart 1 , figure 1a and b ) . 
 ( b ) radiographs before and after removal of the implants twenty - four patients out of 83 ( 28.91% ) needed hardware removal because they had developed infection at the implant site a variable duration after osteosynthesis . 
 their ages ranged from 26 years to 78 years ( mean 49.33 years ) , and the duration since first surgery varied from 2 months to 156 months ( mean 47.57 months ) . 
 one ununited fracture was managed with external fixator ; the other was an infected olecranon which required repeat debridements followed by repeat osteosynthesis and flap coverage . in this group , 
 the implants most commonly removed included distal tibial / ankle plates and screws ( n = 8) , proximal tibial plates ( n = 4 ) and olecranon plates ( n = 3 ) . 
 after the removal , infection subsided in 21 patients out of 24 ( 87.5% ) . 
 one of them had a refracture of the tibial shaft after sequestrectomy ( chart 2 ) ( figures 2 and 3 ) . 
 distribution of infected hardware exposed and infected medial plates in the distal tibia in three patients infected and failed femoral locking nail eight ( 9.52% ) patients required implant removal and revision osteosynthesis for implant failure . 
 their average age was 35 years ( 18 - 50 years ) , and the average time since the primary procedure was 7.6 months ( 2 - 12 months ) . 
 these included 3 femoral i m nails , 2 distal tibial locked plates , 1 humeral shaft dynamic compression plate , and 2 patients with cannulated cancellous screws in the femoral neck ( chart 3 , figure 4 ) . 
 distribution of failed hardware plate failure in the humeral shaft and proximal femur one patient during the routine course of his follow - up after plating of both forearm bones was found to have extensive bone resorption under the plates ( figure 5 ) . 
 these plates were removed . on follow - up , there was no fracture or other complications . 
 bone resorption beneath ulnar plate seventeen ( 20.48% ) patients had their implants removed on demand , despite being asymptomatic . 
 during the course of their follow - up , three of these had persistent pain at the operated site . 
 two developed superficial wound infections which prolonged their hospital stay but responded to intravenous antibiotics and wound lavage . 
 distribution of implants removed on patient s wish the most consistently encountered obstacle during surgery was difficulty in removing the hardware from the bone . 
 this was seen especially in locked plates of the distal humerus and forearm , with ingrowth of bone around the plate / screws . 
 loss of contour (  rounding  ) of the screw head slot was also commonly encountered preventing the engagement of the driver in the screw head . 
 screw heads had to be cutoff to remove the plate in two patients because of this complication , and the shank left in the bone . in one patient who had presented for elective removal of an interlocked tibial nail , we failed to extract the nail despite best efforts . in another patient with a painful femoral nail , 
 fortunately , we did not encounter any major vascular injury or iatrogenic fracture during the removal of any implant . 
 one patient had an ulnar nerve neuropraxia after removal of distal humeral plates , which recovered . 
 sequestrectomy was done , and the patient presented with a refracture in follow - up . 
 these last two , and persistent pain in three previously asymptomatic patients were the only true complications in our series . 
 femoral interlocking nail broken during removal overall , the most frequently removed implants in our series were distal tibial / ankle plates ( 14.45% of implants removed ) , femoral i m nails ( 13.25% ) , olecranon wires and plates ( 12.04% ) , and tibial i m nails and patellar tbws ( 9.53% each ) . 
 pertinent to mention though that 6 of the 8 tibial nail removals were elective ( asymptomatic patients ) ( chart 5 ) . 
 overall distribution of removed implants all implants except one removed in our series were made of stainless steel , and all were made by indian manufacturers . 
 the issue of removing metallic implants used in fracture fixation has been oft discussed , and at length . 
 popular opinion probably still is that implant removal should not be considered a routine procedure.2,3,5 although the ao - association for the study of internal fixation has published recommendations on the timing of hardware removal in recent fractures with uncomplicated healing ( table 2 ) , the clinical indications for implant removal are not well established , and few definitive data exist to guide whether routine implant removal is appropriate . 
 furthermore , the surgical procedures for implant removal are fraught with risks of fracture , neurovascular injury , and infection . 
 various arguments have been made from time to time to justify removal of hardware after fracture union , e.g. , metal allergy , corrosion , carcinogenesis and metal ion toxicity , but for none has concrete proof been produced.2 all the same , a lot of metal is removed from human bones every day . and 
 that depicts that hardware does need to be removed in a significant subset of patients . 
 large studies on implant removal in symptomatic patients are lacking , though many patients get their implants removed for one reason or another . 
 our endeavor was to document the common indications for removal of internal fixation devices and highlight potential issues , even as most specialists are already well versed with them . 
 timing of implant removal in uncomplicated fracture healing ; ao guidelines we found that majority of patients that needed removal of implant were men ( 85.5% ) . 
 shrestha et al . in their retrospective series also found a male preponderance ( 189 out of 275 patients ) to the tune of 68.72%.6 their study , however , also included children . 
 30 of these ( 75% ) were males.7 there definitely seems to be a strong male preponderance in implant removal surgeries . 
 the mean age of patients requiring removal for infection was higher ( 49.33 years ) than those with pain ( 31.6 years ) or implant failure ( 35 years ) . 
 in our study , implant associated pain or discomfort was the most common reason necessitating removal ( 39.75% ) . 
 brown et al . found that 31% patient sunder going open reduction and internal fixation of ankle fractures had persistent lateral pain.8 they also found that only 11 of 22 patients who got their hardware removed had improvement in the pain . 
 prospectively studied 60 patients who had implant removal for hardware pain , and at 1 year follow - up all their patients were satisfied.9 although we did not primarily aim to evaluate the outcome after removal , all our patients had at least some relief in their hardware pain at 4 months follow - up . 
 the next most common indication in our series was implant infection ( about 29% ) . 
 some of these patients had been treated non - operatively for varying lengths of time to control the infection but without success . 
 others presented for the first time at such a stage , e.g. , skin necrosis , that removing the implant was the only logical option . 
 trampuz and widmer estimated that overall about 5% of all internal fixation devices become infected.10 they also impressed the role of biofilms in the resistance of pathogens to systemically administered antibiotics . 
 only one was a delayed infection ( 2 - 10 weeks ) , a woman with an olecranon plate who developed skin necrosis and the plate had to be removed 2 months after surgery . 
 all the others were  late  infections , caused by constant hematogenous seeding of the implant from skin , respiratory , dental and urogenital infections . 
 the incidence of infections is likely to rise as more operations are performed by the day , and longevity increases translating into greater periods of possible bacterial implant seeding in the body . 
 trampuz and widmer recommended stoppage of any antibiotics 2 weeks before the removal surgery , if possible , to get an accurate intraoperative tissue culture . 
 they also suggested that the removed implant be sonicated in saline to dislodge microorganisms from its surface and the resultant sonicated fluid be sent for microbiologic examination . 
 kukla et al . in a study of implants removed from the proximal femur ( dynamic hip screw and gamma nails ) found that the most common indications were avascular necrosis of the femoral head , deep chronic infections , shaft fractures , and screw cut - out.11 implant failure was the next most common indication in our series in symptomatic patients but it was less common than those in whom the hardware was removed at the patient s behest . 
 reasons for failure of the implant include faulty implant ( poor quality ) , faulty operative technique , inadequate implant , delayed healing , and patient noncompliance . 
 akhtar et al . cited the most common cause of failure as poor quality of the implant.12 peivandi et al . 
 they recommended that credible and trusted implant brands should be used in fracture fixation.13 sharma et al . in a restrospective study of 41 failed upper and lower limb implants found that plate failure was more common than nail failure in the lower limb.14 in most cases in their series , the patient had a significant re - trauma causing failure of the implant . 
 several studies have been carried out on the indications of removal of metalwork in asymptomatic patients . although most authors agree that routine removal should not be practiced 
 , they also agree that there is a need for the development of concrete indication guidelines for implant removal . at the same time 
 , there is a paucity of literature assessing the relative frequency of the  usual  indications of implant removal , viz . 
 we believe that routine removal in asymptomatic patients should not be practiced , and if at all necessary , the removal should not require a larger procedure than the index operation . 
 we also agree that implant removal surgeries are fraught with risks , including fractures , bleeding , nerve injuries and infection , and should be done only after explaining to the patient the possibility of all these complications . 
 in addition to the possible new problems , the removal surgery may not entirely fulfill the intended purpose , e.g. , the pain may not completely go away , infection may not resolve , and additional surgeries may be required . 
 all these factors must be borne in mind before embarking on such a process with high hopes of success . 
 our study is limited by a small sample size and a short follow - up period . 
 this may falsely favor titanium implants , although the probable reason for this is the low affordability of patients catered to by our center . 
 more studies with greater number of cases and wider study dimensions are needed to produce concrete literature on the patterns of removal surgeries in symptomatic implants . 
 we found that pain and implant prominence ( mechanical symptoms ) are the most common indications . 
 other indications are implant failure , bone resorption due to excessive stress shielding and patient s will . 
 the average age of patients requiring removal for infection is higher than that for other reasons . 
 carefully done , the removal should be a safe procedure , and there is a low but definite possibility of complications . 
 several factors like bone ingrowth and wear of the implant may make its removal difficult .","introduction : fracture fixation has become advanced with the advent of new and custom metal implants for each type of bone / fracture . after union 
 though , the implant ceases to be important and may be removed . 
 routine removal is advocated by some and opposed by others . 
 nevertheless , some patients require removal of the hardware because of various implant - related problems . 
 our study was aimed at identifying the most common causes for implant removal.objective:to investigate the common indications of orthopedic implant removal surgeries.methods:adult patients admitted for implant removal in our department were included in the study . 
 they were operated in the next ot list . 
 they were then followed for an average 4 months for resolutions of symptoms or appearance of new problems.results:a total of 83 patients were studied . 
 71 of them were males . 
 the mean age was 38 years . 
 the reasons for removal of implants were found to lie in five categories : pain / discomfort / prominent hardware , infected hardware , implant failure , elective ( patient s insistence ) , and other reasons . 
 overall , the most frequently removed implants in our series were distal tibial / ankle plates ( 14.45% of implants removed ) , femoral intramedullary ( i m ) nails ( 13.25% ) , olecranon wires and plates ( 12.04% ) , and tibial i m nails and patellar tension band wirings ( 9.53% each).discussion and conclusion : the clinical indications of implant removal are not well established , and few definitive data exist to guide whether routine implant removal is appropriate . 
 symptomatic hardware frequently needs removal . 
 we found that pain and implant prominence ( mechanical symptoms ) are the most common indications . 
 infection is the next most common , followed by hardware failure . 
 other indications are implant failure , bone resorption due to excessive stress shielding and patient s will . 
 removal is , however , not an easy surgery , and several factors such as bone ingrowth and wear of the implant may make its removal difficult .",pubmed
"glaucoma , a neurodegenerative disease , is characterized by gradual loss of retinal ganglion cells ( rgcs ) and optic nerve atrophy.1 multiple factors are related to glaucoma , such as high intraocular pressure ( iop)2 and low cerebral spinal fluid pressure.3 mller cells are the major type of glial cells in the mammalian retina that can support and nourish retinal neurons , maintain extracellular ion homeostasis , glutamate recycling , and interaction in synaptic transmission.4,5 therefore , mller cells are involved in retinal function . 
 investigations indicate that mller cells not only play an important physiological role , but they are also involved in multiple pathological retinal diseases such as glaucoma.68 it has been reported that reactive mller cells can aggravate retinal damage by releasing cytokines such as tumor necrosis factor- ( tnf-).9 growing evidence supports that increased glial production of tnf- contributes to the neurodegeneration in glaucoma.10 tnf- is a secreted inflammatory cytokine that is responsible for apoptosis , necrosis , and inflammation.11,12 tnf- is increased in the retina following ischemia or damage and other neurodegenerative disorders.1317 -aminoadipic acid ( aaa ) , a six - carbon homolog of glutamate , is a well - known compound that induces specific glial toxicity through blocking the glutamate uptake.1820 the use of aaa to interfere with glial influence on neuronal tissues is reported.21,22 however , the effects of aaa inhibition on mller cell gliosis in glaucoma are unknown . the current study aimed to determine if aaa treatment inhibits mller cell gliosis and protects against retinal abnormalities induced in a rat model of acute ocular hypertension ( aoh ) mimicking glaucoma 
 adult male sprague dawley rats ( weight range , 200250 g , 810 weeks ) were used in this study in accordance with the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research and followed the people s republic of china animal care guidelines . this study was monitored and approved by the animal care committee of the capital medical university , scxk ( jing ) 2012 - 0001 . 
 the animals were housed with a 12-hour light/12-hour dark cycle with standard chow and water . 
 all surgical procedures were performed under anesthesia with intraperitoneal chloral hydrate ( 450 mg / kg ) and topical 0.5% proparacaine hydrochloride eye drops ( alcon , inc . , 
 the iop was increased to 110 mmhg ( 14.63 kpa ) for 60 minutes by using an elevated 500 ml plastic container of sterile physiological saline connected to a 27-gauge needle placed in the anterior chamber of the eye . 
 sham procedure eyes were treated similarly but without the elevation of the bottle ; hence , the normal iop was maintained . 
 time - course examination was performed at 1 day , 3 days , and 5 days after acute iop elevation . 
 animals were divided into sham operation ( ctrl ) group , aoh group , aoh + phosphate - buffered saline ( pbs ) control group , and aoh + aaa - treated group . 
 aaa ( 250 g ) was injected into vitreous humor in 5 l at 50 g / l concentration after 3 hours of acute iop elevation according to the literature.23,24 tissues were fixed with 4% paraformaldehyde . 
 eyeballs were cryoprotected in 30% sucrose overnight at 4c and then embedded in optimal cutting temperature compound ( sakura finetechinical co. ltd . , 
 sections ( 14 m ) were incubated in 3% bovine serum albumin and 0.3% triton x-100 ( sigma - aldrich co. , st louis , mo , usa ) to block nonspecific binding and then incubated with primary mouse monoclonal antibodies against thy-1 ( 1:200 , rgc marker ; abcam , cambridge , uk ) , primary mouse monoclonal antibodies against glutamine synthetase ( gs ; 1:100 , mller glial cell maker ; abcam ) , rabbit polyclonal antibodies against glial fibrillary acidic protein ( gfap ; 1:200 , gliosis maker ; cell signaling technology inc . , danvers , ma , usa ) overnight at 4c , followed by incubation with secondary antibodies for 1 hour at room temperature . 
 sections were mounted with fluoromount - g ( southernbiotech , birmingham , al , usa ) . 
 slides were visualized using a confocal microscope ( leica microsystems , wetzlar , germany ) . 
 the intensity of immunoreactivity from photographs was analyzed using image - proplus 6.0 ( media cybernetics inc . , silver spring , md , usa ) 
 optical densities obtained from immunohistochemistry images were corrected by subtracting the average value of background noise from five image inputs . the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay was conducted according to the manufacturer s protocol . 
 briefly , tissue sections were fixed in paraformaldehyde and were subsequently incubated in a tunel reaction medium . 
 the sections were then mounted on microscope slides and examined under ultraviolet light using an epifluorescence microscope . 
 retinas from sham operation group ( ctrl ) , aoh group , aoh + pbs control group , and aoh + aaa group were homogenized in lysis buffer and sonicated to dissolve the tissue completely . in 
 all , 40 g of total proteins from each sample were loaded per lane for sodium dodecyl sulfate polyacrylamide gel electrophoresis . 
 the proteins were transferred onto the nitrocellulose membrane ( schleicher & schuell , dassel , germany ) and the blocked nitrocellulose membrane was incubated with a primary rabbit polyclonal antibody against tnf- ( 1:1,000 ; abcam ) for 3 hours ( room temperature ) . 
 after washing in tris buffered saline , with tween-20 ( tbst ) , the membranes were incubated with horseradish peroxidase - conjugated goat anti - rabbit or mouse igg prior to detection of the labeled proteins by using ecl - plus kit ( perkinelmer inc . , waltham , ma , usa ) followed by exposure of blots to x - omat ( kodak , rochester , ny , usa ) imaging film . 
 the images were quantified using imagej software ( national institutes of health , maryland , usa ) . the gel doc-2000 imaging system ( bio - rad laboratories inc . , hercules , ca , usa ) was used to perform the quantitative analysis of western blot results . 
 the -actin band density was expressed as 100% , and the other group was expressed as percentage of that from the control group . 
 analysis of variance was used to determine the difference between tnf- and fluorescence intensity among three or more independent ( unrelated ) groups , followed by multiple comparisons using the student  newman 
 wallis test was used to determine the difference between tunel - positive cells and thy-1-positive rgcs among three or more independent ( unrelated ) groups , followed by multiple comparisons using the nemenyi test . 
 statistical analysis was performed using sas 9.2 ( sas institute inc . , cary , nc , usa ) . 
 adult male sprague dawley rats ( weight range , 200250 g , 810 weeks ) were used in this study in accordance with the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research and followed the people s republic of china animal care guidelines . this study was monitored and approved by the animal care committee of the capital medical university , scxk ( jing ) 2012 - 0001 . 
 the animals were housed with a 12-hour light/12-hour dark cycle with standard chow and water . 
 all surgical procedures were performed under anesthesia with intraperitoneal chloral hydrate ( 450 mg / kg ) and topical 0.5% proparacaine hydrochloride eye drops ( alcon , inc . , hnenberg , switzerland ) . 
 the iop was increased to 110 mmhg ( 14.63 kpa ) for 60 minutes by using an elevated 500 ml plastic container of sterile physiological saline connected to a 27-gauge needle placed in the anterior chamber of the eye . 
 sham procedure eyes were treated similarly but without the elevation of the bottle ; hence , the normal iop was maintained . 
 time - course examination was performed at 1 day , 3 days , and 5 days after acute iop elevation . 
 animals were divided into sham operation ( ctrl ) group , aoh group , aoh + phosphate - buffered saline ( pbs ) control group , and aoh + aaa - treated group . 
 aaa ( 250 g ) was injected into vitreous humor in 5 l at 50 g / l concentration after 3 hours of acute iop elevation according to the literature.23,24 
 eyeballs were cryoprotected in 30% sucrose overnight at 4c and then embedded in optimal cutting temperature compound ( sakura finetechinical co. ltd . , tokyo , japan ) . 
 sections ( 14 m ) were incubated in 3% bovine serum albumin and 0.3% triton x-100 ( sigma - aldrich co. , st louis , mo , usa ) to block nonspecific binding and then incubated with primary mouse monoclonal antibodies against thy-1 ( 1:200 , rgc marker ; abcam , cambridge , uk ) , primary mouse monoclonal antibodies against glutamine synthetase ( gs ; 1:100 , mller glial cell maker ; abcam ) , rabbit polyclonal antibodies against glial fibrillary acidic protein ( gfap ; 1:200 , gliosis maker ; cell signaling technology inc . , danvers , ma , usa ) overnight at 4c , followed by incubation with secondary antibodies for 1 hour at room temperature . 
 sections were mounted with fluoromount - g ( southernbiotech , birmingham , al , usa ) . 
 slides were visualized using a confocal microscope ( leica microsystems , wetzlar , germany ) . 
 the intensity of immunoreactivity from photographs was analyzed using image - proplus 6.0 ( media cybernetics inc . , silver spring , md , usa ) . 
 optical densities obtained from immunohistochemistry images were corrected by subtracting the average value of background noise from five image inputs . 
 the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay was conducted according to the manufacturer s protocol . briefly , tissue sections were fixed in paraformaldehyde and were subsequently incubated in a tunel reaction medium . 
 the sections were then mounted on microscope slides and examined under ultraviolet light using an epifluorescence microscope . 
 retinas from sham operation group ( ctrl ) , aoh group , aoh + pbs control group , and aoh + aaa group were homogenized in lysis buffer and sonicated to dissolve the tissue completely . in all , 
 40 g of total proteins from each sample were loaded per lane for sodium dodecyl sulfate polyacrylamide gel electrophoresis . 
 the proteins were transferred onto the nitrocellulose membrane ( schleicher & schuell , dassel , germany ) and the blocked nitrocellulose membrane was incubated with a primary rabbit polyclonal antibody against tnf- ( 1:1,000 ; abcam ) for 3 hours ( room temperature ) . 
 after washing in tris buffered saline , with tween-20 ( tbst ) , the membranes were incubated with horseradish peroxidase - conjugated goat anti - rabbit or mouse igg prior to detection of the labeled proteins by using ecl - plus kit ( perkinelmer inc . , 
 waltham , ma , usa ) followed by exposure of blots to x - omat ( kodak , rochester , ny , usa ) imaging film . 
 the images were quantified using imagej software ( national institutes of health , maryland , usa ) . the gel doc-2000 imaging system ( bio - rad laboratories inc . , hercules , ca , usa ) was used to perform the quantitative analysis of western blot results . 
 the -actin band density was expressed as 100% , and the other group was expressed as percentage of that from the control group . 
 analysis of variance was used to determine the difference between tnf- and fluorescence intensity among three or more independent ( unrelated ) groups , followed by multiple comparisons using the student  newman 
 wallis test was used to determine the difference between tunel - positive cells and thy-1-positive rgcs among three or more independent ( unrelated ) groups , followed by multiple comparisons using the nemenyi test . 
 statistical analysis was performed using sas 9.2 ( sas institute inc . , cary , nc , usa ) . 
 values were reported as mean  sd . a value of p<0.05 was considered as statistically significant . 
 to determine the effects of aaa on rgcs in the aoh model , aoh was induced in sd rats and rats were intravitreally treated with pbs , aaa , aoh + pbs , and aoh + aaa . significantly reduced retinal thickness in the inner plexiform layer ( ipl ) and inner nuclear layer was observed after aoh induction versus control ( figure s1 ) . 
 the mean values of tunel - positive cells per visual field were 0.10.3 in control ( figure 1a ) , 0.20.4 in aaa(figure 1b ) , 4.60.8 in aoh + pbs , and 1.70.7 in aoh + aaa . 
 aoh induced the apoptotic death of cells in the ganglion cell layer(gcl ; figure 1c ) . in the aoh model , intravitreal treatment with aaa 
 attenuated the tunel - positive rgcs more significantly than the intravitreal treatment with pbs ( p<0.05 ; figure 1d ) , indicating that aaa treatment rescued rgcs from the cell apoptosis induced in the aoh model . 
 in addition , retinal sections were immunostained with antibody against thy-1 , a marker for rgcs.25 the mean values of thy-1-positive rgcs per visual field in ctrl , aaa , aoh + pbs , and aoh + aaa groups were 8.20.9 , 8.10.8 , 3.90.7 , and 60.6 , respectively . 
 aaa treatment did not change the number of rgcs in normal conditions ( figure 2a and b ) . when compared with the ctrl group ( figure 2a ) 
 , the number of thy-1-positive rgcs was decreased in the aoh + pbs group ( figure 2c ) , and aaa treatment significantly rescued thy-1-positive rgcs in aoh ( p<0.05 ; figure 2d ) , corresponding to aaa - attenuated apoptosis of rgcs in aoh ( aoh + pbs versus aoh + aaa , p<0.05 ; figure 1 ) . in the rat model of aoh , 
 aaa rescued rgcs . to determine if aaa protects against aoh - induced rgc death through modulating mller cell gliosis , immonohistochemistry for gfap ( a marker for activated glial cells ) and gs ( mller cell - specific marker ) was conducted.26 mller cells cover the whole retina from the nerve fiber layer to the photoreceptor layer,27 and 
 gfap was mainly expressed along nerve fiber layer and gcl and was less or absent at other layers in the control group ( figure s2 ) . in the rat model of aoh , colocalization of gfap and gs was observed across ipl ( figure s2 ) , indicating mller cell activation . 
 after 1 day , 3 days , and 5 days of aoh induction in rat , gfap expression was also observed across ipl ( figure s3 ) , indicating mller cell gliosis . 
 aaa treatment versus pbs control markedly reduced gfap immunoreactivity across ipl ( figure 3 ) , indicating attenuation in mller cell gliosis . as mentioned earlier , aaa rescued rgcs likely through the inhibition of mller cells gliosis in the rat model with aoh . 
 activation of mller cells may induce tnf- production , leading to neurodegeneration in glaucoma.10 to determine if aaa modulates tnf- production , western blot was performed for the investigation of retinal protein levels of tnf-. significantly upregulated protein levels of retinal tnf- were observed in aoh versus sham operation ( ctrl ) group ( p<0.05 ; figure 4 ) . 
 importantly , aaa treatment versus pbs control significantly attenuated tnf- levels in the retinas of the rat model of aoh ( p<0.05 ; figure 4 ) . 
 to determine the effects of aaa on rgcs in the aoh model , aoh was induced in sd rats and rats were intravitreally treated with pbs , aaa , aoh + pbs , and aoh + aaa . significantly reduced retinal thickness in the inner plexiform layer ( ipl ) and inner nuclear layer was observed after aoh induction versus control ( figure s1 ) . 
 the mean values of tunel - positive cells per visual field were 0.10.3 in control ( figure 1a ) , 0.20.4 in aaa(figure 1b ) , 4.60.8 in aoh + pbs , and 1.70.7 in aoh + aaa . 
 aoh induced the apoptotic death of cells in the ganglion cell layer(gcl ; figure 1c ) . in the aoh model , intravitreal treatment with aaa 
 attenuated the tunel - positive rgcs more significantly than the intravitreal treatment with pbs ( p<0.05 ; figure 1d ) , indicating that aaa treatment rescued rgcs from the cell apoptosis induced in the aoh model . 
 in addition , retinal sections were immunostained with antibody against thy-1 , a marker for rgcs.25 the mean values of thy-1-positive rgcs per visual field in ctrl , aaa , aoh + pbs , and aoh + aaa groups were 8.20.9 , 8.10.8 , 3.90.7 , and 60.6 , respectively . 
 aaa treatment did not change the number of rgcs in normal conditions ( figure 2a and b ) . when compared with the ctrl group ( figure 2a ) , 
 the number of thy-1-positive rgcs was decreased in the aoh + pbs group ( figure 2c ) , and aaa treatment significantly rescued thy-1-positive rgcs in aoh ( p<0.05 ; figure 2d ) , corresponding to aaa - attenuated apoptosis of rgcs in aoh ( aoh + pbs versus aoh + aaa , p<0.05 ; figure 1 ) . 
 in the rat model of aoh , aaa rescued rgcs . to determine if aaa protects against aoh - induced rgc death through modulating mller cell gliosis , immonohistochemistry for gfap ( a marker for activated glial cells ) and gs ( mller cell - specific marker ) was conducted.26 mller cells cover the whole retina from the nerve fiber layer to the photoreceptor layer,27 and astrocytes mainly locate along gcl . 
 gfap was mainly expressed along nerve fiber layer and gcl and was less or absent at other layers in the control group ( figure s2 ) . in the rat model of aoh , colocalization of gfap and gs was observed across ipl ( figure s2 ) , indicating mller cell activation . 
 after 1 day , 3 days , and 5 days of aoh induction in rat , gfap expression was also observed across ipl ( figure s3 ) , indicating mller cell gliosis . 
 aaa treatment versus pbs control markedly reduced gfap immunoreactivity across ipl ( figure 3 ) , indicating attenuation in mller cell gliosis . 
 as mentioned earlier , aaa rescued rgcs likely through the inhibition of mller cells gliosis in the rat model with aoh . 
 activation of mller cells may induce tnf- production , leading to neurodegeneration in glaucoma.10 to determine if aaa modulates tnf- production , western blot was performed for the investigation of retinal protein levels of tnf-. significantly upregulated protein levels of retinal tnf- were observed in aoh versus sham operation ( ctrl ) group ( p<0.05 ; figure 4 ) . 
 importantly , aaa treatment versus pbs control significantly attenuated tnf- levels in the retinas of the rat model of aoh ( p<0.05 ; figure 4 ) . 
 glaucoma is a leading cause of irreversible vision loss characterized by progressive death of rgcs , and elevated iop is a major risk factor.28 a rodent model of aoh is well established for acute angle closure glaucoma , and it has been widely used to investigate the pathogenesis of death of rgcs.29 in the current rat model of aoh , we found induced cell apoptosis and decreased number of rgcs with iop induction versus sham operation control . 
 importantly , treatment of aaa versus pbs control significantly attenuated rgc apoptosis and rescued the reduced number of rgcs , demonstrating protective effects on aoh retinas ( figure 5 ) . 
 activated mller cells contribute to the progression of glaucoma.30 mller cells perform a multitude of important regulatory and supportive roles , including secretion of trophic factors , removal of metabolic waste , and neurotransmitter recycling.31,32 we found that mller cells were activated with induced gfap immunoreactivity in the rat model of aoh versus control retinas . 
 gfap , an intermediate filament protein , is considered as a marker of reactive mller cell gliosis,27 which is not or less expressed in mller cells in normal retinas and expressed highly at ischemic,33 light - induced retinal degeneration,34 and retinal detachment.35 activation of mller cells so far was demonstrated to have both protective and detrimental effects.3638 especially early after injury , mller cell gliosis is believed to be neuroprotective and promotes the repair of neurons in response to injury,36 with gfap upregulation to provide additional structural integrity to the retina at the site of injury.39 however , activated mller cells also express tnf-,40 monocyte chemoattractant protein,41 and nitric oxide42 to induce death of rgcs . 
 tnf- induction contributes to inflammation , apoptosis , and necrosis , leading to cell death . 
 we observed that aaa treatment attenuated gfap immunoreactivity in mller cell processes across ipl and reduced retinal tnf- levels in the rat model of aoh . 
 these findings suggested that aaa promoted survival of rgcs in the rat model of aoh , likely through the inhibition of aoh - induced mller cell gliosis and in turn downregulation of tnf- protein production . 
 we found that aaa could effectively protect retina against loss of rgcs and apoptosis in aoh retinas through attenuating mller cell gliosis and downregulating tnf- production . 
 these observations suggested that aaa might be a potential therapeutic target in the treatment of neurodegeneration in glaucoma . 
 ( a ) reprehensive results of hoechst staining in rat retina after 1 d , 3 d , and 5 d of aoh ; ( b ) quantitative analysis of the retinal thickness . 
 notes : the results showed that the thickness of ipl and inl layers decreased significantly following the reperfusion time after aoh treatment ( scale bar = 50 m ) . * p<0.05 compared with the control group ( n=4 per group ) . 
 abbreviations : aoh , acute ocular hypertension ; ipl , inner plexiform layer ; inl , inner nuclear layer ; ctrl , control ; d , day ; gcl , ganglion cell layer ; onl , outer nuclear layer . 
 note : strong expression of gfap was shown in the gs - positive mller cell after 5 d of aoh compared with the control group ( scale bar = 50 m ) . 
 abbreviations : gs , glutamine synthetase ; gfap , glial fibrillary acidic protein ; aoh , acute ocular hypertension ; ctrl , control ; gcl , ganglion cell layer ; ipl , inner plexiform layer ; inl , inner nuclear layer ; onl , outer nuclear layer ; d , days . increased gfap immunoreactivity after aoh induction in rat retinas . 
 notes : ( a ) immunofluorescent staining showed that aoh increased gfap immunoreactivity in the mller cell processes across ipl . 
 abbreviations : gfap , glial fibrillary acidic protein ; aoh , acute ocular hypertension ; ipl , inner plexiform layer ; d , day ; pbs , phosphate - buffered saline ; aaa , -aminoadipic acid .","objectiveocular hypertension is an important risk factor for glaucoma . 
 the purpose of this study was to investigate the gliotoxic effects of -aminoadipic acid ( aaa ) in a rat model of aoh and its underlying mechanisms.materials and methodsin the rat model of acute ocular hypertension ( aoh ) , intraocular pressure was increased to 110 mmhg for 60 minutes . 
 animals were divided into four groups : sham operation ( ctrl ) , aoh , aoh + phosphate - buffered saline ( pbs ) , and aoh + aaa . 
 cell apoptosis in the ganglion cell layer was detected with the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay , and retinal ganglion cells ( rgcs ) immunostained with thy-1 were counted . 
 mller cell activation was detected using immunostaining with glutamine synthetase and glial fibrillary acidic protein . 
 tumor necrosis factor- ( tnf- ) was examined using western blot.resultsin the rat model of aoh , cell apoptosis was induced in the ganglion cell layer and the number of rgcs was decreased . 
 mller cell gliosis in the retinas of rats was induced , and retinal protein levels of tnf- were increased . 
 intravitreal treatment of aaa versus pbs control attenuated these retinal abnormalities to show protective effects in the rat model of aoh.conclusionin the retinas of the rat model of aoh , aaa treatment attenuated retinal apoptosis in the ganglion cell layer and preserved the number of rgcs , likely through the attenuation of mller cell gliosis and suppression of tnf- induction . 
 our observations suggest that aaa might be a potential therapeutic target in glaucoma .",pubmed
"although endoscopic resection can be a curative procedure for such lesions 1 , it still remains technically challenging because of the high risk of severe adverse events ( saes ) including delayed perforation 2 . on the other hand , surgical treatment of such lesions often results in extensive resections , causing significant morbidity . 
 recently , laparoscopic and/or endoscopic full - thickness resection ( ftr ) has been reported to be a useful technique for the treatment of nonampullary sdns 3 
 4 
 5 . despite their minimally invasive nature , all ftr procedures are associated with the possibility of intraperitoneal tumor dissemination . 
 therefore , we developed an alternative ftr method , which we call laparoscopy - assisted endoscopic full - thickness resection with ligation device ( laeftr - l ) . 
 herein we report on resection of nonampullary sdns using laeftr - l in 5 consecutive patients and an evaluation of the efficacy and safety of these procedures . 
 from june 2015 to september 2015 , 5 patients underwent resection of nonampullary sdns using laeftr - l . written informed consent 
 the set up for the laeftr - l procedure was previously described 3 . after mobilizing the duodenum from the retroperitoneum , a forward - viewing endoscope ( gif260j ; olympus , medical systems corp . , 
 tokyo , japan ) was inserted into the duodenum and the target lesion was identified ( fig . 
 endoscopic light was used to help the surgeon identify the tumor location . using an endoclip as a marker , 
 the tumor ( including the seromuscular layer ) was ligated using a ligation device ( pneumo - activate evl device ; sumitomo bakelite corp . , 
 the laparoscopist then temporarily sutured the seromuscular layer , after confirming the presence of a full - thickness aspirated sign ( fig . 
 then , an endoscopist resected the targeted lesion under the ligation band using snare technique ( fig . 
 2b ) . finally , the specimen was retrieved intraluminally using the endoscope ( fig . 
 despite the lack of a seromuscular layer , the provisional and additional sutures prevented exposure of the abdominal cavity to malignant cells . in addition , vessels were ligated during the closure of the defect in the duodenal wall . 
 therefore , the laeftr - l procedure was completed in a closed manner without complications . 
 endoscopic findings of laparoscopy - assisted endoscopic full - thickness resection with ligation device for superficial duodenal neoplasm . a a flat elevated lesion , measuring 15  mm in diameter , located at the opposite side of the ampulla of vater . 
 b using an endoclip as a marker , the targeted lesion was ligated with a ligation device . 
 e laparoscopic temporary suture secures patency of the duodenal wall . f resected lesion after administration of 0.1  % indigo carmine solution . 
 laparoscopic findings of laparoscopy - assisted endoscopic full - thickness resection with ligation device for superficial duodenal neoplasm 
 . a full - thickness aspirated sign , which helps the surgeon accurately identify the tumor location . 
 inhibitors of gastric acid and protease secretion were administered to the patients during their hospitalizations . 
 a normal diet was resumed approximately 2 days after the procedure , and the patients were discharged after a 1-week hospital stay . 
 from june 2015 to september 2015 , 5 patients underwent resection of nonampullary sdns using laeftr - l . written informed consent 
 the set up for the laeftr - l procedure was previously described 3 . after mobilizing the duodenum from the retroperitoneum , a forward - viewing endoscope ( gif260j ; olympus , medical systems corp . , 
 tokyo , japan ) was inserted into the duodenum and the target lesion was identified ( fig . 
 endoscopic light was used to help the surgeon identify the tumor location . using an endoclip as a marker , 
 the tumor ( including the seromuscular layer ) was ligated using a ligation device ( pneumo - activate evl device ; sumitomo bakelite corp . , ltd . 
 the laparoscopist then temporarily sutured the seromuscular layer , after confirming the presence of a full - thickness aspirated sign ( fig . 
 then , an endoscopist resected the targeted lesion under the ligation band using snare technique ( fig .  1c , fig . 
 2b ) . finally , the specimen was retrieved intraluminally using the endoscope ( fig . 
 despite the lack of a seromuscular layer , the provisional and additional sutures prevented exposure of the abdominal cavity to malignant cells . in addition , vessels were ligated during the closure of the defect in the duodenal wall . 
 therefore , the laeftr - l procedure was completed in a closed manner without complications . 
 endoscopic findings of laparoscopy - assisted endoscopic full - thickness resection with ligation device for superficial duodenal neoplasm . a a flat elevated lesion , measuring 15  mm in diameter , located at the opposite side of the ampulla of vater . 
 b using an endoclip as a marker , the targeted lesion was ligated with a ligation device . 
 e laparoscopic temporary suture secures patency of the duodenal wall . f resected lesion after administration of 0.1  % indigo carmine solution . 
 laparoscopic findings of laparoscopy - assisted endoscopic full - thickness resection with ligation device for superficial duodenal neoplasm . 
 a full - thickness aspirated sign , which helps the surgeon accurately identify the tumor location . 
 inhibitors of gastric acid and protease secretion were administered to the patients during their hospitalizations . 
 a normal diet was resumed approximately 2 days after the procedure , and the patients were discharged after a 1-week hospital stay . 
 two of the 5 tumors were located at the duodenal bulb , and the others at the second part of the duodenum ( opposite to the ampulla of vater ) . 
 the mean operative time was 173 minutes ( range 138    217 minutes ) . 
 mean diameter of the resected specimen was 24  mm ( range 18    32  mm ) , and the mean tumor size was 9  mm ( range 7    11  mm ) . 
 complete resection was achieved , and full - thickness resection was histologically confirmed in all cases ( fig . 
 because the malignant potential of sdn is difficult to define from the preoperative endoscopic diagnosis 6 , complete en - bloc resection is necessary to achieve appropriate pathological evaluation and treatment . 
 although conventional endoscopic mucosal resection ( emr ) could be a curative strategy for sdns , complete resection is challenging , especially for flat or non - lifting lesions . in recent years , the indications for esd 
 however , delayed perforation in the duodenum , in particular , may cause potentially fatal peritonitis or retroperitoneal complications , requiring emergency open surgery to counter leakage of bile and pancreatic juices 8 . 
 moreover the technical difficulty of this procedure is extremely high , due to the poor maneuverability of the endoscope and the thin wall . 
 therefore , duodenal esd is not a standard clinical procedure . on the other hand , 
 although short - term clinical outcomes were preferable , we need to consider the risk of intraperitoneal tumor dissemination , given the lack of data on long - term outcomes in patients undergoing conventional ftr procedures . compared with conventional ftr procedures , laeftr - l has two advantages . 
 most importantly , the resected specimen can be collected intraluminally , avoiding contact with the peritoneum . 
 shen et al . conducted an animal model study and revealed that pneumoperitoneum from laparoscopic surgery could be a risk factor for peritoneal dissemination of tumor cells 9 . 
 because long - term outcomes of sdns treated with the conventional ftr method rarely have been investigated , peritoneal exposure of tumor cells should be avoided as far as possible . moreover , laeftr - l does not require proficient experience in esd . 
 indeed , total operative time was not different from previous reports on conventional ftr for sdns 3 
 4 , and endoscopic resection time was even significantly shorter . 
 tumor size is a major limiting factor when considering the indication for laeftr . in the current study , 
 reported that esophageal subepithelial lesions no larger than 13  mm could be successfully resected by emr with a ligation device 10 . 
 considering the size of the ligation device , lesions smaller than 15  mm in diameter may be candidates for this procedure . 
 although additional studies are needed , application of a large oblique cap may enable en - bloc resection of larger lesions . 
 in addition , tumor location is also important to decisions about the indication for laeftr - l . 
 laeftr - l can not to be applied to lesions close to the ampulla of vater , because mobilization of these portions is technically difficult with laparoscopic procedures 
 . in conclusion , laeftr - l may be a promising technique for the treatment of nonampullary sdns which can achieve complete resection without peritoneal exposure of tumor cells or saes . 
 prospective studies are needed to further evaluate the efficacy and safety of laeftr - l procedures .","background and study aims : recently , laparoscopic and/or endoscopic full - thickness resection ( ftr ) has been reported to be a useful technique for the treatment of superficial duodenal neoplasms ( sdns ) . in the current study , we evaluated clinical outcomes in 5 consecutive patients who underwent resection of nonampullary sdns using laparoscopy - assisted endoscopic full - thickness resection with ligation device ( laeftr - l ) , which is an alternative ftr method developed to avoid peritoneal dissemination . using a snare technique with a ligation band 
 , the duodenal lesions were easily resected . 
 the provisional and additional sutures for the resected site prevented delayed perforation and bleeding and they also protected the abdominal cavity from direct exposure to malignant cells . 
 complete resection could be achieved and ftr was histologically confirmed in all cases . 
 the mean operation time was 173 minutes ( range 138 
 217 minutes ) . 
 mean diameter of the resected specimen was 24  mm ( range 18    32  mm ) . 
 no adverse events ( aes ) were observed . 
 laeftr - l , which can achieve complete resection of nonampullary sdns without severe aes and peritoneal dissemination , could be a useful technique for the treatment of such lesions .",pubmed
"congenital adrenal hyperplasia ( cah ) is caused by defects in various enzymes necessary for cortisol synthesis . 
 90 - 95% of cah cases are caused by 21-hydroxylase deficiency ( 21-ohd ) . in patients with 21-ohd , 
 some of these precursors are shunted into the androgen synthetic pathway , causing signs and symptoms of androgen excess , including ambiguous genitalia in females and rapid somatic growth with accelerated skeletal maturation in both sexes1 ) . since the discovery of cortisone in the 1950s , cah has been managed effectively , and the clinical course of the disease has markedly improved2 ) . 
 typical medical treatment consists of oral glucocorticoid and mineralocorticoid administration in order to compensate for adrenal steroid deficiencies and to suppress adrenal androgens . 
 herein , we report four cases of patients who were diagnosed as having cah . they have been managed with hydrocortisone and fludrocortisone . 
 thereafter , adrenal masses were found by abdominal computed tomography ( ct ) scan in all four cases . 
 recent reports reveal a high apprearance of small tumors in patients with untreated cah and most adrenal masses in children with cah are benign3 - 5 ) . 
 two of our four cases were managed with adrenalectomy because of increasing adrenal mass size and virilization . 
 a male neonate who presented with severe vomiting at 15 days of life was brought to medical attention . 
 however severe vomiting recurred after he was discharged and he was brought to kyunghee medical center . 
 laboratory evaluation revealed a ph level of 7.36 , hco3 13 meq / l , base excess -10 meq / l , and na 109 meq / l . 
 electrolytes were normalized and vomiting was resolved . thereafter the level of na was normalized . 
 however at the age of 20 years , his height was 145 cm ( less than 3 percentile ) . 
 abdominal ct performed at the age of 18 years ( july 30 , 2004 ) revealed the presence of multiple bilateral adrenal masses ( possibly adenoma ) . 
 the size of his right adrenal mass was 2.41.3 cm and the size of left adrenal masses were 2.71.7 cm and 2.31.9 cm with adrenal hyperplasia . 
 follow - up abdominal ct was performed at the age of 20 years ( fig . 
 1 ) . abdominal ct showed that both adrenal masses on both sides were increasing in size . 
 the size of his right adrenal mass was 3.01.8 cm , and the size of his left adrenal masses were 3.32.4 cm and 3.02.6 cm . 
 bilateral adrenalectomy was performed at the age of 20 years . a female neonate presented with ambiguous genitalia at birth suggesting salt - losing cah . 
 when she was at the age of 3 years , vaginoplasty and clitoroplasty were performed . at the age of 11 
 abdominal ct performed at the age of 11 years showed a 3.54.0 cm mass in her right adrenal gland ( fig . 
 hydrocortisone and fludrocortisone administration was carried out during follow - up and vaginoplasty and clitoroplasty were performed at the age of 4 years . 
 when she was 11 years old and 12 years old , abdominal cts were performed due to her virilization including hirsuitism and masculine voice . 
 a male neonate was admitted to medical attention at 1 month of age with poor oral intake and diarrhea . 
 he was admitted to kyunghee medical center at 5 months of age with poor oral intake and vomiting . 
 laboratory evaluation revealed a ph level of 7.35 , hco3 14 meq / l , base excess -9 meq / l , and na 119 meq / l . during follow - up , he was diagnosed with cah . 
 after his medication had begun , the level of na was normalized . during follow - up , 
 medication was stopped on his parents ' request and since the age of six , he showed secondary growth such as hyperpigmentation , hirsuitism , large penis , and pubic hair . 
 abdominal ct performed at the age of 14 years revealed the presence of adrenal masses ( left adrenal gland : 2.01.4 cm & 1.21.3 cm , right adrenal gland : 1.11.2 cm ) . since there was no change in the size of adrenal mass , adrenalectomy was not performed . 
 a male neonate who presented with severe vomiting at 15 days of life was brought to medical attention . 
 however severe vomiting recurred after he was discharged and he was brought to kyunghee medical center . 
 laboratory evaluation revealed a ph level of 7.36 , hco3 13 meq / l , base excess -10 meq / l , and na 109 meq / l . 
 electrolytes were normalized and vomiting was resolved . thereafter the level of na was normalized . 
 however at the age of 20 years , his height was 145 cm ( less than 3 percentile ) . 
 abdominal ct performed at the age of 18 years ( july 30 , 2004 ) revealed the presence of multiple bilateral adrenal masses ( possibly adenoma ) . 
 the size of his right adrenal mass was 2.41.3 cm and the size of left adrenal masses were 2.71.7 cm and 2.31.9 cm with adrenal hyperplasia . 
 follow - up abdominal ct was performed at the age of 20 years ( fig . 
 1 ) . abdominal ct showed that both adrenal masses on both sides were increasing in size . 
 the size of his right adrenal mass was 3.01.8 cm , and the size of his left adrenal masses were 3.32.4 cm and 3.02.6 cm . 
 when she was at the age of 3 years , vaginoplasty and clitoroplasty were performed . at the age of 11 
 abdominal ct performed at the age of 11 years showed a 3.54.0 cm mass in her right adrenal gland ( fig . 
 hydrocortisone and fludrocortisone administration was carried out during follow - up and vaginoplasty and clitoroplasty were performed at the age of 4 years . 
 when she was 11 years old and 12 years old , abdominal cts were performed due to her virilization including hirsuitism and masculine voice . 
 a male neonate was admitted to medical attention at 1 month of age with poor oral intake and diarrhea . 
 he was admitted to kyunghee medical center at 5 months of age with poor oral intake and vomiting . 
 laboratory evaluation revealed a ph level of 7.35 , hco3 14 meq / l , base excess -9 meq / l , and na 119 meq / l . during follow - up , he was diagnosed with cah . 
 hydrocortisone and fludrocortisone medication was initiated . after his medication had begun , the level of na was normalized . during follow - up , 
 medication was stopped on his parents ' request and since the age of six , he showed secondary growth such as hyperpigmentation , hirsuitism , large penis , and pubic hair . 
 abdominal ct performed at the age of 14 years revealed the presence of adrenal masses ( left adrenal gland : 2.01.4 cm & 1.21.3 cm , right adrenal gland : 1.11.2 cm ) . since there was no change in the size of adrenal mass , adrenalectomy was not performed . 
 cah describes a group of autosomal recessive condition in which deletions or mutations of the cytochrome p450 21-hydroxylase gene result in glucocorticoid and/or mineralocorticoid deficiency6 ) . in 95% of cases , 
 epidemiology , genetics , pathophysiology , clinical features , and the management of cah will be reviewed as follows . 
 the overall incidence of classical 21-ohd is 1:10,000 - 1:15,000 in most reported populations8 ) . 
 the 21-hydroxylase gene cyp21a2 is located on chromosome 6p21.3 within the hla histocompatibility complex9 ) . 
 two highly homologous 21-hydroxylase genes exist ; cyp21a2 and cyp21a1p , of which cyp21a2 codes for the active 21-hydroxlyase and cyp21a1p is an inactive pseudogene . 
 more than 90% of mutations of the active cyp21a2 gene are generated by recombinations between the active and the inactive pseudogene10 ) . 
 the pathophysiology of cah due to 21-ohd is closely linked to the degree of enzyme deficiency6 ) . 
 the biosynthesis of cortisol is regulated by negative feedback on hypothalamic corticotropin - releasing hormone ( crh ) and pituitary adrenoconticotrophic hormone ( acth ) secretion . 
 a defect in cortisol biosynthesis leads to a compensatory increase in hypothalamic and pituitary secretion of crh and acth . 
 this stimulates the adrenal cortex and results in the pathological correlative of hyperplastic adrenal glands . 
 the consequence of 21-ohd is a state of hypocortisolism , hypoaldosteronism , and hyperandrogenism combined with catecholamine deficiency and several metabolic disturbances . 
 endogenous glucocorticoids are essential for the development and continuing regulation of the adrenal medulla , synthesizing epinephrine and norepinephrine . without cortisol , 
 the organogenesis of the adrenal medulla is severely disturbed and the consequence is catecholamine deficiency , mainly comprising a lack of epinephrine11 ) . 
 cah is classified as classic , the severe form , or non - classic , the mild or late - onset form . 
 classic cah is subclassified as the salt - losing or non - salt - losing ( simple virilizing ) . 
 non - classic cah is subclassified as the late - onset form or cryptic form . 
 salt - losing cah is a severe form with a concurrent defect in aldosterone biosynthesis . 
 non - classic cah may be asymptomatic or may be associated with signs of androgen excess developing during childhood or at puberty . 
 the classic forms are characterized by a congenital decrease of cortisol and aldosterone secretion and an increase in androgen biosynthesis . 
 all female infants with classic cah due to 21-ohd typically have ambiguous genitalia at birth because of exposure to high concentrations of androgens in the uterus . 
 the internal genitalia are normal , including uterus , fallopian tubes , and ovaries ; wolffian duct structures are not present . 
 boys with classic cah have no signs of cah at birth , except subtle hyperpigmentation and possible penile enlargement . 
 thus , the age at diagnosis in boys varies according to the severity of aldosterone deficiency . 
 up to 2/3 of patients suffering from classic 21-ohd have insufficient aldosterone biosynthesis leading to salt - losing episodes after birth and later on12 ) . 
 boys with the salt - losing form typically present at 7 - 14 days of life with vomiting , weight loss , lethargy , dehydration , hyponatraemia , hyperkalaemia , and can present shock . 
 patients with non - classic cah do not have cortisol deficiency , but instead have manifestations of hyperandrogenism , generally later in childhood or in early adulthood13 , 14 ) . 
 manifestations include premature pubarche , tall stature , advanced bone age , menstrual irregularities , infertility , hirsutism and acne . 
 cah due to 21-ohd can be diagnosed in newborn screening programs by screening for 17-hydroxyprogesterone in dried blood spots . today , most cases of classical 21-ohd are diagnosed with this tool . 
 a high concentration of 17-hydroxyprogesterone in a randomly timed blood sample is diagnostic of classic 21-ohd . 
 the gold standard for diagnosis of cah is a corticotropin stimulation test , with measurement of 17-hydroxyprogesterone at 60 min . a stimulated concentration of 17-hydroxyprogesterone higher than 45 nmol / l is diagnostic of 21-ohd . 
 the aim of medical treatment in 21-ohd is to suppress adrenal androgens sufficiently without impairing growth , while allowing for normal pubertal development and fertility . in classic cah 
 , glucocorticoids are given in doses sufficient to suppress excessive secretion of crh and acth without total suppression of the hypothalamic - pituitary - adrenal . 
 physiological cortisol secretion rates are about 6 - 8 mg / m / day15 - 17 ) . to achieve these goals , the glucocorticoid doses have to exceed the physiological cortisol secretion rate , using doses of 12 - 18 mg / m 
 fludrocortisone was administered in cases of salt - losing manifestation and elevated plasma renin activity , to decrease glucocorticoid doses , or according to the genotype19 ) . 
 the use of fludrocortisone in patients with non - salt - losing classic cah allows management with lower doses of glucocorticoid and improves linear growth18 - 20 ) . 
 infants with the salt - losing form of 21-ohd require mineralocorticoid ( fludrocortisone , usually 0.1 - 0.2 mg ) and sodium chloride supplements ( 1 - 2 g daily ; 1 g nacl contains 17 meq of sodium ) in addition to glucocorticoid treatment . 
 treatment is indicated only for those with symptoms and aims to reduce hyperandrogenism18 - 20 ) . 
 cah constitutes a continuum of disorders that affect patients throughout their lives ( table 2 ) . 
 a variety of clinics involving close interaction between paediatric , reproductive , adult endocrinologists , and clinical psychologists experienced in psychosexual counselling will be necessary for patients with cah21 - 24 ) . 
 the development of adrenal adenoma or carcinoma in patients with cah is rare ; the etiology is not clear . 
 however , the incidence of adrenal masses appears to be higher in cah patients and in heterozygotes than in the general population3 ) . 
 recent reports have revealed a high prevalence of small adrenal tumors in patients with untreated 21-ohd cah3 , 4 ) . 
 histological types of adrenal tumor include adenoma , myelolipoma , and hemangioma25 ) . rarely , virilizing adrenal carcinoma has been found in patients with cah , but most adrenal masses in children with cah are benign5 ) . 
 the pathogenesis of adrenal tumor in patients with 21-ohd cah is thought to be related to a consequence of acth hypersecretion , which results from the lack of glucocorticoid synthesis26 ) . 
 avoidance of high steroid dose is likely to result in insufficient suppression of corticotropin stimulation27 ) . 
 chronic poor compliance to therapy appears to be associated with the development of the tumor . 
 therefore patients with 21-ohd cah should maintain steroid medication to avoid the appearance of adrenal tumor . 
 follow - up imaging may help to discover the appearance of adrenal tumor in patients with long - term management of 21-ohd cah . 
 there has been much improvement during the past 50 years in the understanding of cah , and the management with cah continues to improve . furthermore new medical strategies that offer the prospect of an improved outcome continue to evolve . 
 however , patients with 21-ohd cah should maintain steroid medication to avoid the appearance of adrenal tumor , to improve its symptoms , and to normalize electrolyte disorder , particularly hyponatremia .","congenital adrenal hyperplasia ( cah ) caused by 21-hydroxylase deficiency is an autosomal recessive disease , which leads to cortisol and aldosterone deficiency and hyperandrogenism . 
 typical medical treatment includes oral glucocorticoid and mineralocorticoid administration to suppress adrenal androgens and to compensate for adrenal steroid deficiencies . 
 however , some patients stopped taking medicine without the doctor 's consent . among these patients , 
 four cases of cah patients showing the presence of hyponatremia as an initial electrolyte disorder were found with adrenal adenoma . 
 hypersecretion of adrenocorticotrophic hormone and chronic poor compliance to therapy appears to be associated with the development of the adrenal tumor . 
 two cases were managed with adrenalectomy because of increasing adrenal tumor size and virilization . whereas the other two cases did not increase in size and were observed without adrenalectomy . 
 therefore , it is important that patients with cah maintain steroid medication to avoid the appearance of adrenal tumor .",pubmed
"self - evaluation of general health status has been associated with actual health in that what people report about their health has been shown to predict mortality . 
 self - rated health ( srh ) as a single survey question developed by the world health organization ( who )   has been validated as a tool to predict mortality in populations with and without cardiovascular diseases [ 13 ] and functional ability [ 4 , 5 ] . asking participants to describe their overall health on a five - point scale ( ranging from excellent to poor ) 
 has achieved popularity as a health - indicating tool in the united states ( us ) and other countries [ 6 , 7 ] . 
 worldwide , noncommunicable diseases ( cancer , cardiovascular diseases , diabetes , and chronic lung diseases ) are largely attributed to four main behavioral factors : tobacco use , unhealthy diet , insufficient physical activity , and harmful alcohol use . 
 poor health for persons with chronic diseases has been attributed largely to a lack of adherence to medical recommendations which include diet and physical activity . 
 high consumption of fruits and vegetables resulted in a lower incidence of cardiovascular disease in nurses and health professionals in a 14-year follow - up study . 
 reporting poor health may be due , in part , to poor management of chronic disease . 
 symptoms of chronic diseases attributed to poor srh among a swedish population of middle - aged and older adults were tiredness / weakness , depression , and musculoskeletal pains . 
 lifestyle behaviors , particularly diet and physical activity , can contribute to or help prevent cardiovascular diseases such as coronary heart disease , hypertension , and type 2 diabetes . 
 diet and physical activity can have positive effects for persons with preexisting coronary heart disease , as well . 
 reduction in chronic disease burden and increase in quality of life have been well documented with higher daily consumption of fruits and vegetables [ 13 , 14 ] and physical activity . 
 the relative risk of cardiovascular disease events for persons with type 2 diabetes decreased with moderate to vigorous physical activity in 14-year follow - up studies for men   and women   even after adjusting for sociodemographic factors . 
 moreover , srh , as an indicator of health and wellbeing , has been positively associated with fruits and vegetables intake [ 18 , 19 ] . eating at fast - food restaurants   and high - fat diets   were associated with poor / fair srh in african - americans and australian women , respectively . 
 following dietary and physical activity recommendations has been associated with the prevention and lessening of the severity of cardiovascular diseases . 
 dietary recommendations include half your plate being comprised of fruits and vegetables and cutting back on foods high in solid fats . 
 physical activity guidelines specify being physically active most days of the week to attain 150 minutes of moderate or 75 minutes of vigorous physical activity per week 
 . lack of physical activity and unhealthy dietary patterns place a burden on society due to lack of work productivity and increased health care costs . physical health components from the short form 12 ( sf-12 ) 
 were associated with increased physical activity and healthier diet from baseline to five - year follow - up of a european cohort from the inter99 study . 
 several studies have found an association with lifestyle behaviors and self - evaluation of health . in particular , regular physical activity is reported to be significantly associated with srh in various populations [ 2326 ] . 
 adults from the us with and without cardiovascular diseases who reported engaging in healthy dietary and physical activity behaviors had a higher odds of reporting optimal srh ( excellent , very good , or good ) as compared to fair or poor . 
 physical activity , besides lowering the risk for the development of chronic diseases , also improves the health outcomes of such disease conditions . 
 therefore , the objective of this study was to examine the relationship between dietary and physical activity behaviors and srh . 
 it was hypothesized that participants who reported lower fruits and vegetables intake and higher fatty - food intake and perform less physical activity will have srh poor / fair as compared to those in the good , very good , or excellent category , adjusting for covariates . 
 this is a secondary data analysis of the living for health program ( florida heart research institute , miami , florida , us ) . 
 the living for health program was conducted from 2008 to 2012 and collected information on health behaviors from adults 18 years old ( n = 9,453 ) . during these years , 190 health fair events were held in low income minority communities in miami - dade county to screen participants for cardiovascular disease risks . 
 an accredited institutional review board ( irb ) provided a waiver of consent for the analyses . 
 self - rated health assessment was added in the year 2012 and was answered by 2,108 individuals during the remainder of the living for health program . the final sample size ( n = 1,701 ) consisted of the participants who responded to all the questions included in this analysis . 
 self - rated health was measured by the following question :  how would you rate your current health state ?  there were five possible responses : excellent , very good , good , fair , and poor . 
 the responses were categorized into two as follows : ( 1 ) excellent / very good / good or ( 2 ) fair / poor for this study . 
 the sociodemographic questionnaire collected information on age , gender , ethnicity , smoking , health insurance , and comorbidities . 
 age was converted to a categorical variable ( 1830 , 3155 , and > 55 years old ) to assess differences between older and younger adults based on the demographics of the population . 
 smoking status derived from the question  do you smoke ( any tobacco product ) ?  and was categorized as ( yes / no ) . 
 there were insufficient participants to access differences for the original five possible categories for ethnicity . based on the distribution , ethnicity 
 was collapsed into three categories : hispanic , non - hispanic black , and other . 
 other category included white , american indian , pacific islander , indian , and asian . 
 health insurance was categorized as a binary variable ( no insurance / yes insurance ) . 
 presence of comorbidities ( yes / no ) was measured combining two questions : ( 1 )  has any medical professional ever told you that you have high glucose , high blood pressure , high cholesterol , coronary heart disease )  and ( 2 )  have you been prescribed medications for ( diabetes , hypertension , cholesterol , coronary heart disease ) ?  a positive response to either question was categorized as having comorbidities . 
 participant 's height and weight were measured and body mass index ( bmi ) was calculated as weight ( kg)/height ( m ) . 
 bmi was classified as bmi : 18.529.9  m ( nonobese ) and bmi : 30 ( obese ) . 
 there were < 1.0% of participants classified as underweight ( bmi < 18.5 ) ; these participants were included in as part of the  nonobese  group . 
 a single question was used to measure fruits and vegetables intake :  on a typical day in the past month , how many servings of fruits and vegetables did you eat ?  examples of what a serving represents were provided with the question ( i.e. , 1 cup fresh , 1 medium size fruit , etc . ) . 
 the five possible options were 5 or more per day , 4 a day , 3 a day , 2 a day , or 1 a day . 
 the responses were categorized into three as follows : ( 1 ) 5 or more per day ; ( 2 ) 3 - 4 per day ; ( 3 ) 2 or less per day . 
 fat intake was measured by the question :  if you ate fast food in the last month , what type was it ?  examples of high fat foods ( fried food , breaded items , taco salads , nachos , double burgers , pizza , hot dogs , croissant items , donuts , shakes , and cakes ) and low fat foods ( salads ( no creamy dressings ) , single burgers , grilled chicken , fruits , and yogurt parfaits ) were provided . from the five possible options , 
 the responses were categorized into three as follows : ( 1 ) primarily low fat foods , mostly low fat or some high fat ; ( 2 ) both high fat and low fat foods about the same ; ( 3 ) mostly high fat , some low fat or primarily high fat foods . 
 physical activity was measured with the question :  in the last month , how often were you physically active ?  ( performance of at least 30 minutes of fitness walking , cycling , jogging , swimming , aerobic dance , or active sports was considered physical activity ) . 
 physical activity was collapsed from six possible options into three categories as follows : ( 1 ) 57 days a week ; ( 2 ) 14 days a week ; ( 3 ) 13 times a month or never to allow sufficient number in each category . 
 differences between male and female participants were assessed with the chi - square test for categorical variables . 
 logistic regression models were conducted to examine the relationship between the binary dependent variable , srh with fruits and vegetables intake , physical activity , and fat intake adjusting for confounding variables . 
 age and gender differences were found in perceptions of srh [ 11 , 29 ] . 
 poorer physical srh was also associated with lack of health insurance , minority status , and less education for a cohort of randomly selected kentucky adults . 
 comorbidities and obesity are known factors of poorer health . since gender interacted with srh and with physical activity 
 ( fruit and vegetable intake , fat intake , and physical activity ) a reduced model ( unadjusted ) and full model ( adjusted ) including covariates ( age , bmi , smoking status , ethnicity , health insurance , and comorbidities ) were conducted . 
 all statistical analyses were performed using ibm statistical package for the social science version 19 ( spss inc . , chicago il , us ) . 
 females as compared to males had a higher percent classified as obese ( 35.4% versus 27.0% , p = 0.001 ) , reported poor / fair srh ( 23.4% versus 15.0% , p 
 < 0.001 ) , and were less physically active ( 33.9% versus 25.4% , p = 0.001 ) . 
 participants who reported poor / fair srh as compared to those with excellent / very good / good were females ( 78.6% versus 67.9% , p < 0.001 ) ; older > 55 years ( 35.5% versus 29.4% , p = 0.024 ) ; obese ( 46.8% versus 29.2% , p < 0.001 ) ; non - hispanic black ( 38.0% versus 29.4% , p = 0.001 ) ; had comorbidities ( 61.4% versus 40.5% , p 
 < 0.001 ) ; consumed 2 servings of fruits and vegetables per day ( 73.0% versus 59.3% , p < 0.001 ) ; had an intake of primary high fat foods / mostly high fat or some low fat foods ( 17.7% versus 10.8% , p < 0.001 ) ; and were less physically active ( 40.3% versus 29.0% , p < 0.001 ) ( table 2 ) . 
 unadjusted logistic regression models for females showed that those who consumed 2 servings of fruits and vegetables per day were 2.2 times more likely to report poor / fair srh as compared to those who consumed 5 servings per day ( p = 0.001 , 95% ci 1.393.70 ) . 
 logistic regression model adjusted for age , bmi , smoking , ethnicity , health insurance , and comorbidities showed that females who consumed 2 servings of fruits and vegetables per day were 2.1 times more likely to report poor / fair srh as compared to those who consumed 5 servings per day ( p = 0.003 , 95% ci 1.233.54 ) . 
 there was a more modest and nonstatistically significant increase in the odds of poor / fair srh for men with the lowest level of daily fruits and vegetables consumption . 
 unadjusted and adjusted models indicated that males who consumed 3 - 4 servings of fruits and vegetables per day were 2.6 and 2.8 times more likely to report poor / fair srh as compared to those who consumed 5 servings per day ( p = 0.031 , 95% ci 1.096.58 and p = 0.028 , 95% ci 1.127.35 , resp . ) . 
 unadjusted logistic regression models of fat intake for females indicated that those who consumed mostly high fat foods were 1.6 times more likely to report poor / fair srh as compared to those who consumed primarily low fat foods ( p = 0.015 , 95% ci 1.1032.49 ) . 
 females who consumed both high and low fat foods were 1.3 times more likely to report poor / fair srh as compared to those who consumed primarily low fat foods ( p = 0.041 , 95% ci 1.0131.83 ) . 
 logistic regression models adjusted for age , bmi , smoking , ethnicity , health insurance , and comorbidities showed that females who consumed mostly high fat foods were 1.5 times more likely to report poor / fair srh as compared to those who had primarily low fat foods ( p = 0.036 , 95% ci 1.032.43 ) . 
 similarly , females who consumed both high and low fat foods about the same were 1.4 times more likely to report poor / fair srh as compared to those who had primarily low fat foods ( p = 0.018 , 95% ci 1.072.01 ) . 
 unadjusted logistic regression model for males indicated that those who consumed mostly high fat foods were 3.1 times more likely to report poor / fair srh as compared to those who consumed primarily low fat foods ( p < 0.001 , 95% ci 1.705.97 ) . 
 the adjusted model showed that males who consumed mostly high fat foods were 3.2 times more likely to report poor / fair srh health as compared to those who consumed primarily low fat foods ( p = 0.001 , 95% ci 1.672.43 ) . 
 unadjusted logistic regression model for females showed that those who performed 13 times a month or never of physical activity were 1.6 times more likely to report poor / fair srh as compared to those who performed 5 days per week of physical activity ( p = 0.003 , 95% ci 1.182.29 ) . logistic regression model adjusted for age , bmi , smoking , ethnicity , health insurance , and 
 comorbidities showed that females who reported 13 times a month or never of physical activity were 1.6 times more likely to report poor / fair srh as compared to females who performed 5 days per week of physical activity ( p = 0.004 , 95% ci 1.172.35 ) . 
 unadjusted logistic regression model showed that males who performed 13 times a month or never of physical activity were 1.8 times more likely to report poor / fair srh as compared to those who performed 5 days per week of physical activity ( p = 0.043 , 95% ci 1.023.40 ) . 
 the association of physical activity and srh was no longer significant for males once adjustment variables were added ( p = 0.218 , or = 1.50 , 95% ci 0.792.49 ) ( table 4 ) . 
 we found higher odds of poor / fair srh for males and females who consumed the lowest level of fruits and vegetables . 
 no gender differences were found in a longitudinal cohort of canadian young adults who were more likely to report excellent srh if they had a high intake of fruits and vegetables as adolescents . 
 similarly , dietary intake high in fiber was associated with more favorable srh as compared to lower fiber for a cohort of african - american adults , independent of gender . 
 the presence of any chronic disease influences one 's perception of health and wellbeing [ 3335 ] . 
 persons with diabetes may be more likely to consume more fruits and vegetables after their diagnosis . 
 even though diagnosis with chronic disease could result in better health behavior , consuming fruits and vegetables at least five times per day was associated with optimal srh for both individuals with cardiovascular diseases and diabetes as well as for persons without these chronic illnesses . 
 our sample population , who are approximately more than half - hispanic , may have a large proportion of first generation immigrants from caribbean and latin american countries . 
 the perception of good health may differ based on country of origin . for example , foreign - born haitian americans had higher levels of perceived stress as compared to african - americans , but african - americans were more likely to rate their health as poor / fair compared to haitian americans . 
 we found that both males and females , who consumed mostly high fat foods , were more likely to report poor / fair srh as compared to those consuming primarily low fat foods . 
 consistent with our results , collins et al .   reported higher intake of fat and saturated fat to be associated with poorer srh for a large cohort of australian women . 
 frequency of eating at fast - food restaurants was positively associated with poor srh for a cohort of african - americans . 
 our results contradicted barreto and de figueiredo   who reported lower odds of consuming fatty meat and whole milk for those diagnosed with at least one chronic disease and even lower for those diagnosed with two or more chronic diseases compared to absence of disease in a spanish population . 
 they suggested that medical advice to modify lifestyle behavior may be a probable confounder of diet and health when comparing persons with and without chronic disease . 
 similarly , the odds of having poor health increased twofold in a cohort of greek nurses making an effort to avoid fatty foods in their diet . 
 a strong association between physical activity and srh for females was observed in our study . 
 females who engaged in less physical activity were more likely to report poor / fair srh as compared to those who were more physically active . 
 several studies found physical activity to be related to srh regardless of gender for a national sample , for a cohort of older adults , and for african - american adults with a high proportion of chronic diseases . 
 our findings on gender differences in physical activity levels and srh are in agreement with several other studies [ 3841 ] . 
 as expected , the likelihood of rating health as poor / fair was substantially higher for women , as compared to men . 
 considering the impact of physical activity on health status , variation of the effect of physical activity according to gender could also be associated with different proportions of srh . 
 these distinctions could be explained by the biological and sociocultural environment inequalities between men and women . 
 for instance , it was found that gender roles and reduced access to resources and social conditions , such as safe environment , do not foster regular physical activity among women [ 40 , 41 ] . while women generally tend to rate their health worse in health and behavioral studies , this is consistently in line with lack of physical activity . of utmost relevance of the role of physical activity in health 
 is the gradient effect of the levels of physical activity on srh , as demonstrated by evidence from several past studies [ 4244 ] . 
 participants who were highly active and very highly active were twice as likely to have excellent srh as compared to those who were less physically active . 
 the main objective of our study was to assess the relationship of indicators of modifiable cardiovascular disease risk factors as follows : dietary and physical activity lifestyle and their relationship to srh . 
 we examined srh in a half - hispanic population with non - hispanics who were predominately black and we found significant differences in ethnicity for srh . 
 however , it is noteworthy to mention that ethnic differences were adjusted in the final models . 
 a cohort of older hispanics rated their health poorer than non - hispanic whites yet had a lower mortality rate after a 16-year follow - up , based on data from the health and retirement study and adjusting for demographics , socioeconomic status , health status , and health behaviors at baseline . 
 furthermore , level of acculturation in hispanic americans was associated with srh whereby srh of the more acculturated matched native non - hispanic whites and blacks . 
 self - rated health differences were found within african - americans over time that were based on differences in age , education , smoking , and morbidity ( angina , congestive heart failure , diabetes , and kidney disease ) , having been hospitalized in the year prior to baseline , depressive symptoms , mobility limitations , and initial self - rated health . 
 this was a large sample of adults from miami dade county , florida , primarily minorities , half of whom were from a diverse hispanic population . 
 to our knowledge , this is the first study to investigate the relationship between lifestyle behaviors and srh for a population largely comprised of hispanics with a considerable proportion of blacks . nevertheless , there are some limitations . 
 causality of lifestyle behaviors with srh could not be established due to the single time point . 
 other factors that may have influenced srh such as socioeconomic status , neighborhood , housing situation , and psychosocial factors were not collected in this study . 
 modifiable lifestyle behaviors known to reduce cardiovascular disease risk , low fruits and vegetables intake and high fat intake , were associated with poor / fair srh in males and females . 
 poorer srh was significantly associated with low physical activity in females only , and low physical activity was also more common in women than men in this study sample . 
 these results indicate that gender differences may have implications in designing and monitoring lifestyle interventions to prevent cardiovascular diseases .","background . lack of adherence to dietary and physical activity guidelines has been linked to an increase in chronic diseases in the united states ( us ) . 
 the aim of this study was to assess the association of lifestyle behaviors with self - rated health ( srh ) . 
 methods . 
 this cross - sectional study used self - reported data from living for health program ( n = 1,701 ) which was conducted from 2008 to 2012 in 190 health fair events in south florida , us . 
 results . 
 significantly higher percent of females as compared to males were classified as obese ( 35.4% versus 27.0% ) , reported poor / fair srh ( 23.4% versus 15.0% ) , and were less physically active ( 33.9% versus 25.4% ) . 
 adjusted logistic regression models indicated that both females and males were more likely to report poor / fair srh if they consumed 2 servings of fruits and vegetables per day ( or = 2.14 , 95% ci 1.303.54 ; or = 2.86 , 95% ci 1.127.35 , resp . ) and consumed mostly high fat foods ( or = 1.58 , 95% ci 1.032.43 ; or = 3.37 , 95% ci 1.672.43 , resp . ) . 
 the association of srh with less physical activity was only significant in females ( or = 1.66 , 95% ci 1.172.35 ) . 
 conclusion . 
 gender differences in health behaviors should be considered in designing and monitoring lifestyle interventions to prevent cardiovascular diseases .",pubmed
"the role of endoscopic injection of dextranomer / hyaluronic acid copolymer ( dx / ha ) has expanded , as an alternative to open surgery , to a treatment option for observation protocols using prophylactic antibiotics . 
 deflux has been fda approved since 2001 as a treatment option for grade 24 vesicoureteral reflux ( vur ) in single systems with no accompanying urological malformations in children above one year of age . 
 the operative ease of technique , short procedure , and recovery time as well as repeatability of injections have popularized the technique with surgeons and parents alike . 
 reports of expanded use to patients of all ages and grades of vur even in the presence of duplication , ureterocele , or other lower tract anomalies are published worldwide [ 1 , 2 ] .   in a statement of the aua board of directors from october 2007 
 , it was determined that :  it is the current position of the american urological association that endoscopic injection of the dextranomer / hyaluronic compound deflux is an option in the management of pediatric vesicoureteral reflux ( vur )  [ 3 , 4 ] . 
 the initial success rate was quoted as 68%   and is still mentioned on the current website   of the manufacturing company ( q - med , scandinavia inc ) . in the meantime , the original subureteric injection technique ( sting ) has evolved to the intraureteric injection technique introduced by the atlanta group . using this hydrodistention - implantation technique ( hit ) , the authors reported success rates of 89% . 
 however , the reported worldwide success rates differ considerably ( 55%89% [ 7 , 8 ] ) . unpublished data from our institution show an overall radiographic success rate of 75% for patients and 84% for renal units after the first injection . 
 although the success rates continuously improve , the numbers of most institutions still differ significantly from the traditional 98% for open reimplants . since up to 25% of patients will have remaining vur after dx / ha injection , we still perform voiding cystograms 2 months after surgery . 
 positional instillation of contrast ( pic ) cystography was developed to detect occult vur in patients with recurrent urinary tract infections ( uti ) after a negative standard voiding cystogram . 
 it has a higher sensitivity than standard voiding cystogram ( vcug ) and can detect occult vur . 
 parents can be advised that in case of a positive pic , dx / ha injection can be performed during the same anesthesia . in the current study , we compared two different pic techniques and evaluated two different scenarios : first , whether pic cystography or high - pressure cystograms were useful to detect de novo contralateral vur in the case of preoperative unilateral vur and second , whether pic cystography , when performed intraoperatively immediately after dx / ha injection , can predict a successful surgical outcome by correlating the results with a standard 2-month postprocedure vcug . 
 a retrospective chart review was performed on all intraoperative cystograms performed at the time of dx / ha injections at our institution from 20052007 . 
 dx / ha injections were performed for all grades of vur and included complex cases such as duplicated systems . in case of preoperative unilateral reflux , a pic cystogram was performed on the contralateral unaffected side before dx / ha injection . if positive , bilateral injection took place . 
 the injections were done using the hit technique as described by kirsch et al . . 
 dx / ha was injected until a sufficient mound and good ureteral coaptation was observed . at this point 
 the outflow portion of the 10 french off set cystoscope ( wolf , germany ) was placed at the bladder neck filling the bladder with contrast material ( cystografin ) to 100  cm water pressure until voiding occurred around the cystoscope ensuring complete bladder filling . 
 the second group underwent standard pic cystography   with the outflow portion of the cystoscope directed at the ureteral orifice at a 100  cm water pressure for 5 seconds ( figure 2 ) . 
 radiographic views for both tests were performed in anterior - posterior and lateral as well as upper tract views . 
 the intraoperative cystogram was noted to be positive if any grade of vur was present ( figure 3 ) . in cases of a positive study on the contralateral side or for remaining vur after injection , 
 the patients were left on prophylactic antibiotics and followed up at 4 weeks postoperatively with an ultrasound and 2 months postoperatively with a vcug . 
 the correlations of the intra- and postoperative study as well as the detection of de novo vur were noted . 
 altogether 116 patients and 177 renal units ( ru ) were treated and available for evaluation . out of the 116 patients , 
 new contralateral vur ( de novo ) for the 34 preoperative unilateral refluxing patients was found intraoperatively in 27 ru ( figure 4(c ) ) . 
 the high - pressure intraoperative cystogram ( group 1 ) was performed in 86 rus and the standard pic ( group 2 ) in 91 rus . 
 there was no statistically significant difference between groups 1 and 2 for the detection of de novo vur ( figure 4(b ) ) .   for both groups , 
 only 5 ru were found to have remaining vur intraoperatively after dx / ha injection ( 2 in group 1 and 3 in group 2 ) . 
 postoperative pain management , if necessary , consisted of ibuprofen or acetaminophen according to parental preference . in all patients , 
 the postoperative us at four weeks were negative for hydronephrosis or any other new abnormality . 
 the dx / ha deposits were seen in the bladder in all cases . at 2 months 
 postoperative standard vcug detected 20 ru ( 23% ) to be positive for vur in group 1 and 16 ru ( 18% ) positive for vur in group 2 . 
 altogether 36 ru were positive , for an allover success rate of 80% for ru and 75% for patients ( figure 4(a ) ) . 
 the grade of vur , age and gender of patient as well as amount of deflux injected did not influence the result . 
 vesicoureteral reflux remains a common disease , and especially for lower grades the prospect of spontaneous resolution is good . 
 but , the data on the value of daily prophylactic antibiotics remain controversial , and most parents are concerned about their long - term use . 
 the discussion becomes evident by the current multi - institutional nih - funded rivur study , which aims to evaluate the role of prophylactic antibiotic in vur . 
 in addition to prolonged antibiotic use , parents and patients alike dread the yearly necessary voiding cystogram to check for resolution . 
 therefore , to avoid the daily antibiotics and the yearly vcug , many parents consider surgical treatment options . 
 the endoscopic injection of dx / ha is an attractive management alternative for parents . when presenting the surgical options , all parents are enthusiastic about the minimal invasive nature of the procedure with low complication possibilities and fast recovery times . 
 however , the 20%25% lower success rate of the endoscopic procedure in comparison to the open surgery is a concern . 
 although dx / ha can be reinjected , it requires further testing , anesthesia , and time spent away from school or work . 
 therefore , it would be highly desirable to develop a method to check the success of the deflux injection intraoperatively . 
 pic cystography is used to detect occult vur in patients with a negative standard voiding study who suffer from recurrent utis . according to rubenstein 
 , the procedure is a 100% sensitive and 91% specific in identifying vur and can , therefore , detect occult vur . 
 recently confirmed the findings in 5 patients . in a study on 39 patients with febrile utis and negative voiding studies from 4 different institutions , 
 pic cystograms were performed and vur identified in 82% of these patients . additionally , the authors noted that orifices that were patulous , laterally displaced , or positive for hydrodistention were much more likely to be positive for vur than normal orifices . 
 also performed intraoperative pic cystograms and found that 23% of patients had a positive , postoperative vcug despite a negative intraoperative pic study , and palmer confirmed these results . 
 the authors concluded that pic cystography was not useful to raise the success rate of deflux injection .   in the current study , we used two different cystogram techniques : the standard pic study and a high - pressure cystogram , filling the bladder until irrigation fluid leaked around the cystoscope . 
 even with this aggressive cystogram technique only an 80% correlation between a negative intraoperative cystogram and postoperative cystogram was achieved . 
 the success rate of 75% for patients and 80% for ru could not be improved . 
 a relative wound edema might increase the coaptation of the ureteral orifice , temporarily giving a negative intraoperative study result . 
 other possibilities include decrease in the size of the injected dx / ha deposit over time , also thought to be responsible for long - term failure after dx / ha injection . 
 this phenomenon has been noted during cystoscopy at the time of the planned second injection of deflux after initial failure . 
 the dx / ha deposits tend to migrate medially and toward the bladder neck causing long - term failure of injection . 
 elmore et al . reported the onset of new contralateral vur after dx / ha implantation in approximately 13% of 126 patients with preoperatively unilateral vur . in the current study , 
 twenty seven of these 34 patients demonstrated a positive pic cystography intraoperatively and were successfully injected with dx / ha . 
 none of these de novo detected refluxing units were positive for vur in the postoperative cystograms . 
 a negative intraoperative cystogram correlates with the postoperative vcug in only 80% . considering that the success rates of dx / ha injections are commonly between 7580% 
 , the addition of intraoperative cystograms can not replace postoperative studies . in patients treated with unilateral vur 
 , pic cystography can detect occult reflux and prevent postoperative contralateral new onset of vur .","the endoscopic injection of dx / ha in the management of vesicoureteral reflux ( vur ) has become an accepted alternative to open surgery . in the current study we evaluated the value of cystography to detect de novo contralateral vur in unilateral cases of vur at the time of dx / ha injection and correlated the findings of immediate post - dx / ha injection cystography during the same anesthesia to 2-month postoperative vcug to evaluate its ability to predict successful surgical outcomes . the current study aimed to evaluate whether an intraoperatively performed cystogram could replace postoperative studies . but 
 a negative intraoperative cystogram correlates with the postoperative study in only 80% . considering the 7580% success rate of dx / ha implantation 
 , the addition of intraoperative cystograms can not replace postoperative studies . in patients treated with unilateral vur 
 , pic cystography can detect occult vur and prevent postoperative contralateral new onset of vur .",pubmed
"the accuracy to detect fetal pathology depends on the expertise of the sonographer and some limitations inherent to this imaging technique . 
 descent of the fetal head and progressive ossification of the skull bones towards the end of the pregnancy , maternal obesity and oligohydramnios are all well - known limitations affecting the sensitivity to detect central nervous system pathology . 
 the first reported use of fetal magnetic resonance imaging  ( mri ) dates from 1989 . 
 advances in mri technology during the last 2 decades and better knowledge of fetal pathology , have both led to an increasing role for mri in the diagnostic work - up of prenatal anomalies . 
 most fetal mri studies reported so far , have addressed the additional role of mri in the diagnosis and characterization of fetal anomalies detected on ultrasound , with emphasis on central nervous system pathology . in this manuscript 
 we discuss the role of mri as an adjunct to ultrasound for cases diagnosed with ventriculomegaly . 
 the descriptive term cerebral ventriculomegaly is used when the maximal diameter of the atrium of the lateral ventricles  measured in a plane perpendicular to the midsagittal section of the brain  exceeds 10 mm . 
 the prevalence of ventriculomegaly ranges between 0.3 - 1.5 and the severity can be classified in mild ( 10 - 12  mm ) , moderate ( 12 - 15  mm ) and severe ( >  15  mm ) , as illustrated in fig . 1 ( valsky et al . , 2004 ; 
 another classification system is based on measuring the thickness of the overlying brain parenchyma in the category with an atrial diameter of >  15  mm , i.e. >  3  mm thickness indicates a moderate ventriculomegaly and   3  mm a severe form ( levine et al . , 2003 ) . 
 measurement of the lateral ventricle is subject to errors owing to an off - axis image plane of a section , an angled measurement , or improper choice of ventricular boundary giving risk to false - positive test results . 
 therefore , a precise definition of the reference planes and anatomical landmarks is important to avoid inaccurate measurements and facilitate imaging follow - up comparisons ( levine et al . , 2008 ) . 
 ventriculomegaly has a wide range of causes and can roughly be divided in three categories : ( 1 ) an imbalance between the production and absorption of cerebrospinal fluid , of which the obstructive form is most frequently observed , ( 2 )  abnormal cerebral development such as partial or complete agenesis of the corpus callosum and neuronal proliferation / migration disorders and ( 3 )  destructive disease processes leading to loss of neuronal tissue by vascular insults or infectious pathogens . 
 unilateral ventriculomegaly is more often seen in destructive processes , whereas developmental anomalies are more characterized by bilateral broadening of the ventricles ( girard et al . , 2003 ) . 
 the reported incidence of additional anomalies goes up to 70 - 85% , strongly depending on the number of fetuses included in the study and the distribution of the severity of the cerebral ventriculomegaly observed in the study population ( huisman et al . , 2002 ; 
 the term isolated ventriculomegaly is used in case no other structural anomalies are seen at the time of diagnosis . 
 isolated and/or unilateral ventriculomegaly , in particular the mild form , has a lower incidence of peri- and postnatal morbidity and mortality ( senat et al . , 1999 
 fetal mri is mainly performed on 1.5 tesla scanners . to date , no adverse health effects have been demonstrated  neither fetal nor maternal  after short term exposition to electromagnetic fields as used in clinical mri . 
 the haste sequence ( half fourier acquired single shot turbo spin echo ) is nowadays most used and combines short acquisition times ( 1 image in less than 1 second ) with a good signal - to - noise ratio , good t2 contrast and an acceptable spatial resolution ( slice thickness of 3  mm ) . 
 t1 weighted imaging is often used to detect hemorrhages or calcifications . t1-weighted imaging sequences have a lower signal to noise ratio , require longer scanning times ( up to 18 seconds per slice ) and are hence much more susceptible to fetal and maternal motion . 
 novel scanning sequences include the use of diffusion weighted imaging and diffusion tensor imaging for a more functional analysis of the developing brain ( guimiot et al . , 2008 ; 
 mri scans have a big field of view and can be obtained in any given plane 
 . the major limitations of fetal mri are the impact of fetal motion on the image acquisition and its relatively low spatial resolution , in particular compared to ultrasound . 
 contra - indications for mri are the same as for non - pregnant patients ( claustrophobia , metallic brain clips , pacemaker implant ,  ) . 
 opposed to ultrasound , mri has an excellent contrast resolution which enables to differentiate easily between gray and white matter . 
 mri also allows to directly visualize the cortical region and the fossa posterior , without sonographic limiting factors such as maternal obesity , the amount of amniotic fluid , fetal head position or the acoustic shadows of the skull bones . 
 knowledge of the normal appearance and maturation of the developing cerebral gyri and sulci is helpful in the appropriate diagnosis and counseling of anomalous fetuses . at 14 weeks 
 the sylvian fissure and callosal sulcus are respectively visible at 16 and 18 weeks , and the central sulcus is not reliable seen until 24 - 25 weeks of gestation . 
 weeks , numerous new sulci and gyri develop and by the age of 32 - 35 weeks secondary gyri are visible throughout the cerebral cortex ( levine and robson , 2005 ) . 
 an important role in the prenatal diagnostic work - up of ventriculomegaly is the detection and characterization of additional cerebral anomalies ( kubik- huck et al . , 2000 ; 
 , 2002 ; launay et al . , 2002 ; levine et al . , 2003 ; 
 valsky et al . , 2004 ; mehta and levine , 2005 ; zimmerman and bilaniuk , 2005 ; glenn and barkovich , 2006 ; benacerraf et al . , 2007 ; morris et al . 
 the incidence of additional malformations detected on mri following normal ultrasound findings varies greatly between several reported studies and depends on the severity of ventriculomegaly , the expertise of the sonographer and the selected patient population . 
 percentages of detection range between 5% for mild ventriculomegaly and up to 50% for moderate and severe cases ( valsky et al . , 2004 ; salomon et al . , 
 numbers should be interpreted with caution , because of the systematic lack of postnatal / postmortem ( imaging ) correlates , the uncertainty between the time span between prenatal ultrasound and mri in several studies , the a priori knowledge of ultrasound findings by the fetal mri specialist and the lack of data investigating the sonographic detection / confirmation of the fetal anomalies following the mri scan . 
 address several items why the added value of mri is difficult to statistically demonstrate : an overenthusiastic attitude towards new technologies , comparing a first line routine ultrasound exam with a mri exam in a tertiary centre , the lack of description of the ultrasound technique and checklist , suboptimal or insufficient ultrasound images in the reported studies and no information with respect to the time span between the ultrasound and mri exam ( malinger et al . , 2004 ) . 
 at last , it is very important to stress that the interobserver agreement , i.e. the interpretation agreement between 2 observers using the same modality , for both imaging modalities can vary greatly . in a study of levine et al . , 
 in which the agreement between experts using the same imaging modality was investigated , overall consensus was reached with respect to normal and abnormal findings in only 60% of the cases for ultrasound and in 53% for mri ( levine et al . , 2008 ) . the numbers even drop below 50% for the detection of cerebellar and gyral anomalies . 
 important to note is that the readers in this study could not indicate their uncertainty in each finding , but were only allowed to indicate the presence or absence of an anomaly . given the often subtle findings of fetal central nervous pathology , such as cerebellar of gyral anomalies , this probably has led to an overestimate of the discrepancies . 
 another important finding is that the level of subspecialty training of the individuals interpreting the mri examination , in particular the involvement of pediatric neuroradiologists , significantly improved the interobserver agreement . 
 below , we will discuss some disease entities often associated with ventriculomegaly , with emphasis on the added value of mri ( launay et al . 
 , 2002 ; glenn and barkovich , 2006 part 2 ; benacerraf et al . , 2007 ) . 
 mri is a very sensitive technique to detect small deposits of intraventricular blood , which may suggest an intraventricular hemorrhage as cause of the hydrocephaly ( fig . 
 mri has also a high accuracy to detect corpus callosum dysgenesis and allows to screen for additional malformations such as cortical anomalies , periventricular of subependymal nodular heteropia and interhemispheric cysts ( fig . 
 accurate characterization of those additional anomalies is important in terms of prognosis and parental counseling . 
 mri is equally good for a morpho- and volumetric assessment of the fossa posterior in case of suspicious ultrasound findings . 
 it allows a good evaluation of the vermis , the cerebellar hemispheres , the pedunculi , the brainstem , the 4th ventricle , the tentorium cerebelli and the retrocerebellar space , which enables to detect developmental anomalies such as vermis hypoplasia , dandy walker malformations , chiari malformations and other fossa posterior malformations  ( fig . 
 a recurring indication for ventriculomegaly reported in the literature , is the detection of cortical malformations , such as polymicrogyria ( fig .  5 ) , 
 the excellent tissue contrast of mri and the absence of bony interference , makes this technique ideal for cortical evaluation . 
 congenital infections such as cytomegalovirus and toxoplasmosis , are also good candidates to refer for mri , in particular to detect pathological white matter changes and cortical malformations ( fig . 
 focal loss of brain tissue , hemorrhages and sequelae of ventriculitis can also easily be detected on mri . 
 on the basis of reported studies , fetal mri has a limited role over ultrasound in assessing the size of the cerebral ventricles , except for cases where fetal position and calvarial ossification cause problems . in the second trimester , in particular the 20 - 24 weeks group , 
 mri is useful to detect small foci of brain hemorrhage or callosal anomalies in fetuses diagnosed with cerebral ventriculomegaly ( griffiths et al . , 2010 ) . later in pregnancy ( over 25  weeks ) 
 , mri adds information about normal and pathological cortical development and maturation , depicts fossa posterior anomalies and is a more sensitive imaging modality to detect white matter pathology ( griffiths et al . , 2010 ; yi et al . , 
 referring prenatal cases of ventriculomegaly for fetal mri is a topic of debate , not only due to questions related to the diagnostic accuracy of mri and its added value , but also because of practical issues such as the availability of mri , the cost and available expertise of fetal radiologists . 
 most studies reported in the literature advocate the added value of mri in cases of moderate and severe ventriculomegaly , not only to confirm the severity , but mainly to further detect and characterize additional anomalies . in cases of mild unilateral ventriculomegaly , mri might demonstrate additional findings which are difficult to detect with sonography such as leucomalacy and neuronal migration disorders . in both categories ,","the indication for fetal magnetic resonance imaging ( mri ) remains a subject of debate , partly because of questions concerning its diagnostic accuracy compared to ultrasound , partly because of practical factors such as accessibility , high costs and available expertise . 
 most studies advocate an added value for mri in cases diagnosed with central nervous system pathology . 
 mri is a good modality to detect small foci of brain hemorrhage , to depict callosal anomalies , to add information about normal and pathological cortical development , and is a more sensitive imaging method to detect white matter pathology . 
 this manuscript discusses the role of mri as an adjunct to ultrasound for cases diagnosed with cerebral ventriculomegaly .",pubmed
"lung cancer is the third most common cancer diagnosed and the most common cause of cancer - related death among both men and women in the united states ( us ) . 
 the american cancer society estimates that in 2012 , 226,160 patients ( men , 116,470 ; women , 109,690 ) will be diagnosed with lung cancer and 160,340 ( men , 87,750 ; women , 72,590 ) will die from the disease . 
 lung cancer is often diagnosed at an advanced stage of disease , as early - stage disease is often asymptomatic . 
 the stage distribution of lung cancer cases diagnosed in 1999 - 2006 included in the surveillance , epidemiology and end results ( seer ) program were : distant metastasis at diagnosis , 56% ; regional metastasis , 22% ; localized , 15% ; and unstaged , 8% . 
 stage of disease was a strong risk factor for survival , with advanced disease having much lower five - year survival compared to early disease . from 1999 - 2006 , the seer program reported that the five - year relative survival was 3.5% for distant stage at diagnosis , 24% for regional stage and 52.9% for localized stage . 
 bone is one of the most frequent sites of metastasis in patients with lung cancer . 
 these osteolytic lesions undermine the structural integrity of bone placing lung cancer patients at an increased risk for skeletal - related events ( sres ) such as pathological fractures , spinal cord compression and severe pain requiring radiotherapy or surgery for bone lesions.[68 ] these skeletal complications result in impaired mobility and reduced quality of life adding substantially to the overall burden of disease in patients with lung cancer . 
 data are limited on the occurrence and outcomes of bone metastasis and sres among elderly patients who develop lung cancer . 
 a few studies have reported results pertaining to the occurrence of bone metastasis and of sres among lung cancer patients with bone metastases , but none have focused on older patients . 
 we therefore undertook this study to quantify the impact of bone metastasis and sres on mortality among older lung cancer patients by analyzing a combined population - based cancer registry and the medicare claims database . 
 the seer program , supported by the national cancer institute ( nci ) , collects cancer incidence and survival data from population - based cancer registries from selected geographic areas . during the time period of the present study , 
 the seer program covered 14 to 26% of the us population from 9 to 13 geographic areas . 
 the seer data included information on patient demographics , date of cancer diagnosis , tumor stage and other characteristics at diagnosis and summary information on treatments received soon after diagnosis . 
 the medicare program , administered by the centers for medicare and medicaid services , covers 97% of us population ages 65 years . 
 medicare data included demographic information on beneficiaries and claims data from hospitals and other institutional and non - institutional providers . 
 the medicare claims data included dates of service , diagnosis codes ( international classification of diseases ninth revision ( icd-9 ) ) and procedure codes ( current procedural terminology ( cpt ) . 
 we identified a cohort of patients aged 65 years or older and having a seer record of a new diagnosis of lung cancer between july 1 , 1999 and december 31 , 2005 and used seer and medicare data to determine possible occurrence of bone metastasis , sres and deaths occurring through december 31 , 2006 . 
 we required each subject to have a baseline period of at least six months of full fee - for - service medicare coverage before his / her lung cancer diagnosis date . 
 the claims data from this baseline period provided information on the presence of comorbidities , and on the presence of possible bone metastasis and sres at diagnosis ( based on icd-9 codes ) . 
 we excluded patients who did not have both medicare part a and part b coverage and those who were enrolled in a medicare advantage plan during the baseline period because we did not have complete medicare claims data on the services they may have received . in all analyses , we censored subjects if they died or lost full fee - for - service coverage before the end of 2006 ( e.g. , if their coverage changed such that we did not have access to all of their medical claims ) . 
 bone metastasis , with or without evidence of sres , was the time - dependent exposure variable of main interest in our analyses . throughout this paper , we apply the term 
 bone metastasis  and  sres  to patients who have claims - based evidence of these conditions . 
 we used the following medicare claims data as evidence of bone metastasis : a ) at least one inpatient claim with an icd-9 diagnosis code of 198.5 (  secondary malignant neoplasm of bone and bone marrow  ) as the primary or secondary discharge diagnosis ; b ) at least one outpatient claim with a diagnosis code of 198.5 , paired with a code for procedures used to diagnose or treat bone metastasis ; or c ) at least one outpatient physician evaluation and management claim with a diagnosis code of 198.5 ( above algorithm available on request ) . 
 we classified subjects as having concurrent bone metastasis at the time of diagnosis if they had a claim in the month of the lung cancer diagnosis or in the preceding month . among subjects without concurrent bone metastasis at diagnosis , we identified new bone metastasis as the earliest occurrence of one of the above claims patterns at any time during follow - up . for the purpose of this study , we defined sres as one or more of the following conditions , occurring concurrently with or after the first bone metastasis : fractures , radiotherapy to bone , surgery to bone and spinal cord compression . 
 we identified sres using combinations of diagnosis and procedure codes from inpatient and/or outpatient claims ( codes available on request ) . 
 we classified an sre as concurrent with bone metastasis if it occurred within 30 days before or after the earliest bone metastasis date and as subsequent to bone metastasis if it occurred at any time more than 30 days after the bone metastasis date . 
 we excluded from the analysis patients with claims suggesting bone metastases occurring more than 30 days prior to the lung cancer diagnosis ( n = 570 ) . 
 the excluded patients comprising of 2% of overall patients with bone metastases had similar demographic characteristics as the bone metastases patients included in the analysis but were more likely to have unstaged and unspecified histology . 
 we obtained information on date of death using the combined seer registry and medicare claims data . 
 we used the concordant date of death in these two sources , if there was agreement between them . 
 if the seer date of death was missing , we used the medicare date of death . from the seer data 
 , we obtained information on age , gender , race / ethnicity and stage at cancer diagnosis . from the medicare claims data 
 we computed each person 's charlson comorbidity score on the basis of icd-9 diagnosis codes in his / her inpatient records for the 17 medical conditions [ table 1 ] comprising the charlson index in the 12-month period prior to the month of cancer diagnosis . in computing the charlson index , 
 we used the approach described by romano et al , and updated by quan et al . 
 characteristics of patients with lung cancer , total cohort and according to bone metastasis status : seermedicare , july 1 , 1999-december 31 , 2006 descriptive analyses of the cohort included frequency distributions and median values , where applicable . 
 we computed the proportion of patients with lung cancer having claims - based evidence of bone metastasis at one year from diagnosis for the overall cohort of lung cancer cases and for subgroups specified by tumor stage at diagnosis . 
 for these computations , we selected patients diagnosed with lung cancer from july 1 , 1999 through december 31 , 2005 , so that these patients would have a potential observation period of at least one year post lung cancer diagnosis . 
 we used cox regression to estimate mortality hazards ratios ( hrs ) and 95% confidence intervals ( cis ) among patients with bone metastasis with or without sres , compared to patients without bone metastasis , adjusting for covariates . 
 we also estimated mortality hrs for subgroups specified by initial tumor stage , age , gender and race / ethnicity at cancer diagnosis . 
 confounders were categorical forms of age at cancer diagnosis ( 65 - 69 , 70 - 74 , 75 - 79 , 80 - 84 , 85 + ) , gender ( male , female ) , race ( white , african american , hispanic american , other ) , stage at lung cancer diagnosis ( distant , localized , regional , unstaged ) , and charlson comorbidity score ( 0 , 1 , 2 , 3 + ) . 
 this study was approved by the uab institutional review board and by the nci seer - medicare program . 
 the seer program , supported by the national cancer institute ( nci ) , collects cancer incidence and survival data from population - based cancer registries from selected geographic areas . during the time period of the present study , 
 the seer program covered 14 to 26% of the us population from 9 to 13 geographic areas . 
 the seer data included information on patient demographics , date of cancer diagnosis , tumor stage and other characteristics at diagnosis and summary information on treatments received soon after diagnosis . 
 the medicare program , administered by the centers for medicare and medicaid services , covers 97% of us population ages 65 years . 
 medicare data included demographic information on beneficiaries and claims data from hospitals and other institutional and non - institutional providers . 
 the medicare claims data included dates of service , diagnosis codes ( international classification of diseases ninth revision ( icd-9 ) ) and procedure codes ( current procedural terminology ( cpt ) . 
 we identified a cohort of patients aged 65 years or older and having a seer record of a new diagnosis of lung cancer between july 1 , 1999 and december 31 , 2005 and used seer and medicare data to determine possible occurrence of bone metastasis , sres and deaths occurring through december 31 , 2006 . 
 we required each subject to have a baseline period of at least six months of full fee - for - service medicare coverage before his / her lung cancer diagnosis date . the claims data from this baseline period provided information on the presence of comorbidities , and on the presence of possible bone metastasis and sres at diagnosis ( based on icd-9 codes ) . 
 we excluded patients who did not have both medicare part a and part b coverage and those who were enrolled in a medicare advantage plan during the baseline period because we did not have complete medicare claims data on the services they may have received . in all analyses 
 , we censored subjects if they died or lost full fee - for - service coverage before the end of 2006 ( e.g. , if their coverage changed such that we did not have access to all of their medical claims ) . 
 bone metastasis , with or without evidence of sres , was the time - dependent exposure variable of main interest in our analyses . throughout this paper , we apply the term 
 bone metastasis  and  sres  to patients who have claims - based evidence of these conditions . 
 we used the following medicare claims data as evidence of bone metastasis : a ) at least one inpatient claim with an icd-9 diagnosis code of 198.5 (  secondary malignant neoplasm of bone and bone marrow  ) as the primary or secondary discharge diagnosis ; b ) at least one outpatient claim with a diagnosis code of 198.5 , paired with a code for procedures used to diagnose or treat bone metastasis ; or c ) at least one outpatient physician evaluation and management claim with a diagnosis code of 198.5 ( above algorithm available on request ) . 
 we classified subjects as having concurrent bone metastasis at the time of diagnosis if they had a claim in the month of the lung cancer diagnosis or in the preceding month . among subjects without concurrent bone metastasis at diagnosis , we identified new bone metastasis as the earliest occurrence of one of the above claims patterns at any time during follow - up . 
 for the purpose of this study , we defined sres as one or more of the following conditions , occurring concurrently with or after the first bone metastasis : fractures , radiotherapy to bone , surgery to bone and spinal cord compression . 
 we identified sres using combinations of diagnosis and procedure codes from inpatient and/or outpatient claims ( codes available on request ) . 
 we classified an sre as concurrent with bone metastasis if it occurred within 30 days before or after the earliest bone metastasis date and as subsequent to bone metastasis if it occurred at any time more than 30 days after the bone metastasis date . 
 we excluded from the analysis patients with claims suggesting bone metastases occurring more than 30 days prior to the lung cancer diagnosis ( n = 570 ) . 
 the excluded patients comprising of 2% of overall patients with bone metastases had similar demographic characteristics as the bone metastases patients included in the analysis but were more likely to have unstaged and unspecified histology . 
 we obtained information on date of death using the combined seer registry and medicare claims data . 
 we used the concordant date of death in these two sources , if there was agreement between them . 
 if the seer date of death was missing , we used the medicare date of death . 
 from the seer data , we obtained information on age , gender , race / ethnicity and stage at cancer diagnosis . from the medicare claims data 
 we computed each person 's charlson comorbidity score on the basis of icd-9 diagnosis codes in his / her inpatient records for the 17 medical conditions [ table 1 ] comprising the charlson index in the 12-month period prior to the month of cancer diagnosis . in computing the charlson index , 
 we used the approach described by romano et al , and updated by quan et al . 
 characteristics of patients with lung cancer , total cohort and according to bone metastasis status : seermedicare , july 1 , 1999-december 31 , 2006 
 we computed the proportion of patients with lung cancer having claims - based evidence of bone metastasis at one year from diagnosis for the overall cohort of lung cancer cases and for subgroups specified by tumor stage at diagnosis . for these computations , we selected patients diagnosed with lung cancer from july 1 , 1999 through december 31 , 2005 , so that these patients would have a potential observation period of at least one year post lung cancer diagnosis . other analyses , described below , were unrestricted with regard to length of observation . 
 we used cox regression to estimate mortality hazards ratios ( hrs ) and 95% confidence intervals ( cis ) among patients with bone metastasis with or without sres , compared to patients without bone metastasis , adjusting for covariates . 
 we also estimated mortality hrs for subgroups specified by initial tumor stage , age , gender and race / ethnicity at cancer diagnosis . 
 confounders were categorical forms of age at cancer diagnosis ( 65 - 69 , 70 - 74 , 75 - 79 , 80 - 84 , 85 + ) , gender ( male , female ) , race ( white , african american , hispanic american , other ) , stage at lung cancer diagnosis ( distant , localized , regional , unstaged ) , and charlson comorbidity score ( 0 , 1 , 2 , 3 + ) . 
 this study was approved by the uab institutional review board and by the nci seer - medicare program . 
 the overall cohort of 126,123 patients were predominantly white ( 85% ) with a median age at diagnosis of 75 years and a median follow - up of 0.6 years [ table 1 ] . 
 eighty - one percent of patients had non - small cell lung cancer . during the study period , 24,820 ( 19.8% ) patients 
 had claims - based evidence of bone metastasis either concurrently with the diagnosis of lung cancer diagnosis ( n = 9,523 ) or during follow - up ( n = 15,297 ) . 
 the median time from cancer diagnosis to bone metastasis was 5.4 months among patients without a bone metastasis at diagnosis . 
 patients with , compared to those without , a bone metastasis were more likely to have distant stage disease ( 72% vs. 41% ) and to have died by the end of the study period ( 95% vs. 82% ) [ table 1 ] . 
 the two groups ( patients with and without bone metastasis ) were similar with regard to age , race / ethnicity , gender , year of diagnosis , histology , comorbidity score and length of follow - up . 
 figure 1 displays the proportion of patients with evidence of bone metastasis at one year post lung cancer diagnosis , according to stage at diagnosis , among the 126,123 patients ( localized , n = 23,821 ; regional , n = 30,365 ; distant , n = 59,319 ; unstaged , n = 12,618 ) diagnosed with lung cancer from july 1 , 1999 through december 31 , 2005 . at one year post diagnosis of lung cancer , the proportion with evidence of bone metastasis was 21% for all stages combined and was 38% for distant , 12% for regional , 11% for unstaged and 5% for localized stages at primary cancer diagnosis . when restricted to patients with non - small cell lung cancer , the proportion with evidence of bone metastasis at one year post diagnosis was 21% for all stages combined and was 39% for distant , 11% for regional , 13% for unstaged and 5% for localized stages . 
 proportion of 126,123 patients with lung cancer who had evidence of bone metastasis within 1 year by stage of the 24,820 patients with a possible bone metastasis , 12,665 ( 51% ) had evidence of a concurrent ( n = 11,015 , 44% ) or subsequent ( n = 1,650 , 7% ) sre ( data not displayed in a table ) . among the 12,665 patients with an sre , most ( n = 10,598 , 84% ) presented with only one skeletal complication at the first diagnosis of an sre . of the 10,598 presenting with a single skeletal complication , 8,357 ( 79% ) had radiotherapy to bone , 1,509 ( 14% ) experienced a fracture , 645 ( 6% ) had spinal cord compression and 87 ( 1% ) had surgery to bone . 
 of 12,665 patients with sre , 2,067 ( 16% ) had more than one skeletal complication at the first diagnosis of an sre . 
 of these 2,067 patients , 969 ( 47% ) had radiation to bone plus fracture , 617 ( 30% ) had radiation to bone plus spinal cord compression , 183 ( 9% ) had radiation to bone plus fracture plus spinal cord compression , 125 ( 6% ) had fracture plus spinal cord compression ; all other combinations accounted for < 3% each . 
 overall , 81% of patients with sre had radiotherapy to bone either diagnosed as the only sre or in combination with other sres . among the 2,481 patients who developed a fracture , 
 regardless of whether it was an sre by itself or in combination with other sres , 1,625 ( 65% ) had a fracture coded as pathological . 
 the most common site of pathological fracture was the spine ( n = 991 ) , followed by hip ( n = 290 ) , femur ( n = 185 ) , humerus ( n = 141 ) , tibia / fibula ( n = 14 ) , and distal radius / ulna ( n = 4 ) . 
 table 2 displays hrs for death in relation to possible bone metastasis and sres , adjusted for age at cancer diagnosis , race / ethnicity , gender , stage at cancer diagnosis and charlson comorbidity score . 
 the hr for risk of death was 2.4 ( 95% ci = 2.4 - 2.5 ) , both for patients with bone metastasis but no sre and for patients with bone metastasis plus sre , compared to patients without bone metastasis . 
 stage - specific analyses indicated that hrs associated with bone metastasis complicated with sre ranged from 2.0 ( 95% ci = 1.9 - 2.0 ) for distant stage disease at cancer diagnosis to 7.3 ( 95% ci = 6.8 - 7.8 ) for localized disease at diagnosis , and bone metastasis without sre ranged from1.9 ( 95% ci = 1.9 - 2.0 ) for distant stage at diagnosis to 6.1 ( 95% ci = 5.7 - 6.5 ) for localized stage table 3 
 . hazard ratio ( hr ) for death in relation to bone metastasis and other factors among patients with lung cancer : seer - medicare , july 1 , 1999-december 31 , 2006 adjusted hazard ratio ( hr ) for death in relation to bone metastasis and skeletal - related factors ( sres ) among men with lung cancer , by stage at diagnosis : seer - medicare , july 1 , 1999-december 31 , 2006 hrs for death in relation to bone metastasis in white patients were similar to those for african american patients ( white : hr = 2.4 , 95% ci = 2.4 - 2.5 ) ; african american , hr = 2.3 , 95% ci = 2.2 - 2.4 ) ( data not displayed in a table ) . 
 hrs for death among men and women were also similar with regard to bone metastasis ( men , hr = 2.4 , 95% ci = 2.4 - 2.5 ; women , hr = 2.5 , 95% ci = 2.4 - 2.5 ) . 
 we found a positive association between bone metastasis and with bone metastasis complicated with sre and mortality in all categories of age groups ( 65 - 69 , 70 - 74 , 75 - 79 , 80 - 84 , 85 + ) . 
 patients with , compared to those without , a bone metastasis were more likely to have distant stage disease ( 72% vs. 41% ) and to have died by the end of the study period ( 95% vs. 82% ) [ table 1 ] . 
 the two groups ( patients with and without bone metastasis ) were similar with regard to age , race / ethnicity , gender , year of diagnosis , histology , comorbidity score and length of follow - up . 
 figure 1 displays the proportion of patients with evidence of bone metastasis at one year post lung cancer diagnosis , according to stage at diagnosis , among the 126,123 patients ( localized , n = 23,821 ; regional , n = 30,365 ; distant , n = 59,319 ; unstaged , n = 12,618 ) diagnosed with lung cancer from july 1 , 1999 through december 31 , 2005 . 
 at one year post diagnosis of lung cancer , the proportion with evidence of bone metastasis was 21% for all stages combined and was 38% for distant , 12% for regional , 11% for unstaged and 5% for localized stages at primary cancer diagnosis . 
 when restricted to patients with non - small cell lung cancer , the proportion with evidence of bone metastasis at one year post diagnosis was 21% for all stages combined and was 39% for distant , 11% for regional , 13% for unstaged and 5% for localized stages . 
 proportion of 126,123 patients with lung cancer who had evidence of bone metastasis within 1 year by stage 
 of the 24,820 patients with a possible bone metastasis , 12,665 ( 51% ) had evidence of a concurrent ( n = 11,015 , 44% ) or subsequent ( n = 1,650 , 7% ) sre ( data not displayed in a table ) . among the 12,665 patients with an sre , most ( n = 10,598 , 84% ) presented with only one skeletal complication at the first diagnosis of an sre . of the 10,598 presenting with a single skeletal complication , 8,357 ( 79% ) had radiotherapy to bone , 1,509 ( 14% ) experienced a fracture , 645 ( 6% ) had spinal cord compression and 87 ( 1% ) had surgery to bone . 
 of 12,665 patients with sre , 2,067 ( 16% ) had more than one skeletal complication at the first diagnosis of an sre . 
 of these 2,067 patients , 969 ( 47% ) had radiation to bone plus fracture , 617 ( 30% ) had radiation to bone plus spinal cord compression , 183 ( 9% ) had radiation to bone plus fracture plus spinal cord compression , 125 ( 6% ) had fracture plus spinal cord compression ; all other combinations accounted for < 3% each . 
 overall , 81% of patients with sre had radiotherapy to bone either diagnosed as the only sre or in combination with other sres . among the 2,481 patients who developed a fracture , 
 regardless of whether it was an sre by itself or in combination with other sres , 1,625 ( 65% ) had a fracture coded as pathological . 
 the most common site of pathological fracture was the spine ( n = 991 ) , followed by hip ( n = 290 ) , femur ( n = 185 ) , humerus ( n = 141 ) , tibia / fibula ( n = 14 ) , and distal radius / ulna ( n = 4 ) . 
 table 2 displays hrs for death in relation to possible bone metastasis and sres , adjusted for age at cancer diagnosis , race / ethnicity , gender , stage at cancer diagnosis and charlson comorbidity score . 
 the hr for risk of death was 2.4 ( 95% ci = 2.4 - 2.5 ) , both for patients with bone metastasis but no sre and for patients with bone metastasis plus sre , compared to patients without bone metastasis . 
 stage - specific analyses indicated that hrs associated with bone metastasis complicated with sre ranged from 2.0 ( 95% ci = 1.9 - 2.0 ) for distant stage disease at cancer diagnosis to 7.3 ( 95% ci = 6.8 - 7.8 ) for localized disease at diagnosis , and bone metastasis without sre ranged from1.9 ( 95% ci = 1.9 - 2.0 ) for distant stage at diagnosis to 6.1 ( 95% ci = 5.7 - 6.5 ) for localized stage table 3 . 
 hazard ratio ( hr ) for death in relation to bone metastasis and other factors among patients with lung cancer : seer - medicare , july 1 , 1999-december 31 , 2006 adjusted hazard ratio ( hr ) for death in relation to bone metastasis and skeletal - related factors ( sres ) among men with lung cancer , by stage at diagnosis : seer - medicare , july 1 , 1999-december 31 , 2006 hrs for death in relation to bone metastasis in white patients were similar to those for african american patients ( white : hr = 2.4 , 95% ci = 2.4 - 2.5 ) ; african american , hr = 2.3 , 95% ci = 2.2 - 2.4 ) ( data not displayed in a table ) . 
 hrs for death among men and women were also similar with regard to bone metastasis ( men , hr = 2.4 , 95% ci = 2.4 - 2.5 ; women , hr = 2.5 , 95% ci = 2.4 - 2.5 ) . 
 we found a positive association between bone metastasis and with bone metastasis complicated with sre and mortality in all categories of age groups ( 65 - 69 , 70 - 74 , 75 - 79 , 80 - 84 , 85 + ) . 
 we found that about 19.8% of patients 65 years or older with newly diagnosed lung cancer included in the seer program from july 1999 to december 2005 presented with or subsequently had evidence of a possible bone metastasis as indicated by medicare claims . 
 sres were common , occurring in 51% of these patients . among patients with a potential follow - up time of one year post lung cancer diagnosis , the proportion with bone metastasis was 21% overall and 38% for distant stage of lung cancer . having a possible bone metastasis was associated with mortality among patients with lung cancer ; the association was similar for bone metastasis complicated with an sre and for bone metastasis without an sre . 
 the skeleton is a frequent site of metastasis in patients with non - small - cell lung cancer which is the most common type of lung cancer . 
 about 85% of lung cancer cases included in the seer program have a non - small cell histology . in our data , 
 bone metastasis may be asymptomatic at onset and thus , are likely to be under - diagnosed resulting in inaccurate staging of lung cancer at diagnosis . 
 studies report that the prevalence of bone metastasis among patients who report bone pain is almost similar to patients who did not report bone pain ( 27 - 32% vs. 16 - 27% ) . prior to 1991 , 
 several studies found skeletal metastases in non - small cell lung cancer to range from 8% to 34%.[2531 ] with the advent of newer imaging techniques such as the positron emission tomography ( pet ) in the 1990s , bone metastasis has been noted to range from 24% to 30% for non - small cell lung cancer . 
 the preceding results are consistent with our finding that 21% of medicare non - small cell lung cancer patients had possible bone metastasis within a year of lung cancer diagnosis . 
 , the seer program reports that 56% of lung cancer patients were classified as having distant stage at the time of disease diagnosis . in the present study , 
 bone metastases are common in patients with distant stage of lung cancer . in the present study 
 , we found that 38% of patients with distant stage lung cancer had possible bone metastasis within a year of their cancer diagnosis . 
 this result is consistent with findings from other studies which have reported that 30 - 40% of lung cancer patients with distant metastatic disease had a bone metastasis at diagnosis or subsequently developed a bone metastasis . 
 in addition , autopsies of patients dying of lung cancer indicated that up to 30 - 40% had evidence of metastatic bone disease . in the present study 
 , we found that 51% of patients with possible bone metastasis had one or more sres . 
 this result agrees with findings from a retrospective observational study using data from a health insurance claims database in the us which reported that sres occurred in 55% of 534 lung cancer patients with bone metastasis . 
 clinical trials of lung cancer have found that sres occurred in 48% to 50% of patients with bone metastasis . in our study , 
 radiotherapy ( 81% of patients with sre ) to bone was the most common sre followed by fracture , spinal cord compression and surgery to bone . 
 we observed that among patients with fractures , 65% ( or 13% of patients with sre ) had a pathological fracture with the spine as the most common site of occurrence followed by pelvis and extremities . 
 other studies report that 7 - 10% of non - small lung cancer patients with sres developed pathological fractures . 
 our results indicated that having a bone metastasis was associated with mortality among lung cancer patients . 
 the lack of an additional effect of sre on mortality among patients with bone metastasis may be due to the short survival time in lung cancer patients compared to other tumor types . 
 our companion studies of breast and prostate cancer , cancers with relatively longer survival time , found stronger associations for bone metastasis complication by sre than for bone metastasis without sre . 
 our finding of a stronger association of bone metastasis and for bone metastasis complicated with sre for patients with localized stage lung cancer than for those with distant metastatic lung cancer at diagnosis may reflect the low mortality among patients with early stage disease and no bone metastasis . 
 the strengths of the study include the use of the large population - based seer cancer registry data with pathologically confirmed cancer and the focus on lung cancer in the elderly . the combined cancer registry and medicare data provide a unique opportunity to evaluate the clinical progress of lung cancer . 
 other advantages of the seer data include the continuity of data over time and the quality control measures instituted to ensure completeness of case ascertainment in the participating cancer registries . 
 the main limitations of our study stem from the characteristics of the seer - medicare data . 
 our study was limited to patients aged 65 years or older diagnosed with lung cancer between 1999 and 2005 , who had medicare part a and b coverage and who resided in one of the seer geographic areas . 
 medicare data do not include claims of health maintenance organization enrollees , care provided in other settings like the veterans administration , reimbursement of covered services not captured by the medicare data such as out - of - pocket expenditures or coverage provided by medigap , or long term care at home or nursing homes . 
 therefore , our results will not be generalizable to patients less than 65 years of age and may not represent patients aged 65 years and older who resided in geographic areas not included in the seer program who had only had medicare part a coverage or who were enrolled in medicare advantage plan . 
 second , we have not validated the procedures used to identify bone metastasis in this study . 
 we identified possible bone metastasis from inpatient claims or outpatient claims paired with selected procedure codes , an approach designed to reduce false positives . despite this approach 
 it is also possible that we may have missed true cases in instances where claims were not obtained from the relevant facilities or where claims were obtained but not coded as bone metastasis . 
 third , it is possible that the mortality differences we noted for bone metastasis or bone metastasis plus sre compared to those without these outcomes may be due to other confounding factors such as treatment factors or concurrent comorbid conditions that were not included in our analysis . 
 finally , the data do not include information on functional status of patients , a predictor of outcomes . 
 in conclusion , the present study found that having a bone metastasis as indicated by medicare claims , was associated with mortality among patients with lung cancer . 
 we found no difference in mortality between patients with bone metastasis complicated by sre and patients with bone metastasis but without sre .","background : to quantify the impact of bone metastasis and skeletal - related events ( sres ) on mortality among older patients with lung cancer.materials and methods : using the linked surveillance , epidemiology and end results - medicare database , we identified patients aged 65 years or older diagnosed with lung cancer between july 1 , 1999 and december 31 , 2005 and followed them to determine deaths through december 31 , 2006 . 
 we classified patients as having possible bone metastasis and sres using discharge diagnoses from inpatient claims and diagnoses paired with procedure codes from outpatient claims . 
 we used cox regression to estimate mortality hazards ratios ( hr ) among patients with bone metastasis with or without sre , compared to patients without bone metastasis.results:among 126,123 patients with lung cancer having a median follow - up of 0.6 years , 24,820 ( 19.8% ) had bone metastasis either at lung cancer diagnosis ( 9,523 , 7.6% ) or during follow - up ( 15,297 , 12.1% ) . 
 sres occurred in 12,665 ( 51% ) patients with bone metastasis . 
 the hr for death was 2.4 ( 95% ci = 2.4 - 2.5 ) both for patients with bone metastasis but no sre and for patients with bone metastasis plus sre , compared to patients without bone metastasis.conclusions:having a bone metastasis , as indicated by medicare claims , was associated with mortality among patients with lung cancer . 
 we found no difference in mortality between patients with bone metastasis complicated by sre and patients with bone metastasis but without sre .",pubmed
"fiber - reinforced posts have superior advantages as compared with cast posts , generally regarding their esthetics , physical properties and their modulus of elasticity , which is similar to root dentin.1 posts are generally cemented with resin cements . proper selection of resin cements influence the retention of posts and the success of restoration.2,3 the studies indicate that failure of glass fiber - reinforced post - and - core restorations often occurs because of debonding at the post - resin interface and/or resin - dentin interface as a result of bond deficiencies.4,5 self - adhesive resin cements are the least investigated group among the resin cements . 
 application steps are reduced by removing the pre - treatment procedures of the dentin.6 the organic matrix consists of newly developed phosphoric acid methacrylate . 
 the adhesive mechanism is based on the chemical reaction between the hydroxyapatite crystals of the tooth and phosphoric acid monomers.7 self - adhesive cements are also resistant to moisture and able to release fluoride comparable to glass ionomer cements.8 also , they have satisfactory esthetic and mechanical properties.9 because of these positive features , they are used for the adhesive cementation of fixed prostheses and posts . removing the smear layer that contains gutta - percha remnants , microorganisms , and infectious dentin is necessary for the penetration of the adhesive system and resin cement into the dentin tubules.10,11 self - adhesive resin cements can not completely remove the smear layer.12,13 this remaining smear layer could cause a lower bond strength for self - adhesive resin cements as compared with an etch and rinse adhesive system.14 several chemical substances that are used as irrigants during the biomechanical preparation can remove the smear layer and alter the characteristics of the dentin substrate . 
 sodium hypochlorite ( naocl ) has the capacity to dissolve organic tissues and neutralize toxic products and also has an antimicrobial action15 whereas ethylene diamine tetra acetic acid ( edta ) chelates calcium ions and promotes dentin demineralization and smear layer removal.16 sodium hypochlorite , ethylene diamine tetra acetic acid , orthophosphoric acid ( h3po4)10,17 and different laser systems18 are used to increase the micromechanical retention of the cement by removing the smear layer . 
 no study has been conducted on comparing traditional irrigation systems and photodynamic diode laser performance on the bond strength of self - adhesive cements using fiber posts . 
 lasers can be used to vaporize tissues , remove and modify the smear layer and eliminate residual tissue in the main canal.19,30 diode lasers can make alterations on dentin and may increase the bond strength to root canal dentin.20 alfredo et al.20 found increased bond strength using with 980 nm diode laser and attributed to the alterations caused by the laser on dentin surface , such as fissures and topographic changes . 
 laser treatment can be a valuable instrument for the removal or modify of the smear layer in root dentin , as a debridement device during post space preparation . 
 the aim of this study was to compare the effect of the photodynamic diode laser and other smear layer removal methods on bond strength of the self - adhesive cement . 
 the null hypothesis was that no significant differences would be found among push - out bond strength values after different root canal surface treatments . 
 in this study , 55 maxillary central incisors , which were extracted due to periodontal problems , were used . 
 the teeth were cleaned of both calculus and soft tissues and stored in physiological saline before proceeding . 
 the crowns of each tooth were removed at the level of the cemento - enamel junction with a high - speed diamond bur ( zr diamonds , brasseler gmbh , lemgo , germany ) under water spray . 
 the working length of each root canal was established 1 mm short of the apical foramen with a size 15 k - type file ( mani inc . , 
 the canals were prepared with a rotary system ( x - smart , dentsply , maillefer , ballaigues , switzerland ) . 
 all teeth were instrumented using a set of rotary instruments ( pro taper , dentsply , switzerland ) to the size of f3 ( multi tapered # 30 ; finishing file ) by the same operator . 
 the canals were irrigated at every change of instrument with 2 ml 5% hypochlorite solution and 5 ml for the final irrigation . 
 the prepared canals were filled with matching tapered gutta percha points ( dentsply , maillefer , france ) and a resin sealer ( ah plus , dentsply , konstanz , germany ) . 
 the coronal aspect of the gutta percha was removed with a heated probe ( gutta cut , vdw gmbh , munich , germany ) . after storing the teeth in a humid medium for 1 week at 37 , 
 14 ( diameter 1.4 mm ) glass fiber reinforced composite post ( snowlight post , abrasive technology , oh , usa ) with matching drill . 
 after the post space preparations , the teeth were randomly divided into 5 experimental groups of 11 teeth each . 
 the post spaces were treated with the following : 
 group 1 : 5-ml 0.9% physiological saline ( nacl ) , with a disposable syringe for 1 minutegroup 2 : 5-ml 5.25% sodium hypochlorite ( naocl ) , with a disposable syringe for 1 minutegroup 3 : 5-ml 17% edta with a disposable syringe for 1 minutegroup 4 : etching with 37% orthophosphoric acid for 30 secondsgroup 5 : photodynamic diode laser ( helbo theralite laser , helbo photodynamic systems gmbh & co. , germany ) . 
 group 1 : 5-ml 0.9% physiological saline ( nacl ) , with a disposable syringe for 1 minute group 2 : 5-ml 5.25% sodium hypochlorite ( naocl ) , with a disposable syringe for 1 minute group 3 : 5-ml 17% edta with a disposable syringe for 1 minute group 4 : etching with 37% orthophosphoric acid for 30 seconds group 5 : photodynamic diode laser ( helbo theralite laser , helbo photodynamic systems gmbh & co. , germany ) . 
 bacteria - sensitive , light - active dye solution helbo endo blue ( phenothiazine-5-ium , 3 , 7-bis ( dimethylamino)- , chloride ) ( helbo photodynamic systems gmbh & co. , germany ) photosensitizer was applied for 1 minute into the post spaces and rinsed with water in group 5 . after rinsing in group 5 , 
 a laser probe ( helbo 3d endo probe , helbo photo - dynamic systems gmbh & co. , germany ) was placed near the apex and irradiated for 1 minute with helbo theralite laser with a wavelength of 660 nm , an output power of 75 mw . 
 the laser was delivered through a flexible fiber optic tip curved at an angle of 60. the fiber design permits a 3-dimensional exposure of the area , emitting light at the tip and from the lateral sides , thus leading to even light distribution both vertically and horizontally . 
 ( r = 1.4 mm ) snowlight posts were luted with self - adhesive resin cement ( clearfil sa cement , kuraray medical inc . , japan ) . 
 each root was sectioned perpendicular to its long axis to create 1 mm thick specimens with a 0.3 mm thickness slow - speed diamond saw ( buehler , usa ) at the coronal third of the root . 
 one section of the 1 mm thick specimen was prepared from each tooth ( fig . 
 push - out bond strength was tested using a universal testing machine ( ags - x , schimadzu co. , kyoto , japan ) at a crosshead speed of 1 mm / min until post debonding occurred . 
 the specimens were loaded with a 1 mm diameter cylindrical tip in an apical - coronal direction to push - out the post toward the wider part of the root slice to avoid the taper limitation . 
 the loads at failure were recorded as n and the bond strengths were expressed in mpa ; n was divided by the area of the bonded interface , which was calculated using the following formula : a = 2rh ( where a is the area of the bonded interface ,  = 3.14 , r is the post radius , and h is the thickness of the specimen in mm ) . 
 the debonded specimens investigated under the stereomicroscope at original magnification 40 for the failure interface as between dentin - cement , post - cement and both dentin - cement and post - cement ( fig . 3 and fig . 
 one tooth from each group was processed for sem analysis to observe the changes on root canal surfaces after the post space treatments . 
 the two halves of the tooth were dehydrated and coated with gold for sem analysis . 
 all calculated bond strengths were analyzed using one - way analysis of variance ( anova ) and tukey honestly significant differences ( hsd ) tests with spss 16.0 ( spss , inc . , chicago , il , usa ) ( =.05 ) . 
 the mean values and standard deviations of the test groups are presented in table 1 . 
 bond strength values showed that the higher results were obtained for the helbo theralite laser and 37% orthophosphoric acid groups . 
 although nacl , naocl , edta , and h3po4 groups showed post - cement and cement - dentin failure both ( mixed failure ) ; helbo theralite laser showed post - cement failure at the cross sectional image of the dentin discs ( fig . 
 the null hypothesis was rejected because the results of this study revealed that different smear layer removal methods from root canal dentin affect the bond strength of a fiber post cemented with adhesive resin to dentin ( p<.05 ) . the smear layer produced by post space preparation 
 should be removed according to some authors owing to the increase of antibacterial effects21,22 and also adhesive effectiveness23 as it becomes porous and the authors insisted that the smear layer causes a weak adherent interface between cement and dentin . on the other hand , 
 other authors insist upon retaining smear layer for adaptation of the materials to root surface.24,25 interestingly , in photodynamic diode laser group , bond strength was higher than the other groups except h3po4 group without the removal of smear layer . 
 it may arise from the fact that collagen fibrils structure does not change after lasing . by conserving most of their intrafibrillar mineral , 
 they are more stable and less affected by dehydration , which improves the infiltration of the luting agent . 
 sodium hypochlorite increases the penetration of the monomers into the dentinal tubules by removing organic components , and breaks down to sodium chloride and oxygen . 
 this is because broken down oxygen inhibits interfacial polymerization of resin bonding materials.24,26 on the other hand , 5% naocl alone did not remove the smear layer as effectively as found in other studies.27,11 also the residual naocl solution affect the change in redox potential of the bonding agent.11 others have found naocl and nacl could not remove the smear layer , which can act as a diffusion barrier on the dentin and reduce dentin permeability for bonding agents.2,28 results from another study with 5.25% naocl and 0.9% saline did not increase bond strength of luting cement,29 as shown in this study . 
 a study conducted by takeda et al.30 mentioned that 17% edta alone was not effective in removing smear remnants as it did in this study ; in the same study , authors used 6% orthophosphoric acid to remove the smear layer , but in contrast to this study , it was not useful . 
 , other authors showed that 37% orthophosphoric acid improved retention by eliminating dentin mineral content.31 dual irrigation methods , such as a combination of 17% edta and 5.25% naocl are effective methods for removing the smear layer,32 but this method results in sporadic erosion and dissolution of the dentin.33,34 moreover , it was shown that these solutions did not have a good ability in preparing complicated root canals.35 - 37 therefore , dual irrigation methods were not used for this study . when the push - out tests were examined , generally , failures of bonding were observed between resin and dentin.38,39 in the present study , only the diode laser group showed post - cement failure , the other groups showed mixed failures ( fig . 3 and fig . 
 it was shown that using the photodynamic diode laser in post space increased the bond strength of resin cement to dentin interface , as compared with other surface treatment methods . 
 it is important to improve the bonding effectiveness of the resin and dentin for post restoration success with removal of the smear layer and formation of the resin - dentin interdiffusion zone.2,40 on the contrary , demiryrek et al.27 used acetone based agent , which could not remove the smear layer and sealer remnants effectively on radicular dentin surfaces , to treat post space treatment and found to be more effective compared with edta , citric acid and orthophosphoric acid . 
 it was concluded that removal of the smear layer and opening of dentinal tubules are not recommended when a self - etching / self - priming adhesive system is used.27 laser devices have been used in root canal treatment since the early 1970s,41 but their acceptance has been very slow . 
 general causes of dissatisfaction have included thermal damage caused by the laser photonic energy.42 therefore , the photonic energy should be at its lowest possible level to eliminate thermal damage . it has been reported that as the diode - laser treatment (  = 810 nm ) partially opened dentinal tubules , signs of fusions and temperature increase at the root surfaces were observed.43 on the other hand , a study conducted by altundasar et al.42 after irradiation with a 980-nm diode laser ( gaalas ) ( with 2 times more power ) had opposite results and two - fold increase in temperature at the root surface was observed . to avoid the temperature rise , 
 lower irradiation power was used for this study (  = 660 nm ) , and it was enough to improve bonding effectiveness at the cement - dentin interface . 
 the results of this study suggest the following : ( 1 ) naocl ( 5.25% ) and naci ( 0.9% ) were not effective in removing the smear layer when used as a final irrigation . 
 ( 2 ) according to the sem evaluation , orthophosphoric acid ( 37% ) and edta ( 17% ) were more effective methods in removing the smear layer than naocl , naci irrigants , and helbo theralite laser . however , helbo theralite laser and orthophosphoric acid were more effective at cement - dentin interface than edta for bond strength of self - adhesive cements 
 . therefore , it may be more effective to modify the smear layer when a self - adhesive system is used .","purposethe purpose of this study was to compare the effect of a diode laser and traditional irrigants on the bond strength of self - adhesive cement.materials and methodsfifty - five incisors extracted due to periodontal problems were used . 
 all teeth were instrumented using a set of rotary root canal instruments . 
 the post spaces were enlarged for a no.14 ( diameter , 1.4 mm ) snowlight ( abrasive technology , oh , usa ) glass fiber reinforced composite post with matching drill . 
 the teeth were randomly divided into 5 experimental groups of 11 teeth each . 
 the post spaces were treated with the followings : group 1 : 5 ml 0.9% physiological saline ; group 2 : 5 ml 5.25% sodium hypochlorite ; group 3 : 5 ml 17% ethylene diamine tetra acetic acid ( edta ) , group 4 : 37% orthophosphoric acid and group 5 : photodynamic diode laser irradiation for 1 minute after application of light - active dye solution . 
 snowlight posts were luted with self - adhesive resin cement . 
 each root was sectioned perpendicular to its long axis to create 1 mm thick specimens . 
 the push - out bond strength test method was used to measure bond strength . 
 one tooth from each group was processed for scanning electron microscopic analysis.resultsbond strength values were as follow : group 1 = 4.15 mpa ; group 2 = 3.00 mpa ; group 3 = 4.45 mpa ; group 4 = 6.96 mpa ; and group 5 = 8.93 mpa . 
 these values were analysed using one - way anova and tukey honestly significant difference test ( p<.05 ) . 
 significantly higher bond strength values were obtained with the diode laser and orthophosphoric acid ( p<.05 ) . 
 there were no differences found between the other groups ( p>.05).conclusionorthophosphoric acid and edta were more effective methods for removing the smear layer than the diode laser . 
 however , the diode laser and orthophosphoric acid were more effective at the cement dentin interface than the edta , therefore , modifying the smear layer may be more effective when a self - adhesive system is used .",pubmed
"in recent years , concerted efforts to identify , describe , and quantify the bacterial communities of the mammalian gastrointestinal tract have begun to bear fruit . many international consortia and collaborations 
 have been established to answer the question :  who is there? hot on the heels of this is the query :  what are they doing there? this second , crucial , and rather more complex question is yet to be satisfactorily tackled , but fascinating glimpses of what lies ahead have been caught . 
 the human microbiome project in the usa and the metagenomics of the human intestinal tract ( metahit ) consortium in europe are only two of the major initiatives with the purpose of characterizing the microbial communities that inhabit multiple sites in the human body ( skin , nasal , oral , urogenital , and intestinal flora ) and to look for correlations between the changes in the microbiome and prevalence of diseases . 
 collaborations to investigate if there is a common core of bacterial species shared among all or the majority of human beings have also been initiated , resulting in some interesting paradigms . in the uterus , the developing fetus is believed to be completely devoid of bacterial flora . 
 the same can not be said of bacterial influence on the growing fetus . upon birth , the intestinal tract of 
 the microbiota is first acquired in the birth canal during delivery and then through breast milk . 
 fecal microbial profiles of infants show a striking similarity to maternal vaginal and breast milk bacterial profiles ( 4 ) . during the course of early childhood , microbial composition changes with age and diet ( 5 , 6 ) . from the evolutionary point of view 
 , the character of gut microbiota strongly depends on the main nutritional source , separating bacterial profiles of omnivores , carnivores , and herbivores ( 7 ) several groups have been studying the convergence and variability of the human gut microbiome . 
 these studies were normally performed on a small group of healthy individuals . however , even though the sample size has been minimal , there were some major accomplishments , setting the scene for further large - scale metagenomic approaches . 
 one initial observation was that the human gut flora belongs mainly to only two phyla  firmicutes ( mostly represented by clostridia ) and bacteroidetes , with a smaller representation of bacteria belonging to proteobacteria and actinobacteria ( 8) . the second observation brought about the discovery of archaea , mostly represented by the methanogenic methanobrevibacter smithii ( 8 , 9 ) . 
 what is interesting is that the diversity of archaea is but a fraction of the expansiveness of the bacterial species colonizing the gut . 
 nevertheless , it has been argued that archaea may yet contribute to the health status of the human gut , due to the unique nature of their metabolic prowess ( 10 ) . 
 the composition of the bacterial flora seems to strongly depend on the environment and the maternal input during delivery and upon breast feeding . in humans 
 , members of the same family were shown to have bacterial communities that were more similar to each other than to unrelated individuals ( 11 ) . moreover , this feature was host genotype independent because there was no significant difference detected in the degree of similarity between samples collected from mono- and dizigotic twins . 
 this likely suggests that the environment and maternal flora , rather than genetic factors alone , have greater impact on establishing bacterial communities in a new individual . 
 this observation confirmed the statement that the largest amount of variability between the gut bacterial flora could be explained by intersubject differences ( 8) . 
 recent second generation , high throughput studies have revealed more details about the human bacterial communities . 
 one major study specifically aimed at assessing whether a common bacterial core shared by all or at least a majority of humans could be determined ( 12 ) . 
 fecal material collected from a large cohort of individuals in different countries was subjected to metagenomic analysis . 
 the bacterial groups that were present in the samples in vast numbers were bacteroidetes and dorea / eubacterium / ruminococcus . 
 only 18 species were present in all individuals examined , while 57 species were seen in more than 90% of samples and around 50% shared 75 species . 
 although these numbers are higher than routinely observed , they are indicative of the diversity of the human microbiome , while at the same time emphasizing the extent of the interindividual differences . to obtain a better picture of the role or function of the bacterial communities , 
 failing this , concerted efforts have been made to first identify genes that are encoded by intestinal microbiota and to place their relevance within the context of gut function . 
 more than 3 million open reading frames ( orf ) of bacterial genes have been , thus , identified , potentially coding for as many genes . 
 however , the majority of these genes could only be found in very few samples , again confirming the huge variability between individuals . 
 only around 300,000 genes were found in more than half of the individuals queried , denoting commonly shared microbial genes . 
 each individual subject carried around 500,000 bacterially encoded genes , among which the majority were rare genes , shared by less than half of the group sampled ( 12 ) . around 2.5% of the microbially encoded genes 
 could be assigned to the functional group of enzymes involved in carbohydrate metabolism , and again these sets of genes were more similar within individuals of the same family ( 11 ) . 
 attention has also been drawn to the minimal gut microbiome  genes that are present in most bacteria and code for functions that are necessary for a bacterium to survive in the gut . 
 this set of genes can be divided into housekeeping genes ( amino acid synthesis , central carbon metabolism , and protein complexes such as rna / dna polymerases ) and those involved in gut - specific functions ( fermentation and adhesion ) . however , large portions of the orf / genes were of unknown functions ( 12 ) . 
 on the other hand , efforts have been made to identify the minimal gut metagenome coding for functions involved in the homeostasis of the whole ecosystem , which are present in most individual host samples . 
 most of these common bacterial genes are involved in the digestion of complex sugars and their subsequent fermentation . 
 these genes were coded not by the most abundant bacteria but rather by species present in low number , suggesting that even a relatively small bacterial group might be crucial for the establishment of a well functioning gut , as exemplified by archaea . 
 one must , however , note that the minimal gut microbiome and minimal gut metagenome are not mutually exclusive , and there are many genes that will belong to both groups , being necessary both for the survival of the bacterium as well as for gut function 
 . a higher degree of redundancy at the gene level rather than taxonomic level suggests that the core microbiome should exhibit shared bacterial functions that need to operate to ensure survival and successful symbiosis in the gut ( 11 ) . in a more recent study , inching closer to marrying microbial profiles and defined functions , arumugam et al . 
 ( 2011 ) have proposed a relatively new concept in the classification of the gut microbiome ( 13 ) . in this study , using stool samples representing different ethnicities , countries , and continents , the authors chanced on a perplexingly simple finding : despite a large and dynamic bacterial community , the combination of the microbes in each individual appeared to be systematically organized into clusters termed enterotypes . 
 even more surprising was that only three enterotypes were identified based on different clusters of bacterial species . 
 . a unique set of properties have been assigned to each enterotype , notably that there is no geographical demarcation , and further , no correlation between presence of a given enterotype with sex , age , or body mass index ( bmi ) could be shown . 
 the three enterotypes can be identified and viewed on various levels  prevalence of bacteria , methods of harvesting energy , and overrepresentation of certain enzymatic pathways . 
 for instance , enterotype 1 is dominated by bacteroides that derive energy mostly from fermentation of sugars and is enriched in genes coding for biotin biosynthesis . 
 the second enterotype is driven by prevotella and can be characterized by enrichment of genes coding for thiamine biosynthesis and harvesting energy from biodegradation of mucin glycoproteins . 
 enterotype 3 is mostly enriched in ruminococcus , also known to be able to degrade mucins . 
 this enterotype is , however , enriched in genes coding heme biosynthesis in contrast to the prevotella enterotype . 
 the phylogenetic structure in this instance appears to be indicative of specific functional properties ( summarized in fig . 
 the contemporary concept of enterotypes was given a boost recently when another study independently confirmed the existence of two enterotypes in a population of 98 individuals ( 6 ) . 
 the study further found that the enterotype was associated with long - term diet as shown by transient feeding experiments . 
 hence , although significant changes in the microbiota can occur rapidly on dietary change , no temporary shifts in enterotypes are forthcoming , that is the enterotype does not seem to budge easily . 
 the human gut flora has recently been classified as belonging to one of three different clusters termed enterotypes . 
 this diagram postulates that properties of the microbiome unique to each enterotype ( hatched - hexagon , hatched - triangle and hatched - rhombus ) give rise to enterotype - specific effects . each enterotype 
 likely also harbors common essential functions that may then facilitate bacterial survival in the gut . 
 the role of the individual 's immune system , nutritional input during early childhood , lifestyle , and the exposure to other gut microbial ecologies during the formative years of the microbiome may play a role in the establishment of the individual 's enterotype . 
 the use of genetically identical individuals ( twin registries ) , patient cohorts , and materials collected from longitudinal studies may aid in answering some of these queries . finally , although exciting , the enterotype postulate does require further , robust proof for universal acceptance and use . 
 it is altogether heartening to see that the findings of this study do not preclude the existence of additional enterotypes . 
 the results obtained from analyzing the bacterial communities of the human gut and their roles in host physiology suggest transience in the gut microbiota , mechanisms of which remain to be discovered ( 4 , 6 ) . 
 the 16s rna - based methods of identification still have a number of limitations including incomplete coverage of the sequenced gene fragments and problems of correct alignment to known bacterial genomes . 
 one predictable problem in trying to fuse function and identity is of course aligning the bacterial presence with respective gene expression patterns . 
 this conundrum has yet to be seriously tackled , although limited steps to redress this problem are underway . 
 ( 2011 ) in a metatranscriptomic study of 10 volunteers found microbial mrna expression to have a stable representation of genes involved in nutrient processing , energy harvest , and biosynthesis of cellular components . 
 finally , this study also found expression of small rnas that may have higher regulatory properties . 
 although in no way conclusive , this study highlights an additional criterion of gut health assessment to complement with the concept of enterotype that is presently available only at the metagenomic level ( 14 ) . to facilitate rapid , easy , and non - invasive procurement of samples , fecal material 
 the fecal microbiota is used to infer the entire gut microflora , and the lack of information about the potential difference between specific anatomical sites and viability of the bacteria , although glaringly obvious , is usually ignored . 
 microbiota composition has been to compare microbial communities residing on intestinal mucosal surfaces with those found in the feces . 
 the conclusions from such studies point toward variabilities in microbial composition between sample sites , indicating that looking at bacterial communities in the fecal sample may not be as representative for the gut microbiome as we would like to see it ( 8) . 
 the results obtained from fecal - derived studies have , nevertheless , given added credence to the long - held view that different physiological properties may be influenced by different microbial manifestations , for example varied microbiomes or enterotypes . 
 this may lend itself to some plasticity in the bacterial clusters , while retaining core enzymatic and biochemical functions that will reflect the individual enterotypes . 
 the current definition of enterotypes will no doubt undergo further refinement over the next few years . 
 this is likely to provide a better picture of microbial properties that impact on health status , xenobiotic , and nutritional metabolism and perhaps act as an indicator of disease susceptibilities . 
 ideally , this information may in the future aid the choice of preferred therapeutic modules for selected diseases . 
 although our current knowledge of enterotypes is far from complete , the potential uses of this new microbiome classification are tantalizing and set a standard to which the disease - associated bacterial flora or microbiome may be compared . 
 the results obtained from analyzing the bacterial communities of the human gut and their roles in host physiology suggest transience in the gut microbiota , mechanisms of which remain to be discovered ( 4 , 6 ) . 
 the 16s rna - based methods of identification still have a number of limitations including incomplete coverage of the sequenced gene fragments and problems of correct alignment to known bacterial genomes . 
 one predictable problem in trying to fuse function and identity is of course aligning the bacterial presence with respective gene expression patterns . 
 this conundrum has yet to be seriously tackled , although limited steps to redress this problem are underway . 
 ( 2011 ) in a metatranscriptomic study of 10 volunteers found microbial mrna expression to have a stable representation of genes involved in nutrient processing , energy harvest , and biosynthesis of cellular components . 
 finally , this study also found expression of small rnas that may have higher regulatory properties . 
 although in no way conclusive , this study highlights an additional criterion of gut health assessment to complement with the concept of enterotype that is presently available only at the metagenomic level ( 14 ) . to facilitate rapid , easy , and non - invasive procurement of samples , fecal material 
 the fecal microbiota is used to infer the entire gut microflora , and the lack of information about the potential difference between specific anatomical sites and viability of the bacteria , although glaringly obvious , is usually ignored . 
 microbiota composition has been to compare microbial communities residing on intestinal mucosal surfaces with those found in the feces . 
 the conclusions from such studies point toward variabilities in microbial composition between sample sites , indicating that looking at bacterial communities in the fecal sample may not be as representative for the gut microbiome as we would like to see it ( 8) . 
 the results obtained from fecal - derived studies have , nevertheless , given added credence to the long - held view that different physiological properties may be influenced by different microbial manifestations , for example varied microbiomes or enterotypes . 
 this may lend itself to some plasticity in the bacterial clusters , while retaining core enzymatic and biochemical functions that will reflect the individual enterotypes . 
 the current definition of enterotypes will no doubt undergo further refinement over the next few years . 
 this is likely to provide a better picture of microbial properties that impact on health status , xenobiotic , and nutritional metabolism and perhaps act as an indicator of disease susceptibilities . 
 ideally , this information may in the future aid the choice of preferred therapeutic modules for selected diseases . 
 although our current knowledge of enterotypes is far from complete , the potential uses of this new microbiome classification are tantalizing and set a standard to which the disease - associated bacterial flora or microbiome may be compared . 
 the former is a program to extract , preserve , and build energy reserves , whereas the latter will identify and eliminate elements detrimental to the long - term survival of the organism . in superorganisms such as ourselves ( and other eukaryotes ) , the resident microbiota is able to influence both types of metabolic programs . here 
 , we would like to discuss the current conundrums facing researchers exploring the microbial basis of adipocity . 
 we think that it is wise to discuss the phenomenal progress made thus far , and emphasize the legitimate concerns that still plague the interpretation of the results . 
 the role of the microbiota in regulation of host energy balance and metabolism can be studied at various levels . by introducing a semi - synthetic high fat diet ( hfd ) , containing a large amount of fat as well as sugars , one can induce weight gain and subsequent development of metabolic diseases ( 1517 ) . 
 the composition of the bacterial flora can then be monitored simultaneously with changes in the animal 's body weight . 
 one very powerful tool to address this question is germ - free mice raised in an environment completely devoid of bacteria . by simply comparing the physiology of germ - free mice with that of conventionally raised animals 
 more interrogative methods rely on using germ - free mice that have been colonized with one specific species of bacteria ( monocolonization ) , a whole group of bacteria , or the entire bacterial flora isolated from conventionally raised animals ( coventionalization ) . 
 experiments have shown that germ - free animals seem to be protected from diet - induced obesity ( 18 ) . however 
 , this protective effect was later shown to be strongly dependent on the sugar compositions , that is type of sugar of these diets , not just the amount of fat - derived calories ( 19 ) . 
 this may well be because germ - free animals lack the bacterial enzymes needed to digest polysaccharides , leading , therefore , to a lower calorie intake . 
 complex polysaccharides are processed in the gut and fermenting microbes produce short - chain fatty acids ( scfas ) . 
 butyrate , propionate , and acetate are scfas that can be directly used by colonocytes as an energy source or be further transported to the liver where they can be used as substrates for lipid synthesis ( 20 , 21 ) . 
 the levels of scfa can be measured directly in the caecum but more often than not fecal material is used because it is readily available . 
 however , one must remember that neither ceacal nor feacal levels of scfas are necessarily true indicators of the amounts produced in the gut because they are continuously used and absorbed by colonocytes . 
 genes involved in scfa synthesis were identified in several metagenomic screenings as being overrepresented and highly abundant in the gut flora , strongly suggesting that carbohydrate fermentation is one of the most conserved and important functions of the gut microbiome ( 9 , 12 ) . 
 genes governing scfa biosynthesis were also shown to be stably represented in at least one metatranscriptomic analyses ( 14 ) . given these observations , a series of experiments were performed to show that the ability of carbohydrate fermentation together with the production of scfas was linked to induction of obesity . when germ - free mice were colonized either by whole bacterial flora ( 22 ) or by saccharolitic fermenting bacteria such as bacteroidetes thetaiotamicron together with m. smithii , which facilitates fermentation ( 23 ) , an increase in body weight and adiposity was observed . 
 the effect of fermenters on host adiposity was strongly reduced in mice lacking the major scfa receptor , gpr41 ( g - protein coupled receptor 41 ) , hence suggesting a link between scfa producer and host cell signaling ( 23 ) . in other studies , 
 introduction of hfd in conventionally raised animals was accompanied by a shift toward fermenters in gut flora . 
 this manifested as the appearance of bacterial genes taking part in absorption , transportation , and utilization of simple sugars as well as polysaccharide fermentation leading to scfa production . 
 the authors then referred to this phenomenon as an  obesity - associated microbiome with increased capacity for energy harvest ( 24 ) . 
 animals on hfd had a microbial community that was characterized by a general decrease in microbial diversity and a phylogenetic shift from bacteroidetes toward firmicutes ( 24 ) . 
 this could further be attributed to an extensive bloom of one of the families within firmicutes , namely mollicutes , belonging to erysipelotrichaceae ( 25 , 19 ) . here 
 , it appears that noticeable phylogenetics shifts need not be a result of changes of many different species but rather the selective overgrowth of one or two members . 
 the change in the proportional abundance of two major bacterial phyla was then confirmed in another set of experiments . 
 however , in this instance , although the obese mice , as before , had more firmicutes , the levels of fecal scfas were not significantly different ( 26 ) . 
 intriguingly , one study showing a decrease in fecal scfa content in obese mice , compared to lean ones , has also been published ( 19 ) . 
 a more detailed approach to determine the pathways and routes of scfa absorption and use in these mouse models would shed more light on the relationship between hfd , scfa , and the microbiome . 
 one of the most intensely studied models of obesity and metabolic syndrome are animals with disrupted leptin signaling . 
 this is achieved in two ways , either by using mice lacking leptin receptor ( lep mice ) or by deleting the gene for the hormone itself ( ob / ob mice ) . 
 . both genotypes of leptin signalling deficiency are characterized by uncontrolled food intake that results in morbidly increased body weight and development of metabolic problems ( 27 , 28 ) . 
 comparison of intestinal microbiota compositions of ob / ob mice and their lean wild - type littermates revealed a decrease in bacteroidetes and a corresponding increase in firmicutes in obese animals , accompanied by a general decline in microbial diversity in this group ( 29 ) . 
 this shift was probably responsible for the observed increase in abundance of bacterial genes involved in sugar metabolism in another study ( 24 ) . 
 it is tempting to speculate that the increased food intake in those animals favored colonization by bacteria that had a higher metabolic capacity to extract energy , thanks to which both host and microbiota can benefit from otherwise lost calories . 
 interestingly , the phenotype was somewhat transferable : wild - type germ - free mice colonized with flora from ob / ob mice ( 24 ) as well as mice that received hfd ( 25 ) were better at storing fat compared to those colonized with a normal flora . 
 apart from effects on host metabolism arising through changes in the gut microbial community or the abundance of metabolites such as short chain fatty acids , the microbiome can also interact with the host tissues by tweaking host gene expression . among the many recently discovered genes 
 transcriptionally regulated by bacteria is fasting - induced adipose factor ( fiaf ) also known as angiopoietin - like protein 4 ( 22 , 30 ) . 
 fiaf is a lipoprotein lipase ( lpl ) inhibitor that in effect blocks lipid storage mediated through lpl activity . 
 2008 showed that fiaf mrna in epithelial cells increased in the presence of bacteria such as enterococcus faecalis ( 30 ) . 
 a putative mechanism through the activity of the metabolically important nuclear receptor peroxisome proliferator - activated receptor gamma ( ppar ) was suggested to govern this increase . in a recent study , 
 increases in the serum level of fiaf caused by administration of lactobacillus paracasei ssp paracasei f19 were associated with reduced weight gain in hfd - fed mice ( 31 ) . however , other studies reported lowered levels of mrna expression of fiaf in the intestinal tissue on bacterial exposure ( 22 , 19 ) . 
 these seemingly confounding results may be explained as reflecting two different situations : ( 1 ) in which exposure of mice to the whole bacterial community results in a decrease in intestinal fiaf levels ; ( 2 ) , while introduction of a single defined bacterial species has the opposite result . because e. faecalis ( 30 ) or f19 ( 31 ) seem to be able to upregulate fiaf expression , it is , therefore , tempting to speculate that certain bacterial species could have a therapeutical role in weight management . 
 another probiotic strain lactobacillus gasseri sbt2055 ( lg2055 ) was shown to impact health in small human cohort study . 
 volunteers with higher bmi and visceral fat when consuming formula milk containing lg2055 lost weight and had reduced abdominal adiposity . 
 this suggests that it has a positive effect on human metabolic health ( 32 ) . 
 the mechanism(s ) of action is unknown , but another study in rodents postulated that milk fermented by lg2005 was able to restrict dietary fat absorption in rat intestine ( 33 ) . in humans , however , the correlation between changes in the gut flora and weight gain are much less clear because all the confounding factors ( sex , age , diet , exercise , living environment , antibiotic treatments , disease history , and finally genetic heterogeneity ) have to be taken into account . 
 some groups claim that the shift from bacteroidetes toward firmicutes that was observed in mice was also associated with weight gain in humans ( 34 ) . 
 a later study outrightly presents the opposite finding showing a lower ratio of firmicutes to bacteroidetes in overweight subjects ( 35 ) . in other reports 
 , the decrease in the abundance of bacteroidetes could be confirmed , but no difference in firmicutes was observed , instead a bloom of actinobacteria carrying the majority of genes overrepresented in obese volunteers was apparent ( 11 ) . in another study , 
 no significant differences were seen in the proportion of firmicutes versus bacteroidetes in relation to bmi ( 36 ) . 
 ( 2010 ) also suggested that rather than major phylogenetic shifts , discrete , yet metabolically functional changes at the species level may occur in response to dietary changes similar to that observed for mollicutes in mice ( see above ) ( 35 ) . 
 presently , we do not fully understand the role of microbiota in the regulation of host adiposity . in mice , 
 the situation in human looks much more complicated , and it is very difficult to reach a consensus on the influence of a specific group of microbiota on changes in body weight . 
 the variability seen in different studies built on microbial composition may be due to a multitude of factors , including diet , volunteer cohort , duration of study , methods of sample preparation , and storage as well as methods of detection . 
 additionally , concentrations of bacterial metabolites such as scfa might vary due to increased microbial production , changes in absorption , shifts in microbial cross feeding patterns , or even the rate of transit ( reviewed in ( 37 , 38 ) ) . due to the complex nature of the gut environment 
 , it is quite unlikely that a single factor , such as a phylogenetic change in the microbioata , could be responsible for the development of obesity . 
 rather , changes in eating habits may favor bacterial species that now have to accommodate to a different nutritional environment . using germ - free mice , faith et al . 
 ( 2011 ) have attempted to formulate a model to predict microbiota changes that are dependent on diet ( 38 ) . 
 varying the abundance of 10 defined bacterial species that were introduced to the guts of germ - free mice that in turn received different refined diets ( protein , sugars , complex carbohydrate , and fat rich ) , faith and coworkers were able to infer a relationship between diet and microbiota . 
 it appeared that in general , bacterial presence relied on availability of a single component in the food , indicating that the bacterial community may predominantly depend on the diet and can be changed by manipulating the type of food ingested . within this limited study at least 
 however , as the authors rightly point out , one challenge ahead is to determine how the complex interplay between the human microbiome and the inordinate variability of the human diet affects parameters of gut health . presently , it is difficult to show whether the changes in the bacterial flora preceded weight increase , or if the changes that occur in the presence of each bacterial species are strongly associated with the ingested food . introducing more processed foods and antibiotics has led and will lead in the future to the reduction in the diversity of the microbial flora , simply because there will be a gradual decrease in nutrients available for microbial use ( reviewed in ( 39 ) ) . 
 it seems likely that a balanced bacterial flora is needed for maintaining good health , and perturbing this balance could disrupt controlled energy harvest leading to problems such as obesity . 
 inflammatory bowel disease ( ibd ) is an example of a complex disease postulated to arise following a disharmonious relationship between the immune system and the commensal flora . 
 genetic , microbial as well as dietary and environmental cues together seem to play a role in the development of pathology . 
 there are two major forms of ibd  ulcerative colitis ( uc ) affecting mostly colon and crohn 's disease ( cd ) affecting colon as well as the distal part of the small intestine ( reviewed in ( 40 , 41 ) ) . 
 previous studies have demonstrated that intestinal dysbiosis , characterized mainly by altered bacterial composition and loss of diversity , is strongly associated with the development of the disease ( 4244 , 12 ) . 
 there are two major models for experimental colitis , chemically induced colitis , and spontaneous colitis occurring in genetically modified animals . two methods to chemically induce colitis are extensively used : the administration of either dextran sulphate sodium ( dss ) in drinking water or 2 , 4 , 6-trinitrobenzenesulfonic acid intrarectally . both chemically induced and spontaneous models have advantages and disadvantages that have been extensively reviewed elsewhere ( 45 ) . 
 one posit that perturbed balance between the commensals bacteria and the host may play a role in the development of obesity is equally applicable to the pathogenesis of ibd . 
 studies performed on animals depleted of certain genes involved in immune regulation of gut homeostasis show that specific genetic mutations harbored by the host may cause an uncontrolled growth of otherwise underrepresented bacteria , which leads to development of colitis ( reviewed in ( 46 ) ) . in recent studies , 
 this altered microbiota was transmitted to wild - type animals that then develop the disease , suggesting that the microbiota retained the disease - causing shift that can be transmitted between individuals ( especially from mother to pups ) ( 47 , 48 ) epithelial cells have the ability to sense microbes by recognizing the microbe - associated molecular patterns ( mamps ) . 
 several families of receptors expressed both on the cell surface as well as in the cytoplasm are involved in recognition of those mamp molecules , such as toll - like receptors ( tlrs ) ( 49 ) , nucleotide - binding oligomerization domain ( nod ) , ( 50 ) and nod - like receptors ( nlrs ) ( 51 , 52 ) . in this review , we would like to briefly highlight the involvement of all three of these recognition molecules in the pathogenesis of ibd , and how this may be connected to shifts in microbial communities . 
 ligands recognized by tlrs are not only specific for pathogens but are also present on commensal microbiota , and it remains to be understood how the host distinguishes danger signals and homeostatic signals . 
 most tlrs use an adaptor molecule myd88 to transmit the signals within the cell ( 49 ) . 
 myd88- , tlr2- , and tlr4-deficient mice show a more severe phenotype of dss - induced colitis compared to their wild - type littermates . 
 similarly , animals with depleted microbiota ( following treatment with antibiotics ) show increased susceptibility to experimental colitis . on the other hand , administration of tlr4 or tlr2 
 ligands , in microbiota - depleted animals , was able to ameliorate the disease to a level observed in animals harboring normal flora . 
 these results strongly suggest that commensal microbiota play an important role in stimulating tlrs and setting the threshold of immune response needed for homeostasis ( 53 ) . 
 additionally , spontaneous colitis observed in conventionally raised , but not germ - free il10   mice is dependent on myd88 signaling . 
 deletion of myd88 in il10   animals abolishes tlr signaling and is protective against the development of the disease . 
 these results suggest that tlr signaling , although necessary for establishing and maintaining balance between pro- and antiinflammatory signals , might also be responsible for development of the disease if other factors , such as il10 , are missing or when the balance is otherwise disrupted ( 54 ) . 
 in addition to tlrs , microbial molecules can be recognized by members of the nod family that reside in the cytoplasm 
 mutations in the nod2 gene have been associated with increased susceptibility to ibd , especially cd , but the precise role of this protein in the pathogenesis of the disease remains unknown ( 55 ) . 
 nod2   mice display abnormal paneth cell functions and develop symptoms similar to cd ( 56 , 57 ) . 
 microbial samples coming from patients harboring mutations in the nod2 gene display a shift in bacterial composition not seen in individuals not carrying these mutations ( 58 ) . 
 additionally , nod2 animals harbor a microflora that is different from that of wild - type animals ( 59 ) . 
 the third important group of defense molecules is nlrs that form cytoplasmic complexes known as inflammasomes that are able to sense both endo- and exogenic stress or damage - associated signals ( reviewed in ( 60 ) ) . 
 apoptosis - associated speck - like protein ( asc ) , into multiprotein complexes that activate caspase 1 and subsequent cleavage of il-1b and il-18 ( 61 , 62 ) . 
 mice that lack components of the inflammasome ( nlrp6 , asc ) or downstream targets of active complex ( caspase 1 , il-18 ) suffer from more severe dss - induced colitis than their wild - type littermates ( 48 ) . 
 inflammasome loss resulted in the development of more colitogenic microflora that could be transferred to wild - type mice that were cohoused or cofostered with knock - out mice , resulting in more severe phenotype in these animals . 
 two groups of bacteria were associated with the colitogenic microbiome : family prevotellaceae in the phylum bacteroidetes as well as a genus belonging to tm7 phylum . 
 both groups are capable of disrupting the mucus barrier through its degradation and have been previously associated with development of ibd and periodontal disease . 
 the prevotella cluster has been detected on mucosal surfaces taken from patients suffering from uc or cd but not in the guts of healthy subjects ( 63 ) . 
 2006 also highlighted the abundance of the members of the prevotella genera in mucosal biopsy specimens from uc patients ( 64 ) . 
 on the species level , prevotella denticola is much more prevalent in subjects suffering from periodontitis than in healthy subjects ( 65 ) . 
 antibiotic resistant bacteria belonging to prevotella genus has also been identified among periodontal bacteria ( 66 ) . 
 tm7 phylum members ( gram - positive , non - cultivable bacteria ) could be found in equal numbers in biopsies obtained from uc , cd , and non - ibd patients ; however , the composition of the cloned 16s rrna sequences differed significantly between diseased and healthy samples ( 67 ) . 
 one of the clones showed very high similarity to a clone suggested to play a causative role in periodontal disease ( 68 ) , and many members of that phylum have been associated with oral inflammation ( 69 , 65 , 70 ) . hence , there appear to be commonalities in mucosal inflammation , be it manifesting in the oral or intestinal cavities . 
 klebsiella pneumoniae and proteus mirabilis as highly associated with colitogenic phenotype of t - bet rag2 mice ( 71 ) . 
 these animals harbor a deficiency in innate immunity relying on t - cells responses , and they spontaneously develop colitis ( referred by authors as t - bet rag2 uc  truc mice ) ( 47 ) . 
 the colitic phenotype was transmittable to cohoused or cross - fostered wt mice , akin to the inflammasome - deficient mice discussed above ( 71 ) . 
 neither of the identified species was , however , able to cause colitis when transferred to germ - free mice , indicating a need for other bacterial signals for a full - blown inflammatory reaction . 
 klebsiella pneumoniae together with other drug - resistant enterobacteriacae has been previously reported to be present in oral cavities of patients with periodontal disease . 
 it is postulated that the hydrolytic enzymes produced by these bacteria play a role in the destruction of the tooth - supporting tissues ( 72 ) . 
 frank et al . ( 2007 ) identified additional shifts in microbiota in uc and cd patients compared to non - ibd samples ( 73 ) . 
 uc and cd patients had fewer sequences representative of bacteroides and lachnospiraceae ( a subgroup of firmicutes ) and an overrepresentation of proteobacteria and also bacillus  another subgroup of firmicutes . in the small intestine , proteobacterial sequences were more abundant and bacillus sequences less so , in uc and cd patients , relative to non - ibd samples . 
 the bacterial profiles belonging to some cd and uc patients were clustered separately from the non - ibd samples and were characterized among the others by a decreased bacterial load . 
 ibd patients recruited to this study harbored on average 25% less bacterial genes than healthy subjects , and principal component analysis clearly separated ibd patients from healthy subjects and cd patients from uc patients . 
 one speculation , built on the hygiene hypothesis , is that decreasing diversity of the bacterial ecology may be a causative factor in diseases such as ibd . 
 a new player in ibd pathogenesis is atg16l1 ( autophagy - related 16-like 1 ) that codes for a protein that is a part of a large complex necessary for autophagy  a process in which cell components are targeted to degradation . 
 the disease variant of the gene can be found in around 50% of european population and confers a twofold increase in disease susceptibility ( 7476 ) . 
 people harboring such mutations also have altered microbial flora ( 58 ) . in mice with deleted atg16l1 , a reduced capacity for autophagy 
 is seen ( 77 , 78 ) . on viral infection with a murine norovirus ( mnv ) , 
 agt16l1 mice display additional features with strikingly abnormal paneth cells , resulting in aberrant packing and exocytosis of antimicrobial peptides . 
 on dss treatment , these mice developed colonic ulcers with various hallmarks of cd - associated lesions . 
 administration of antibiotics in these mice prevented the abnormal dss - induced pathologies suggesting the necessity of the microflora to induce a phenotype resembling cd ( 79 ) . 
 interestingly , although antibiotics have been used to treat cd and uc , it seems to be more effective in the case of the former , as they could also prevent the relapse of the disease ( 80 ) . 
 apart from the inflammatory disorder , ibd also predisposes to colorectal cancer ( crc ) . around 18% of patients 
 changes in bacterial composition seen prior to the manifestation of colitis may also be associated with the pathology of colorectal cancer because truc mice ( described above ) spontaneously develop colonic dysplasia and rectal adenocarcinoma , resembling human colonic cancers associated with ibd ( 83 ) . 
 toxin produced by this bacterium ( bft ) was identified as being a causative factor for both colitis and very early tumorigenesis in the apc mouse model of colon cancer ( harboring a heterozygous mutation in the apc gene ) ( 84 ) . 
 bft acts as an oncogene by cleaving e - cadherin , a tumor suppressor protein triggering the nuclear localization of b - catenin and colonic cells proliferation , through the activation of the wnt pathway . 
 it also activates the nf-b signaling pathway in the cells that results in secretion of proinflammatory cytokines . 
 interestingly , btf exerts its oncogenic functions via the th17 cells ( 84 ) , the development of which is solely dependent on the presence of commensal bacteria in the gut ( 85 ) . 
 hence , the pathologies linked to klebsiella ( 71 ) and b. fragilis appear to be dependent on the presence of the whole microbial community . 
 although many bacterial species or groups are associated with the pathology of ibd , other prokaryotic partners may play protective roles . 
 interestingly , b. fragilis implicated earlier in crc may have protective effects in infectious colitis caused by helicobacter hepaticus . 
 further research could link the protective effects of psa with the function of il10 in response to commensal bacteria . 
 psa was shown to not only prevent but also cure experimental colitis in mice , by actively inducing mucosal tolerance ( 87 ) . 
 the presence of whole bacterial flora , as well as the fraction of fermenting microbiota , has also been suggested to play a protective role against colitis and helps exclude lethal invasions of pathogenic bacteria . 
 germ - free animals are characterized by a more severe phenotype of dss - induced colitis compared to conventionally raised animals ( 88 , 89 ) . 
 this phenotype could be reverted on conventionalization of gf animals , or by the administration of acetate in drinking water prior to dss treatment . 
 g - coupled receptor 43 ( gpr43 ) is one of the cell surface receptors that is activated on binding of acetate . 
 gpr43 mice showed greater morbidity and reduced potential to overcome the disease ( 89 ) . 
 acetate produced by certain species of bifidobacteria has also been found recently to protect mice from colitis and subsequent death caused by infection with escherichia coli o157:h7 ( 90 ) . 
 this clearly indicates that intestinal bacterial metabolites , garnered through microbial processing of available nutrients , do have a significant effect on gut health . in conclusion , 
 recent studies have prompted researchers to peer deeply into the altered microbial composition in patients suffering from ibd . 
 this may be associated with a general reduction of bacterial diversity as well as shifts toward certain bacterial groups and the appearance of specific , hitherto unidentified species . 
 the changes might be driven by environmental cues and genetic predispositions . as elegantly shown by cadwell et al . 
 ( 2010 ) , ibd is a very complex disease and to understand it , we have to look at more than one factor and be able to link microbial changes , presence of viruses with environmental conditions , such as diet and small changes in the host dna , that are not able to cause a disease when present alone ( 79 ) . 
 this observation only serves to emphasize the importance of understanding the cross talk between different microbial communities thriving in the intestinal milieu . 
 the dynamic communities of intestinal bacterial biofilms are likely to play a major role in setting the stage for host  microbe interactions and subsequent physiological outcomes . 
 the vast numbers of bacteria in the gut have over the years been postulated to directly extend their realm of influence beyond the immediate gut environment . here , we briefly list the role of the gut flora liver function , glucose metabolism , and the control of behavior . 
 the gut microbiota has been attributed a role in the development of non - alcoholic fatty liver disease ( nalfld ) ( 91 , 92 ) . 
 urinary metabolites of the nafld - susceptible mice strain 129s6 when fed hfd were enriched in microbiota - derived methylamines : dimethylamine , trimethylamine ( tma ) , and trimethylamine - n - oxide ( tmao ) . 
 tma is synthesized exclusively by symbiotic bacteria ( 93 ) and can be further transformed into tmao by the microbiota themselves or at least in humans by the liver enzyme fmo3 . 
 the production of methylamines by microbiota results in decreased bioavailability of choline for the host and seems to trigger nafld in mice . 
 ( 2011 ) , who could link high levels of tmao in plasma with increased risk for cardiovascular disease in humans ( 92 ) . 
 the interdependence between microbiota , metabolism , and inflammation as well as the host genotype has also been elegantly shown . 
 mice lacking tlr5 suffer from spontaneous colitis ( 94 ) and at the same time have higher body weights ( 95 ) than their wild - type littermates . 
 interestingly , the increase in weight could be attributed to higher chow consumption and indeed , the trait disappeared when mice were subjected to antibiotic treatment . 
 the obese phenotype could be transferred from tlr5 animals to wild - type germ - free mice through the gut flora , suggesting that microbiota may be a factor responsible for hyperphagia and increased body weight in tlr5 mice . 
 hyperphagia is also very much associated with satiety signals emanating from the gut and directed to the brain . 
 increasingly , the gut flora is being recognized as a factor in the regulation of the central nervous system ( cns ) . 
 germ - free mice display increased motor activity and reduced anxiety , compared with conventionally raised ones . 
 this phenotype could be reversed if germ - free animals were exposed to bacteria early in life ( 96 ) . 
 lactobacillus rhamnosus has been recently shown to possess antiinflammatory properties and has been suggested as a candidate to interfere with the cns signaling through the vagus nerve , which allows communication between the viscera and the brain . 
 this probiotic was shown to reduce plasma levels of stress - induced corticosterone as well as anxiety- and depression - related behavior . 
 these results strongly suggest that modulation of intestinal microbiota in healthy animals may lead to neurochemical changes affecting animal behavior ( 97 ) . 
 these examples are telling in that they have attracted the attention of researchers who have traditionally been working away from the gut environment to seriously consider the gut microbial milieu when studying a variety of diseases ( see fig . 
 the scope of this review unfortunately precludes the discussion of microbiome as a source or target for therapies and treatments of various ailments ( reviewed in ( 98 ) ) the gut microbiota can affect a variety of host responses . in this schematic diagram , host genotype , the environment , and diet 
 the microbiota exerts its effects on the intestine through bacterial signaling molecules and metabolic products ( microbiome ) . 
 components of the microbiome can also enter the circulation , be transported to various organs , and potentiate multiple effects in these organs ( gray arrow ) . at the same time , the gut  brain axis can circumvent intestinal absorption and may allow the microbiome to directly affect the brain . 
 once touted as the forgotten organ ( 99 ) , the microbiota is slowly beginning to reveal many of its secrets . having been under close scrutiny for many decades 
 , the gut microbiota is now said to dictate important traits in the human host . 
 future research delineating properties of the oral microflora should desist from chasing purely metagenomic queries . 
 the oral cavity has one distinct advantage over the gi tract : accessibility , a trait that should be exploited to the fullest . 
 crucially , information thus accrued would be important to those of us aspiring to understand the workings of the microbiota in the gi tract and beyond . 
 painstakingly , slow research of yesterday that lay much of the groundwork for today 's ideas has given way to meta - analyses of high throughput findings . nevertheless , as we begin to answer the questions of  who is there and  what are they doing there, we inadvertently arrive at the inquiry  how and why the superorganism functions . herein lies the potential to alter the course of many a pathology . 
 what is obvious is the notion that microbial influence permeates into nearly every aspect of our existence . as a component species of a superorganism 
 , we should now universally acknowledge the importance of the microbial world in which we live and , crucially , that which lives on us . 
 there is no conflict of interest in the present study for any of the authors .","the gut microbiota consists of trillions of prokaryotes that reside in the intestinal mucosa . 
 this long - established commensalism indicates that these microbes are an integral part of the eukaryotic host . 
 recent research findings have implicated the dynamics of microbial function in setting thresholds for many physiological parameters . 
 conversely , it has been convincingly argued that dysbiosis , representing microbial imbalance , may be an important underlying factor that contributes to a variety of diseases , inside and outside the gut . 
 this review discusses the latest findings , including enterotype classification , changes brought on by dysbiosis , gut inflammation , and metabolic mediators in an attempt to underscore the importance of the gut microbiota for human health . 
 a cautiously optimistic idea is taking hold , invoking the gut microbiota as a medium to track , target and treat a plethora of diseases .",pubmed
"onychomycosis is a chronic fungal infection of the nail , characterized by nail discoloration , thickening , and defect . 
 the involved pathogens are dermatophytes , yeasts ( candida ) , and nondermatophytes.1 dermatophytes such as trichophyton rubrum and trichophyton mentagrophytes are the most common agents of onychomycosis , accounting for 50%90% of cases , with prevalence of the infection higher in toenails than in fingernails.2 
 candida and nondermatophytic molds are frequent in countries with hot and humid climate.3 if untreated , nail fungal infections can extend to other nails and potentially cause further complications , especially in at - risk patients . 
 aging and reduced peripheral circulation are among the major risk factors for onychomycosis ; diabetes weakens the immune function , favoring slow nail growth , thickening , and susceptibility to infections and nail injury.47 onychomycosis sometimes is associated with nail psoriasis , favored by abnormal capillary units and onychodystrophy.8 differential diagnoses of onychomycosis include chronic nail eczema , bacterial infections , traumatic onychodystrophies , pachyonychia congenita , nail bed tumors , yellow nail syndrome , and idiopathic onycholysis.9 the treatment of onychomycosis is sometimes difficult because the predisposing factors are not influenced by therapy.10,11 onychomycosis is usually treated with topical or systemic antifungals.12,13 local treatment requires strong patient adherence because the treatment time is generally long ; moreover , onychomycosis - related subungueal hyperkeratosis is generally thick and this limits the penetration of the antimycotic drug . 
 consequently , long - term treatments can discourage or induce stopping of the therapy before healing is achieved . 
 the cochrane meta - analysis of crawford et al14 reports limited success in the treatment of onychomycosis with topical agents . 
 although many therapeutic options are available , new drugs with a greater safety and relatively rapid efficacy are required . 
 one of the most used topical drug for nail psoriasis is tazarotene.15,16 its efficacy is classically related to its immunomodulating properties and anti - inflammatory activity , with consequent inhibition of the infective process.17 tazarotene is also successfully used for acne treatment , given its anti - inflammatory and immune - modulating effectiveness that protect keratinocytes from infection by propionibacterium acnes.17 epidermal penetration of tazarotene depends on its ability to reduce hyperkeratinization , favoring keratinocyte differentiation , as also documented by the reduction of expression of hyperproliferation - associated keratins 6 and 16.18 the latter appear dysregulated in genetic diseases , such as pachyonychia congenita and focal palmoplantar keratoderma.18 as a matter of fact , fungal infections could exacerbate or predispose an individual to pachyonychia congenita.19,20 in light of our previous studies reporting the clinical efficacy and the simplicity of the use of tazarotene in the nails of psoriatic patients,15,16 we investigated its effect on distal and lateral subungual onychomycosis in an open preliminary study of immunocompetent patients and its fungistatic activity in vitro . 
 we report an observational open clinical trial in 15 patients affected by onychomycosis of the toenail treated with topical tazarotene ( zorac / tazorac ; allergan inc , irvine , ca , usa ) 0.1% gel in an off - label use . 
 the enrolled patients were of both sexes and aged between 14 years and 70 years ( table 1 ) . 
 patients eligible were those affected by distal and lateral subungual onychomycosis without associated skin or systemic diseases , nave or in washout for other treatments . 
 exclusion criteria were immunosuppression , treatment for other systemic diseases , and the presence of other diseases causing nail dystrophy such as psoriasis , eczema , and lichen planus . 
 the drug was applied once a day on the affected nail plates and nail folds for 12 weeks ( end point ) . 
 we evaluated onycholysis , nail bed discoloration , and subungueal hyperkeratosis ( thickening of the subungueal region ) at baseline and end point , assigning the following semiquantitative scores for onycholysis and discoloration : absence ( 0 ) , 0%25% ( 1 ) , 25%50% ( 2 ) , and > 50% ( 3).14 for subungual hyperkeratosis , the scores were as follows : absent ( 0 ) , 1 mm ( 1 ) , 2 mm ( 2 ) , and > 3 mm ( 3).21 we also evaluated changes of the affected area comparing baseline and end point appearance of the affected target areas , as reported.22 briefly , the response rate was assessed by the analysis of digital photographs of the affected nails taken at the baseline and at the end of the study.23 data were analyzed by means of analysis of variance and student s t - test for continuous data and by mann 
 whitney test for ordinal data ; p - values < 0.05 were considered statistically significant . 
 mycological cultures and potassium hydroxide stains were also performed on nail samples taken by scraping , clipping , or curetting the affected nail site and subungual region at the baseline and at the end point . 
 staining of fungi was performed with lactophenol cotton blue solution ( sigma - aldrich , st louis , mo , usa ) . 
 fungistatic activity of tazarotene in vitro was investigated by disk diffusion assay ( kirby  bauer method).24 briefly , 1 g of 0.1% tazarotene gel was dissolved in 3 ml of physiological solution in a sterile tube . to test the effect , 
 the fungi analyzed were candida albicans , c. glabrata , t. mentagrophytes , and t. verrucosum . 
 all statistical analysis was performed using the statistical package spss16.0 ( spss inc , chicago , il , usa ) . 
 mycological cure in target nails ( ie , those with mycological confirmation of onychomycosis at baseline ) was observed in subungual samples collected at the first follow - up visit ( 4 weeks ) in 40% of patients , and complete cure with complete clinical healing in all patients was observed at the end point ( 12 weeks ) . 
 direct microscopy and in vitro laboratory culture of the sampled material collected after 12 weeks were negative for infections . 
 representative clinical images of four patients at baseline and end point after tazarotene treatment are reported in figure 1 . 
 therapeutic results were good , with no side effects except for mild erythema on the perionychia in two patients ; nail growth and tropism were also restored . as shown in figure 2 , 
 the percentage of affected area and the proportion of affected patients decreased ( p<0.001 ; figure 2a and b ) . 
 onycholysis , subungual hyperkeratosis , and discoloration completely disappeared in all patients at the end point ( p<0.04 ; figure 2c ) . 
 the nails were completely healed at the follow - up visits up to 6 months . 
 patients fungal culture test11 was performed at baseline ( figure 3 ) ; 66.7% of patients were positive for t. rubrum ( figure 3a ) , 13.3% for t. mentagrophytes , 13.3% for e. floccosum , and 6.7% for t. tonsurans . 
 disk diffusion assay ( figure 3b and c ) document the inhibition of t. mentagrophytes growth , starting with 2 ml of tazarotene solution , which became almost complete with 5 ml ( figure 3b ) , as well as the reduced growth of c. albicans ( figure 3c ) starting with 1 ml of tazarotene solution . 
 similar results were observed with plates with c. glabrata and t. verrucosum , with greater prominence in the presence of 4 ml and 5 ml of the solution ( not shown ) . 
 in this study , we have reported the therapeutic efficacy of tazarotene 0.1% gel for the topical treatment of distal and lateral subungual onychomycosis . 
 t. rubrum , t. mentagrophytes , and e. floccosum are the most common agents implicated in onychomycosis.12 these dermatophytes are hyaline - septated molds , whose growing fungal hyphae penetrate into the stratum corneum of the skin and nails . 
 the fungal infection produces keratinolytic proteases , creating a gap in the living cells.25 some fungi have developed the ability to digest keratinous debris in soil and the faculty of parasitizing keratinous tissues in animals.25 compared to the normal skin , the nail apparatus has a different immunological profile , a strong innate immunity , and an increased expression of antimicrobial peptides.26 on the other hand , the nail apparatus has a lower level of major histocompatibility complex class-1a antigen expression , production of immunosuppressive agents , anomalies of antigen - presenting cells , and inhibition of natural killer cell activity,27 all aspects that favor onychomycosis . 
 successful topical treatment of nail onychomycosis is fraught with difficulty due to several factors , including slow nail growth , the physical presence of nail plate interfering with nail bed access , and the difficulty in finding compounds with adequate pharmacologic profile for nail penetration.28 these difficulties are reflected in the high incidence of recurrence after topical therapeutic clearance.29 at present , the most frequently used topical therapies for onychomycosis are amorolfine 5% , ciclopirox 8% nail lacquer , and tioconazole 28% solution , which need to be applied for prolonged periods and require strong adherence by the patients.13 amorolfine 5% nail lacquer is effective as monotherapy for the treatment of onychomycosis , but with limited efficacy when distal nail involvement is greater than 50%.30 up to june 2014 , ciclopirox was the only topical treatment approved by the us food and drug administration for onychomycosis in the us.28 the action of ciclopirox differs from that of other topical antifungal drugs , which generally act through ergosterol inhibition . 
 the high affinity of ciclopirox for trivalent metal cations results in the inhibition of the metal - dependent enzymes responsible for the degradation of peroxides within the fungal cell.31 efficacy of ciclopirox 8% as a topical monotherapy ( ranged from 20% to 36% ) has been disappointing and limited to the mildest cases of toenail onychomycosis.28 for the remaining cases , a combined therapy with a systemic antifungal is recommended.28 nevertheless , oral antifungals have general adverse effects , including intestinal and respiratory complaints.32 tioconazole is an imidazole antifungal agent recommended for local infections caused by candida28 and can be used for the topical treatment of pachyonychia . 
 efinaconazole 10% and tavaborole 5% solutions are new topical antifungals specifically developed for the treatment of dermatophytic onychomycosis . in phase iii clinical trials , 
 both newer agents were applied once daily for 48 weeks without concomitant nail debridement . reported mycologic cure rates with efinaconazole are superior to that achieved with ciclopirox 8% nail lacquer.33 in our study , complete healing after topical treatment with tazarotene was obtained at the first follow - up visit after 4 weeks in 40% of patients and complete healing in all patients after 12 weeks , with a rapid and progressive amelioration of all evaluated clinical parameters . 
 sometimes , the ineffectiveness of topical treatment is due to the subungual strong hyperkeratosis , which prevents drug penetration.34 hyperkeratosis also represents one of the most frequent features of nail psoriasis , and nail psoriatic patients also suffer from onychomycosis.35 in our patients , tazarotene has proven quite effective in reducing hyperkeratinization in fungus - infected nails . concerning the therapeutic effects of tazarotene , our results in vitro document a direct fungistatic activity . 
 tazarotene is a synthetic third - generation retinoid derived from vitamin a , which has proved to be beneficial in modulating keratinocyte proliferation and in reducing inflammation . 
 tazarotene is used for the treatment of acne , nonmelanoma skin tumors , and inflammatory skin diseases such as psoriasis.3638 in particular , tazarotene s effect is mediated by the intracellular upregulation of cellular retinoic acid - binding protein - ii expression,39 which transactivates nuclear retinoic acid receptors ( rars ) , in particular rar and rar , by binding to specific dna sequences on their promoter gene regions , leading to reduced proliferation of normal and neoplastic cells and favoring differentiation and apoptosis.3638,40 the reduction in the excessive v - akt murine thymoma viral oncogene homolog 1 activation supporting cell growth41 may also explain the beneficial effects of tazarotene on ungual hyperkeratosis . 
 moreover , tazarotene can exert a protective action on the microvascular bed against fungal infection , similarly to that reported for psoriasis patients.16 finally , the activity of tazarotene against inflammation through immunomodulation may induce the blocking of fungal keratinolytic proteases , thus contributing to its efficacy . 
 a limitation of our preliminary study results is the absence of a randomized placebo control group . 
 nevertheless , a recent analysis published in 2014 reveals that in 21 randomized controlled trials of dermatophytic nail onychomycosis , present in the pubmed database , placebo cure varied from 8.7% to 1.2% , documenting that the pooled placebo cure mean rate is very low.42 
 our preliminary results documented the safety and rapid efficacy of topical treatment with tazarotene on distal and lateral subungual onychomycosis , thus representing an alternative new promising therapeutic approach . treated and cured patients were followed every 4 weeks , with no evidence of recurrence after 6 months . 
 we also documented the fungistatic activity of tazarotene in t. mentagrophytes , t. verrucosum , c. albicans , and c. glabrata , supporting the theoretical assumption about its direct action on fungus replication . 
 our clinical evidence of efficacy suggests that tazarotene could be useful in those patients with treatment - resistant forms of onychomycosis or in those with evident enhanced nail thickening , wherein the therapeutic reducing action of the retinoid can be providential 
 . further studies concerning the specific effects of tazarotene and in a larger cohort of patients with a longer follow - up are needed to confirm the efficacy in terms of relapse and reinfection .","backgrounddistal and lateral onychomycoses are the most frequent forms of onychomycosis , causing subungual hyperkeratosis that usually limits local penetration of antimycotic drugs . 
 tazarotene exerts anti - inflammatory and immune - modulating activities toward both infective agents and damaged keratinocytes . 
 given the well - documented efficacy of tazarotene on hyperkeratotic nail psoriasis , we investigated its therapeutic use in onychomycosis.patients and methodswe designed a preliminary open clinical trial in patients affected by distal and lateral subungual onychomycosis of the toenails and verified the fungistatic activity of tazarotene in vitro . 
 fifteen patients were treated with topical tazarotene 0.1% gel once per day for 12 weeks . 
 mycological cultures and potassium hydroxide stains of nail samples were performed at the beginning and at the end of the study . 
 treatment was considered effective when clinical healing and negative mycological culture were obtained . 
 onycholysis , nail bed discoloration , and subungual hyperkeratosis were measured using standardized methodologies and analyzed by means of mann  whitney test and analysis of variance . 
 fungistatic activity of tazarotene was evaluated by disk diffusion assay.resultssix patients ( 40% ) reached a mycological cure on target nail samples already after 4 weeks of treatment . 
 complete clinical healing and negative cultures were reached in all patients at week 12 , with a significant improvement of all clinical parameters of the infected nails . 
 disk diffusion assay after 48 hours of incubation with tazarotene solution showed a central area of inhibition in all examined fungal cultures.conclusionour results documented a good clinical outcome using topical tazarotene 0.1% gel in distal and lateral subungual onychomycosis and its fungistatic activity of tazarotene in vitro . 
 the majority of patients appeared cured at a 6-month follow - up . the efficacy and safety of tazarotene 
 must be confirmed on a larger number of patients , although already documented in nail psoriasis patients often affected by onychomycosis .",pubmed
"cardiac resynchronisation therapy ( crt ) has become an important treatment for patients with heart failure and left ventricular ( lv ) dyssynchrony [ 13 ] . 
 however , clinical non - response to crt is reported in 2535  % of patients . 
 although a large variety of causes for a suboptimal response have been cited , most attention has been focused on the selection of patients eligible for crt , and less on inadequate delivery of crt therapy . however , even with growing experience and improved materials and tools , the optimal position can not always be reached in one of the tributaries of the coronary sinus . 
 this can be due to the absence of suitable side branches in the posterolateral area , coronary vein stenosis , lead instability , high stimulation threshold , phrenic nerve stimulation , or a combination of the above [ 57 ] . 
 we studied the acute haemodynamic response ( ahr ) of the implanted system and alternative left ventricular endocardial pacing sites in patients clinically not responding to crt . 
 we asked patients who remained in new york heart failure class iii or iv despite at least 6 months of crt and adequate medical therapy and with no therapeutic options left to undergo an acute haemodynamic study in search of improving existing crt , as previously described [ 8 , 9 ] . 
 twenty - four non - consecutive patients , 23 males , age 72.8    9.1 years , mean ejection fraction 22.5    7.1  % , agreed to undergo this test ( table  1 ) . 
 the ecg prior to implantation showed left bundle branch block ( lbbb ) in 12 , non - lbbb in 4 , and right ventricular ( rv ) pacing in 8 patients . 
 table 1characteristics of clinical non - responders ( 24 patients)patientgenderage ( years)nyha classicm / dcmejection fraction ( % ) qrs morphologyqrs width ( ms)pt . 01m56ivdcm13lbbb135pt . 
 ivicm17rvp210average23m/1f72.8    9.13.2    0.36dcm22.5    7.112lbbb/8rvp175    22patient characteristics of 24 clinical non - responders to crt . 
 nyha class new york heart association class , dcm dilated cardiomyopathy , icm ischaemic cardiomyopathy , lbbb left bundle branch block , non - lbbb non left bundle branch block , pt . 
 characteristics of clinical non - responders ( 24 patients ) patient characteristics of 24 clinical non - responders to crt . 
 nyha class new york heart association class , dcm dilated cardiomyopathy , icm ischaemic cardiomyopathy , lbbb left bundle branch block , non - lbbb non left bundle branch block , pt . 
 a 6f multipurpose angioplasty guiding catheter was introduced into the lv after a standard transseptal catheterisation . in one patient 
 , the guiding catheter was introduced through the radial artery following coronary angiography . through this guiding catheter , endocardial pacing and measurements of lvdp / dtmax 
 were accomplished by roving a medtronic 6416 temporary bipolar screw - in lead ( medtronic inc . 
 minneapolis , mn . ) and a radi pressure wire ( radi medical , a st jude medical company , st . 
 initially the atrioventricular and interventricular intervals of the implanted system were optimised to obtain the maximal lvdp / dtmax . 
 lv pacing was performed from basal posterolateral , mid - posterolateral , lv apical and lv septal locations , and optimal ahr obtained after optimisation of the crt system for all positions . 
 if none of the 4 endocardial positions were opposite the position of the coronary sinus lead , an additional measurement was done at this location . a rise in lvdp / dtmax 
 15  % from baseline was considered to be a positive haemodynamic response . in all patients with an ahr of   15  % during lv endocardial pacing , the interval between the onset of the qrs complex and the intrinsic activation at the lv electrode ( q - lv interval ) was measured and the ratio between q - lv / qrs width interval calculated . 
 this ratio expresses the relation between q - lv interval and qrs width , which is a better indicator for late or early lv sensing than the absolute value of q - lv . 
 notwithstanding that all patients were clinical non - responders , in 5 patients lvdp / dtmax with the implanted coronary sinus system increased   15  % after optimisation : average 30.2  % , 15.644.5  % ( table  2 ) . in 3 out of these 5 patients with the coronary sinus lead in a posterolateral position , endocardial pacing did not increase the lvdp / dtmax substantially ( ahr less than 3  % and even an adverse effect from lv endocardial pacing was observed in one patient ) . in one of the two remaining patients with an apical position of the coronary sinus lead , 
 the ahr could be increased during lv endocardial pacing from 19.766  % and we considered the increase sufficient to justify the upgrade to lv endocardial pacing ( fig .  1 ) . 
 table 2individual haemodynamic results of all clinical non - responders ( 24 patients)patientqrs morphologybaseline lvdp / dtmax ( mmhg / s)position cs leadahr cs lead ( % ) ahr cs - level - endo(%)lv endo optimal positionahr lvendo ( % ) pt . 
 07non - lbbb893pl - mid  23.4lv septal  21.5pt . 08lbbb827pl - bas44.951.0pl - bas51.0pt . 
 24rvp489lv apical12.717.2pl - mid24.1average891    2478.7    15.810.5    8.919.6    17.5haemodynamic measurements of 24 clinical nonresponders to crt . 
 baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . 
 grey shaded patients have an ahr from the cs lead   15  % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . 
 individual haemodynamic results of all clinical non - responders ( 24 patients ) haemodynamic measurements of 24 clinical nonresponders to crt . 
 baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . 
 grey shaded patients have an ahr from the cs lead   15  % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . 
 all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . 
 arrows indicate the position of the bipolar pacing lead angiographic pictures of the haemodynamic study in patient no . 
 all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . 
 arrows indicate the position of the bipolar pacing lead in the 19 patients with an ahr from the implanted system <  15  % , 9 patients had an increase in ahr above the 15  % limit . 
 the results of the total group of 24 patients showed a positive response in 11 patients with an apical lead position in 7 , anterolateral in 2 and mid - posterolateral in 2 patients . 
 a negative response of endocardial pacing was observed in 13 patients with a basal posterolateral lead position in 3 , mid - posterolateral in 8 and apical in 2 patients . in 5 out of the 9 acute haemodynamic non - responders 
 ( ahr <  15  % ) with lbbb , the ahr could be increased above the 15  % level by lv endocardial pacing . 
 the average ahr with the coronary sinus system was 9.6  % ( 5.814.9  % ) and at the lv endocardial optimal position 25.8  % ( 19.034.3  % ) . 
 the average qrs width in these patients was 169  ms ( 135198  ms ) and the average q - lv at the optimal lv endocardial pacing site 157  ms ( 128176  ms ) , resulting in a q - lv / qrs ratio of 93  % . 
 the position of the original coronary sinus lead was apical in 3 , anterolateral in 1 and mid - posterolateral in 1 patient ( fig .  2 ) . 
 2recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions recordings of the temporary study of patient no . 
 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions in the remaining 4 patients , the average ahr at the optimal endocardial position increased <  15  % , with an average of 7.3  % ( 1.79.9  % ) . 
 the average qrs width in these patients was 176  ms ( 149209  ms ) and the average q - lv 136  ms ( 100174  ms ) , resulting in a q - lv / qrs ratio of 83  % . 
 of these , only one patient with rbbb , combined with left anterior hemiblock , showed an increase in the ahr , from   9.1  % ( apical coronary sinus position ) to 25.8  % at an endocardial basal posterolateral position . 
 3 ) ; one had a non - specific intraventricular conduction delay with a qrs complex of 175  ms . 
 the coronary sinus lead position was mid - posterolateral in 2 and apical in 1 patient . 
 3recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . 
 this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . 
 this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect three out of the 6 patients with rv pacing became haemodynamic responders ( lv dp / dtmax from average 9.7  % , ( 9.312.7  % ) increased to 21.8  % ( 16.624.8  % ) ) . 
 in the other 3 patients the average ahr increased from 2.9  % ( 1.14.0  % ) to 9.7  % ( 7.811.4  % ) . in 2 patients the coronary sinus lead was in a mid - posterolateral position , one in an apical position . 
 individual details are summarised in table  2 and a flow chart overview in fig .  4 . 
 4flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . 
 ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular when pacing endocardially , opposite the epicardial coronary sinus lead , we found that lvdp / dtmax did not differ significantly for any of the patients : lvdp / dtmax from the coronary sinus averaged 1046    292  mmhg / s vs. 1068    296  mmhg / s from the corresponding lv endocardial site ( p  =  0.37 ) . 
 we found that basal or mid - posterolateral segments had the highest ahr of the tested endocardial sites in all 14 patients who showed an ahr   15  % rise in lvdp / dtmax compared with baseline . because either the basal or mid - posterolateral segment had the highest ahr , we also calculated the combined best results from basal posterolateral and mid - posterolateral ( pl - best ) . 
 pl - best had a rise to 30.2    13.6  % in lvdp / dtmax , basal posterolateral 27.7    15.2  % , mid - posterolateral 24.6    12.6  % , lv apical 14.8    9.1  % and lv septal 17.0    11.2  % . 
 we also found that in 12 out of the 14 patients ( 85  % ) with an ahr   15  % , the lv endocardial site with the longest time interval between the onset of the qrs complex and lv sensing ( q - lv interval ) resulted in the best haemodynamic response . 
 the average qrs width was 133    22  ms ( range 120205  ms ) and average q - lv interval 155    26  ms ( 128205  ms ) . 
 the average q - lv / qrs width ratio , which is q - lv expressed as a percentage of qrs width , was 90  % . 
 table 3haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax   15  % from endocardial pacingpts.ahr pl - best ( % ) ahr pl - basal ( % ) ahr pl - mid ( % ) ahr lv apical ( % ) ahr lv septal ( % ) qrs width ( ms)max q - lv interval ( ms)location longest q - lvpt.0124.224.22.71.72.1135128pl - baspt.0331.631.625.84.826.1174165pl - baspt.0424.818.724.814.219.6196205pl - midpt.0520.120.118.018.111.8165163pl - midpt.0851.051.046.022.029.2154138pl - baspt.1219.013.519.017.613.7198176pl - midpt.1316.55.316.68.89.6152128pl - midpt.1566.066.050.231.347.3200181pl - midpt.1625.825.814.1  0.612.2175147pl - baspt.2019.719.716.78.916.3156124pl - midpt.2134.928.034.916.96.9174132pl - midpt.2234.334.320.315.614.4175155pl - baspt.2331.026.231.026.718.6160138pl - midpt.2424.122.924.117.211.5210192pl - mid30.2    13.627.7    15.224.6    12.614.8    9.117.1    11.2173    22155    26 
 ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . 
 grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . 
 haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax   15  % from endocardial pacing 
 ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . 
 grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . 
 ten patients were considered possible candidates for lv endocardial pacing : 9 patients in whom a rise in lvdp / dtmax   15  % level was only obtained by endocardial pacing , and the patient that showed a substantial additional rise in ahr with lv pacing compared with coronary sinus pacing ( patient 15 ) . 
 of the remaining 9 patients , and after deliberation with the patients about the possible benefits and risks , 5 of them finally agreed to lv endocardial implant at the optimal lv site as indicated from the acute study . 
 all five patients improved clinically with a reduction of at least 1 class in the nyha score after 6 months of follow - up . 
 two patients with a positive response in the temporary study refrained from implantation of an lv endocardial lead . 
 one patient with a positive ahr from endocardial pacing also improved significantly after changing stimulation from the distal to the proximal electrode with a more basal position , and became a clinical responder after optimisation of the atrioventricular and interventricular interval . 
 in one patient , the deterioration of his condition after initial improvement proved to be the result of a blunted chronotropic response after increasing beta - blocker dosage following an episode of atrial fibrillation resulting in an inappropriate icd shock . 
 notwithstanding that all patients were clinical non - responders , in 5 patients lvdp / dtmax with the implanted coronary sinus system increased   15  % after optimisation : average 30.2  % , 15.644.5  % ( table  2 ) . in 3 out of these 5 patients with the coronary sinus lead in a posterolateral position , endocardial pacing did not increase the lvdp / dtmax substantially ( ahr less than 3  % and even an adverse effect from lv endocardial pacing was observed in one patient ) . in one of the two remaining patients with an apical position of the coronary sinus lead , 
 the ahr could be increased during lv endocardial pacing from 19.766  % and we considered the increase sufficient to justify the upgrade to lv endocardial pacing ( fig .  1 ) . 
 table 2individual haemodynamic results of all clinical non - responders ( 24 patients)patientqrs morphologybaseline lvdp / dtmax ( mmhg / s)position cs leadahr cs lead ( % ) ahr cs - level - endo(%)lv endo optimal positionahr lvendo ( % ) pt . 
 07non - lbbb893pl - mid  23.4lv septal  21.5pt . 08lbbb827pl - bas44.951.0pl - bas51.0pt . 
 24rvp489lv apical12.717.2pl - mid24.1average891    2478.7    15.810.5    8.919.6    17.5haemodynamic measurements of 24 clinical nonresponders to crt . 
 baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . 
 grey shaded patients have an ahr from the cs lead   15  % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . 
 individual haemodynamic results of all clinical non - responders ( 24 patients ) haemodynamic measurements of 24 clinical nonresponders to crt . 
 baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . 
 grey shaded patients have an ahr from the cs lead   15  % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . 
 all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . 
 arrows indicate the position of the bipolar pacing lead angiographic pictures of the haemodynamic study in patient no . 
 all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . 
 arrows indicate the position of the bipolar pacing lead in the 19 patients with an ahr from the implanted system <  15  % , 9 patients had an increase in ahr above the 15  % limit . 
 the results of the total group of 24 patients showed a positive response in 11 patients with an apical lead position in 7 , anterolateral in 2 and mid - posterolateral in 2 patients . 
 a negative response of endocardial pacing was observed in 13 patients with a basal posterolateral lead position in 3 , mid - posterolateral in 8 and apical in 2 patients . 
 in 5 out of the 9 acute haemodynamic non - responders ( ahr <  15  % ) with lbbb , the ahr could be increased above the 15  % level by lv endocardial pacing . 
 the average ahr with the coronary sinus system was 9.6  % ( 5.814.9  % ) and at the lv endocardial optimal position 25.8  % ( 19.034.3  % ) . 
 the average qrs width in these patients was 169  ms ( 135198  ms ) and the average q - lv at the optimal lv endocardial pacing site 157  ms ( 128176  ms ) , resulting in a q - lv / qrs ratio of 93  % . 
 the position of the original coronary sinus lead was apical in 3 , anterolateral in 1 and mid - posterolateral in 1 patient ( fig .  2 ) . 
 2recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions in the remaining 4 patients , the average ahr at the optimal endocardial position increased <  15  % , with an average of 7.3  % ( 1.79.9  % ) . 
 the average qrs width in these patients was 176  ms ( 149209  ms ) and the average q - lv 136  ms ( 100174  ms ) , resulting in a q - lv / qrs ratio of 83  % . 
 four patients did not have lbbb . of these , only one patient with rbbb , combined with left anterior hemiblock , showed an increase in the ahr , from   9.1  % ( apical coronary sinus position ) to 25.8  % at an endocardial basal posterolateral position . 
 3 ) ; one had a non - specific intraventricular conduction delay with a qrs complex of 175  ms . 
 the coronary sinus lead position was mid - posterolateral in 2 and apical in 1 patient . 
 3recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . 
 this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . 
 this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect 
 three out of the 6 patients with rv pacing became haemodynamic responders ( lv dp / dtmax from average 9.7  % , ( 9.312.7  % ) increased to 21.8  % ( 16.624.8  % ) ) . 
 in the other 3 patients the average ahr increased from 2.9  % ( 1.14.0  % ) to 9.7  % ( 7.811.4  % ) . in 2 patients the coronary sinus lead was in a mid - posterolateral position , one in an apical position . 
 individual details are summarised in table  2 and a flow chart overview in fig .  4 . 
 4flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . 
 ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . 
 ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular 
 when pacing endocardially , opposite the epicardial coronary sinus lead , we found that lvdp / dtmax did not differ significantly for any of the patients : lvdp / dtmax from the coronary sinus averaged 1046    292  mmhg / s vs. 1068    296  mmhg / s from the corresponding lv endocardial site ( p  =  0.37 ) . 
 we found that basal or mid - posterolateral segments had the highest ahr of the tested endocardial sites in all 14 patients who showed an ahr   15  % rise in lvdp / dtmax compared with baseline . because either the basal or mid - posterolateral segment had the highest ahr , we also calculated the combined best results from basal posterolateral and mid - posterolateral ( pl - best ) . 
 pl - best had a rise to 30.2    13.6  % in lvdp / dtmax , basal posterolateral 27.7    15.2  % , mid - posterolateral 24.6    12.6  % , lv apical 14.8    9.1  % and lv septal 17.0    11.2  % . 
 we also found that in 12 out of the 14 patients ( 85  % ) with an ahr   15  % , the lv endocardial site with the longest time interval between the onset of the qrs complex and lv sensing ( q - lv interval ) resulted in the best haemodynamic response . 
 the average qrs width was 133    22  ms ( range 120205  ms ) and average q - lv interval 155    26  ms ( 128205  ms ) . the average q - lv / qrs width ratio , which is q - lv expressed as a percentage of qrs width , was 90  % . 
 table 3haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax   15  % from endocardial pacingpts.ahr pl - best ( % ) ahr pl - basal ( % ) ahr pl - mid ( % ) ahr lv apical ( % ) ahr lv septal ( % ) qrs width ( ms)max q - lv interval ( ms)location longest q - lvpt.0124.224.22.71.72.1135128pl - baspt.0331.631.625.84.826.1174165pl - baspt.0424.818.724.814.219.6196205pl - midpt.0520.120.118.018.111.8165163pl - midpt.0851.051.046.022.029.2154138pl - baspt.1219.013.519.017.613.7198176pl - midpt.1316.55.316.68.89.6152128pl - midpt.1566.066.050.231.347.3200181pl - midpt.1625.825.814.1  0.612.2175147pl - baspt.2019.719.716.78.916.3156124pl - midpt.2134.928.034.916.96.9174132pl - midpt.2234.334.320.315.614.4175155pl - baspt.2331.026.231.026.718.6160138pl - midpt.2424.122.924.117.211.5210192pl - mid30.2    13.627.7    15.224.6    12.614.8 
 9.117.1    11.2173    22155    26 
 ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . 
 grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . 
 haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax   15  % from endocardial pacing 
 ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . 
 grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . 
 ten patients were considered possible candidates for lv endocardial pacing : 9 patients in whom a rise in lvdp / dtmax   15  % level was only obtained by endocardial pacing , and the patient that showed a substantial additional rise in ahr with lv pacing compared with coronary sinus pacing ( patient 15 ) . 
 of the remaining 9 patients , and after deliberation with the patients about the possible benefits and risks , 5 of them finally agreed to lv endocardial implant at the optimal lv site as indicated from the acute study . 
 all five patients improved clinically with a reduction of at least 1 class in the nyha score after 6 months of follow - up . 
 two patients with a positive response in the temporary study refrained from implantation of an lv endocardial lead . 
 one patient with a positive ahr from endocardial pacing also improved significantly after changing stimulation from the distal to the proximal electrode with a more basal position , and became a clinical responder after optimisation of the atrioventricular and interventricular interval . 
 in one patient , the deterioration of his condition after initial improvement proved to be the result of a blunted chronotropic response after increasing beta - blocker dosage following an episode of atrial fibrillation resulting in an inappropriate icd shock . 
 this observational study showed that endocardial lv pacing , guided by acute haemodynamic studies , could improve clinical outcome in patients not responding to conventional crt . 
 it also demonstrated the importance of lead location , as the clinical response improved with a posterolateral position of the lead instead of the original apical or anterolateral lead location . 
 conversely , none of the patients who already had a coronary sinus lead in a posterolateral position improved sufficiently with endocardial pacing at any site during the acute haemodynamic study to justify endocardial lead implant . 
 this is in line with the madit - crt trial , where the apical and anterior positions of the lv lead had a lower clinical response . 
 noteworthy , and similar to what has previously been reported , endocardial pacing opposite the coronary sinus pacing site did not improve the ahr in our study [ 1113 ] . 
 this is in contrast with animal experiments that show significantly better haemodynamic results with endocardial vs. epicardial pacing at the same location . 
 there is no clear single cause for this difference and it is most probably related to variance in the anatomical and electrophysiological substrate . 
 besides this , in animal experience the lead configuration guarantees an exact position of the endocardial lead opposite to the epicardial position ; this in contrast with human studies in which the endocardial lead is placed as close as possible to its epicardial counterpart . 
 therefore , although not examined in this and other studies , one either might speculate that epicardial pacing opposite the optimal endocardial sites , via the coronary sinus or surgically by thoracoscopy , might have resulted in similar benefits . if feasible , this might be a good alternative to avoid the uncertainty of thromboembolic complications with lv endocardial pacing . 
 our observational study also showed some limitations in the use of acute haemodynamic studies to predict clinical outcome . 
 first , five clinical non - responders showed a clear ahr according to the accepted criteria ( even with the higher than usual cut - off value of 15  % increase in lvdp / dtmax to exclude borderline cases ) but failed to respond clinically . 
 still , when a substantial further increase in lvdp / dtmax from endocardial pacing at a posterolateral position compared with the apical coronary sinus lead ( 66 vs. 19.7  % ) was obtained in one patient , endocardial lead implantation resulted in a positive clinical response . 
 this illustrates the limitation of cut - off values in crt studies of the 15  % level as surrogate for differentiating between clinical response and non - response , and that what is considered a positive haemodynamic response can still be significantly improved in the presence of an anterior or apical lead position . 
 a similar discrepancy is found with echocardiography as the observed presence or absence of decrease in lv end - diastolic volume or end - systolic volume and clinical response does not always correlate . to err on the safe side , we therefore proceeded to a permanent lv endocardial implantation when the final increase in ahr was at least 25  % . 
 the best ahr correlates well with the longest time interval between onset qrs and lv sensing in relation to the qrs width in the individual patient . 
 the results of the group of patients with non - lbbb are in line with what could be expected according to the literature . 
 the absence of response correlated well to the lack of conduction delay in any of the lv segments on the intracardiac lv electrogram . 
 the only responder to endocardial pacing in this group had a significant lv conduction delay due to the left anterior hemiblock that accompanied rbbb . 
 it is therefore questionable if one should proceed with endocardial mapping in this cohort of patients given the disappointing results . 
 this observational study showed that a temporary acute haemodynamic study could be useful in non - responders to establish the chance of improvement with alternative pacing sites before embarking on complex procedures as endocardial lv pacing . 
 it remains , however , uncertain what the optimal cut - off value for increase in lvdp / dtmax is before the improvement justifies intervention . 
 the longest q - lv interval is a reliable indicator for the optimal electrode position in the individual patient . 
 endocardial pacing opposite epicardial sites does not show acute haemodynamic improvement , so epicardial pacing at the optimal site may be a good alternative for endocardial lv lead placement . 
 although all patients who had a lv endocardial system implanted after a positive ahr improved clinically , larger randomised series are necessary to justify this technique before definitive conclusions can be drawn . 
 van gelder has received training and education on pacing and crt from st jude medical , is a consultant with radi pressure wire systems , a st jude medical company , and consultant for pacing and crt with sorin group crm sas . 
 b.m . van gelder has received training and education on pacing and crt from st jude medical , is a consultant with radi pressure wire systems , a st jude medical company , and consultant for pacing and crt with sorin group crm sas .","introductionnon response to cardiac resynchronisation therapy ( crt ) may be related to the position of the coronary sinus lead.methodswe studied the acute haemodynamic response ( ahr ) from alternative left ventricular ( lv ) endocardial pacing sites in clinical non - responders to crt . 
 ahr and the interval from qrs onset to lv sensing ( q - lv interval ) from four different endocardial pacing sites were evaluated in 24 clinical non - responders . a rise in lvdp / dtmax   15  % from baseline 
 was considered a positive ahr . 
 we also compared the ahr from endocardial with the corresponding epicardial lead position.resultsthe implanted system showed an ahr   15  % in 5 patients . in 9 of the 19 remaining patients , ahr could be elevated to   15  % by endocardial lv pacing . 
 the optimal endocardial pacing site was posterolateral . 
 there was no significant difference in ahr between the epicardial and the corresponding endocardial position . 
 the longest q - lv interval corresponded with the best ahr in 12 out of the 14 patients with a positive ahr , with an average q - lv / qrs width ratio of 90  % .conclusionsacute haemodynamic testing may indicate an alternative endocardial pacing site with a positive ahr in clinical non - responders . 
 the q - lv interval is a strongly correlated with the optimal endocardial pacing site . 
 endocardial pacing opposite epicardial sites does not result in a better ahr .",pubmed
"autosomal dominant polycystic kidney disease ( adpkd ) affects between 1 in every 500 and 1 in every 1000 individuals and is found in all racial and ethnic groups 
 . the disease may not be clinically apparent until the third or fourth decade of life and only about half of the patients will progress to renal failure . in young patients , there is no way to predict whether or when renal failure will develop , although several risk factors such as hypertension , renal hemorrhage , multiple pregnancies , male gender , and pkd1 disease genotype have been associated with a more rapid course . 
 complications associated with pregnant subjects with chronic renal failure undergoing dialysis include maternal hypertension , preeclampsia , anemia , infected renal and hepatic cysts , pyelonephritis , and rupture of cerebral artery aneurysm during labor with subarachnoid hemorrhage 
 . however , with careful and effective perioperative monitoring spinal anesthesia can be safely performed for cesarean section in patients undergoing hemodialysis . 
 an 80 kg 35-year - old full term primigravida with oligohydramnios was scheduled for emergency lower segment cesarean section ( lscs ) . 
 she was diagnosed to have adpkd in the seventh month when she complained of flank pain , generalized swelling , and decreased urine output . 
 she was hospitalized and hemodialyzed thrice a week for the next 2 months to maintain blood urea nitrogen ( bun ) , serum creatinine , and potassium values within acceptable limits . 
 the nephrologists and obstetricians decided to proceed with an emergency lscs as she was full term . 
 clinical parameters were heart rate of 102/ min regular , systemic blood pressure of 120/74 mm hg in supine position , central venous pressure ( cvp ) 1 - 2 cm h2o . 
 preoperative investigations showed hemoglobin ( hb ) of 10.2 gm% , normal leucocyte count , and mild proteinurea and pus cells in urine . 
 her biochemical profile revealed bun of 30 mg / dl , serum creatinine 3.7 mg% , serum sodium 140 mmol l , serum potassium 5 mmol l , and normal liver function tests . 
 coagulation profile was bleeding time 3 min , clotting time 9 min , activated partial thromboplastin time ( aptt ) 30 s , platelet count 150  10l , prothrombin time 13 s , and international normalized ratio 1:2 . 
 arterial blood gas on air showed ph 7.41 , pco2 30.8 mm hg , po2 93.3 mm hg , spo2 98% , and hco3 22 meq . after obtaining informed written consent , patient 
 monitoring included pulse oximeter , cardioscope , noninvasive blood pressure ( nibp ) , cvp , and urine output measurement . 
 a peripheral line of 18 g in the left arm was used to preload her with 500 ml of normal saline till cvp was 5 cm h2o . 
 spinal anesthesia was successfully given in the l3 - 4 intervertebral space using 1 ml of 0.5% hyperbaric bupivacaine ( 5 mg ) with 25 mcg fentanyl in the left - lateral position with a 25 g spinal needle . 
 intraoperatively , bp was in the range of 100 - 120 mm hg systolic , hr 70 - 100/ min , cvp 5 - 6 cm h2o and urine output of approximately 1 
 a 2.0 kg live male child was delivered ( apgar score of 9 at 5 min ) . 
 patient did not complain of pain for about 4 h ; thereafter , she was given intravenous tramadol 8 hourly . 
 postoperatively , she maintained normal hemodynamic and biochemical parameters and did not require any hemodialysis . 
 there was no neurological deficit ; she was shifted to the nephrology unit after 72 h and discharged on day 7 . 
 women with moderate or severe antenatal renal insufficiency often experience deterioration of their renal functions during pregnancy . the pathophysiology by which pregnancy exacerbates renal disease is unknown . 
 one hypothesis is that increased glomerular perfusion which normally accompanies pregnancy paradoxically causes further injury to the kidneys in patients with preexisting impairment of function . 
 however , this hypothesis is not supported by published data , which reveal no evidence of hyperfiltration . 
 epstein outlined an alternative hypothesis in which preexisting renal disease may induce a cascade of platelet aggregation , microvascular fibrin thrombus formation , and endothelial dysfunction that leads to microvascular injury in the already tenuous kidneys . increased cardiac output and decreased intrarenal vascular resistance in pregnancy cause an 80% increase in renal blood flow and a 50% increase in glomerular filtration rate ( gfr ) . 
 because of the increased gfr , a serum creatinine concentration greater than 0.8 mg / dl and a bun concentration greater than 13 mg/ dl ( which are normal values for the nonpregnant patient ) suggest renal insufficiency . 
 tubular sodium reabsorption and osmoregulation are reset , allowing a  physiologic hypervolemia  during gestation . 
 modest proteinuria also occurs during pregnancy ( up to 300 mg in 24 h ) . 
 when serum creatinine concentration exceeds 2.0 mg / dl there is a one in three chance of developing dialysis - dependent end stage renal disease during or shortly after pregnancy . our patient presented with symptoms of renal insufficiency only in the seventh month of gestation , it is difficult to opine whether her disease progressed during pregnancy or the diagnosis was missed earlier . on presentation , 
 the blood erythrocyte count and hematocrit may be increased above normal possibly because of abnormal erythropoietin production by the cysts ; hence , they do not have anemia as profound as that occurring in other types of terminal renal disease . for ambulatory chronic renal failure patients , hb of 11 - 12 g / dl 
 blood coagulation studies and platelet counts are usually normal in patients of adpkd , as was the case in our patient . 
 hypertension an early symptom , occurring in approximately 60% of patients secondary to stretching of arterioles across expanding cysts leading to increased secretion of renin and angiotensin . 
 although the patient was not anuric and did not have ecg changes due to hyperkalemia , it was decided to dialyze her at least thrice a week . during pregnancy early dialysis 
 is recommended ( when the gfr is approximately 10 ml / min , rather than waiting until it falls further ) and treating with more frequent and longer hemodialysis sessions . 
 problems occurring in a pregnant patient on hemodialysis are need for vascular access , cardiovascular instability , large fluid , and electrolyte shifts , need for anticoagulation of extracorporeal circuit and risk of hepatitis . 
 fetal heart rate monitoring is recommended , even when hypotension and major fluid shifts are avoided , as doppler examination of uterine and umbilical artery flow during hemodialysis suggest redistribution of arterial flow away from the uteroplacental vascular bed . 
 bun levels are kept below 80 mg / dl predialysis and 30 mg / dl postdialysis . at birth , 
 the increase in concentration of most coagulation factors is associated with a shortening of the prothrombin and aptt during pregnancy . 
 there are reports of safe administration of epidural anesthesia , without any neurologic complications , in thrombocytopenic ( platelet count < 100 000/cumm ) healthy pregnant women , preeclampsia , and women with autoimmune thrombocytopenic purpura . 
 they state that in the absence of clinical evidence of bleeding , regional anesthesia should not necessarily be withheld in pregnant women with a platelet count of less than 1 00 000/cu mm . 
 no hemorrhagic complications were observed in 24 women with unsuspected thrombocytopenia ( platelet counts of 15 - 99  10/l ) who received spinal anesthesia or in 24 children with acute lymphoblastic leukemia and platelet counts below 20  10/l . 
 intravenous unfractionated heparin should be stopped 4 - 6 hrs before neuroaxial blockade and a normal aptt should be confirmed . since our patient had been dialyzed the previous day and her electrolytes , aptt , platelet counts , bleeding time and hemoglobin values were within acceptable limits , we decided to proceed with spinal anesthesia . 
 high degree of suspicion for any neurological complications should be maintained in the postoperative period . 
 preservation of renal function intraoperatively depends on maintaining an adequate intravascular fluid volume and minimizing drug - induced cardiovascular depression . 
 we used low dose of local anesthetic with intrathecal opioid in an effort to preserve renal and uterine blood flow . 
 low dose of spinal anesthesia has the advantage of providing cardiovascular stability and it is advocated that 8 mg of 0.5% hyperbaric bupivacaine is the optimal dose for cesarean section . 
 we were able to achieve a sensory level of t6 with 5 mg of 0.5% bupivacaine combined with fentanyl 25 mcg . 
 bogra j et al . studied the synergistic , potentiating effect of fentanyl on bupivacaine in spinal anesthesia for cesarean section , on comparing the hemodynamic stability of equipotent doses of bupivacaine and bupivacaine fentanyl , they found the latter to be more stable .","renal disease , either preexisting or occurring during gestation may impair maternal and fetal health . a 35-year - old primigravida with autosomal dominant polycystic kidney disease on hemodialysis was scheduled for emergency cesarean section . 
 she was managed successfully with low - dose intrathecal bupivacaine and fentanyl . in the case of pregnancy in such a patient , early involvement of the nephrologists along with the obstetrician 
 can improve maternal and fetal outcome .",pubmed
"it also depicts summary of the pattern of milk yield determined by the biological efficiency of the cow . 
 the cost of milk production depends to a large extent on the persistency of lactation , that is , the rate of decline in production after peak milk yield . 
 high persistency is associated with a slow rate of decline in milk production , whereas low persistency is associated with a rapid rate of decline in milk yield . in general declining rate of milk production 
 estimates of heritabilities for milk yield and persistency traits in hf cows have been reported [ 3 , 4 ] . 
 the lactation curve models have been used to predict the milk yield at any point of the lactation [ 5 , 6 ] . 
 various models have been tried by different researchers to fit the lactation curve in indigenous as well as exotic cattle [ 710 ] . 
 further getting the test - day milk yield information from the field conditions is not easy and there is every chance of missing the data due to certain inevitable circumstances . in such case mathematical models may prove beneficial for prediction of milk production performance using . in view of this 
 the lactation curve was composed for the normal vis -  - vis mastitis cows using gamma type function during different parity to find out the accuracy of model in predicting the milk yield with minimum error in normal and mastitis crossbred cows . 
 the data on 11728 fortnightly test - day milk yields ( ftdmys ) of 733 karan fries cows for a period of 12 years ( 20002011 ) was collected from livestock farm of national dairy research institute , karnal , india . 
 the climate of the farm is subtropical in nature with the lowest temperature reaching 2c during winter and the highest temperature up to 45c in the summer months . 
 the annual rainfall is about 760 to 960  mm , out of which most of the rainfall is received during the months of july and august . 
 the amount of milk recorded in 24-hour duration on any particular day is called the test - day ( td ) milk yield and is expressed in kilograms . 
 the fortnightly test - day intervals of 15 days were considered starting from days 5 , 20 , 35 , 50 , 65 , 80 , 95 , 110 , 125 , 140 , 155 , 170 , 185 , 200 , 215 , 230 , 245 , 260 , 275 , 290 , and 305 of lactation . 
 twenty - one ( 21 ) fortnightly test - day milk yield ( ftdmy ) records were considered per lactation ( from the 6th to the 305th day of lactation ) . 
 the data was grouped as mastitis ( cows suffering from clinical mastitis ) and nonmastitis ( normal cows ) . 
 statistical analysis of data was carried out using gamma type function   with the following equation : 
 ( 1)yt = atbect , 
 where yt is the average daily yield in the tth fortnight , a is the initial milk yield after calving , b is the ascending slope parameter up to the peak yield , and c is the descending slope parameter . 
 the constants were derived by solving the above equation after transformation on the log scale : 
 ( 2)ln(yt)=ln(a)+bln(t)ct . the milk yield up to week  t 
 the fortnightly test - day milk yield was less in td-1 ( 9.78  0.29  kg / d ) and increased ( p < 0.01 ) gradually to attain peak in td-3 and td-4 ( 14.34  0.38 and 14.48  0.37  kg / d ) in normal cows ; however , in mastitis cows , the peak milk yield was attained in td-3 ( 13.15  0.37  kg / d ) and was maintained only for this period ( figure 1 ) . 
 the cows of both of the groups exhibited a steady decline ( p < 0.01 ) in milk yield with increase in fortnights of lactation . 
 in general the fortnightly test - day milk yield increased till td-3 and declined steadily with advancement of lactation . in the second parity the ftdmy showed an increasing trend from td-1 to td-4 and decreased thereafter gradually in both of the groups . 
 a minimum ftdmy of 11.31  0.39  kg / d and 9.18  0.44  kg / d and a maximum ftdmy of 17.19  0.44  kg / d and 15.53  0.46 
 kg / d were observed on td-4 in both of the groups ( figure 2 ) . 
 kg / d to 9.55  0.47  kg / d in td-21 and the maximum ftdmy of 19.08  0.53  kg / d and 16.44  0.47 
 an increase in ftdmy was observed from 14.36  kg and 12.34  kg in td-1 to a peak yield of 20.50  0.59  kg / d in td-4 and 17.63  kg / d in td-3 which subsequently declined ( p < 0.05 ) to 10.50  0.54  kg / d and 9.54  0.54  kg / d in td-21 in normal and mastitis kf cows ( figure 3 ) . 
 the ftdmy milk yield in the 4th parity was maximum in comparison to the first , second , and third parity in both normal and mastitis cows ( figure 4 , table 2 ) . 
 an increase in milk production was observed up to the 4th fortnight and milk productions decreased ( p < 0.01 ) during the 5th parity in normal cows . in mastitis cows 
 increase in milk yield was noticed during the first three fortnights and milk production decreased subsequently ( p < 0.05 ) until the end of lactation ( figure 5 ) . 
 the parity - wise estimated lactation curve parameters for ftdmy indicated higher average initial milk yield ( a ) in normal cows as compared to mastitis cows in different parities ( table 1 ) . 
 the peak milk yield was observed in the 4th parity ( 16.45  kg and 14.42  kg ) while minimum yield was observed in the 1st parity in both normal and mastitis cows ( 11.28 versus 10.35  kg / d ) . 
 lactation curve parameter of ascending slope parameter up to peak yield ( b ) was similar in both the groups in the 1st parity , declined in the 2nd parity , and increased in the subsequent parities . 
 the descending slope parameter ( c ) was lower in the 1st parity ( 0.041 ) and higher in the 4th parity ( 0.072 ) in normal cows , but , in mastitis cows , it was minimum in the 1st parity ( 0.043 ) and maximum in the 5th and above parity ( 0.070 ) . 
 further higher persistency of lactation was observed in the 1st parity ( 54.34 and 51.40 ) which declined till the 5th and above parity in all of the cows . 
 the fortnights of peak milk yield ranged from averagely 4.53 to 6.15 fortnights in normal cows and from 4.49 to 5.88 fortnights in mastitis cows . 
 the cows in the first parity took more time ( fortnight ) to attain peak yield in comparison to subsequent parity ; however , peak milk yield increased ( p < 0.05 ) with increase in number of parity in both of the groups . 
 the peak yield was minimum in the 1st parity ( 13.89  kg / d and 12.58  kg / d ) and maximum in the 4th parity ( 19.67  kg / d and 16.61 
 the coefficient of determination of variation ( r ) ranged from 84.88% to 98.18% in normal cows and from 83.93% to 97.68% in mastitis cows during different parities . 
 the rmse value was minimum in the 3rd parity ( 0.09  kg ) and maximum in the 5th and above parity ( 0.16  kg ) in normal cows ; however , in mastitis cows rmse was minimum in the 5th and above parity ( 0.09 ) and maximum in the 4th parity ( 0.13  kg ) . 
 the pattern of change in the ftdmy observed in this study was in agreement with the earlier reports in normal cattle and buffaloes [ 1217 ] 
 . however comparable literature on fortnightly test - day milk yield ( ftdmy ) and fortnightly average daily milk yield ( fadmy ) in mastitis crossbred kf cows is scanty . 
 the peak milk yield attained during the 3rd and 4th fortnights in both of the groups corroborates with the values reported earlier in cows [ 10 , 18 , 19 ] . 
 the trend in ftdmy in both of the groups suggested that peak yield duration is not affected by incidence of mastitis in spite of significant decline in milk yield ( p < 0.01 ) 
 . the lower initial milk yield ( a ) in the first parity and an increasing trend in subsequent parity have also been reported in normal kf cows   and butana dairy cows . 
 many research findings indicated an increase in initial milk yield value with increase of parity order and the highest initial milk yield reaches the 5th lactation in friesian and ayrshire crossbred and kf cows [ 2125 ] ; however , such trend was evident up to the 4th parity in this study . 
 the lower initial yield , peak milk yield , and greater persistency in the first parity than the subsequent parity in kf cows as observed in this study corroborate earlier reports [ 6 , 2631 ] . 
 this is expected because lactation curves of the first parity are characterized by a less peak milk production and a greater persistency in ruminants [ 11 , 20 , 24 ] . 
 the decline in persistency of lactation with increase in parity order is attributed to age factor as older animals start their lactation at a higher level and had a rapid rate of decline in milk production due to regression of alveolar cells with advancement in age . 
 further , first - calving animals have less body weight and maturation of mammary tissue is still active , which counterbalances decline in milk production . during the first lactation , animal encounters unfamiliar situations , including the atmosphere of the milking parlor , presence of dairy farmer , and the milking procedures . 
 thus variation in the initial milk yield in different parities could be due to addition of more number of alveolar cells ( secretory cells ) at each successive pregnancy which reach their maximum numbers at about the 5th calving and diminish gradually thereafter . 
 the value of ascending slope parameter up to peak yield ( b ) in the present study was lower than reported earlier in different parity of friesian and ayrshire crossbred cows due to difference in milk yield of cows , management practices followed in a farm , and breed difference . 
 bwith advancement of parity in both of the groups was also reported in butana dairy cows , hf cows , and crossbred cows 
 b  was lower than the reported values in hf , kf , and sahiwal cows [ 10 , 21 , 36 ] than the value of parameter 
 the higher descending slope parameter ( c ) observed in this study was in agreement with the findings of previous reports [ 33 , 35 ] in crossbred cows and was lower than reported values in sahiwal cows   and holstein cows . 
 c  in the first parity than the rest of the parities indicated better utilization of feeds and less susceptibility of cows to metabolic and reproductive disorders . the marginal differences in parameters  a ,   b ,  and 
 c  in mastitis cows rather than the normal ones were due to disturbed milk secretion and apoptosis of mammary secretory cells that is solely responsible for the decline in milk yield after peak lactation [ 38 , 39 ] . 
 it has been found that turn of lactation curve was unimportant on the level of persistency of lactation in brown swiss cows . 
 further occurrence of atypical shapes of lactation curve characterized by the absence of the lactation peak varied from 25 to 42% . 
 previous report also supports the fact that peak yield is lower in the first parity and increases in subsequent parity . 
 the lower initial and peak milk yield in the first parity and more time to attain peak yield in the first parity than the remaining parities were in agreement with previous reports in norwegian mastitis cows [ 25 , 33 ] . 
 it has been found that holstein x zebu cows require 71 days to reach peak yield , which was less than the predicted 6.15 fortnights observed in crossbred cows . however , predicted fortnightly peak milk yield was nonsignificantly different in both mastitis and normal cows ( 5.88 versus 6.15  kg / d ) . 
 further moderate to high heritability estimates for different lactation curve shape parameters suggest that these traits can be included in selection schemes . 
 it has been reported that the milk secretory tissue requires more time for peak activity in primiparous cows than multiparous cows due to reduced udder size , less digestive capacity , and directs partitioning of nutrients [ 24 , 41 ] . 
 the high persistency is associated with a slow rate of decline in milk yield after peak production , while low persistency is associated with a rapid rate of decline in milk yield due to less feed intake . 
 further persistent lactations are characterized by lower peak yield   and reduced metabolic stress in early lactation . 
 the high score of goodness of fit ( r value ) of gamma type function corroborates the earlier reports in cows [ 10 , 19 , 21 , 22 , 36 ] . 
 the gamma function has been reported as the best model because of the small error variance and high determination coefficient . from gamma function , approximately one third ( 31.28% percent ) of lactation curves were named atypical . 
 atypical curve percentages were found as follows : 44.19% downhill , 45.08% concave , 7.32% lna negative , and 3.39% c negative . 
 further first lactation goodness of fit in normal and mastitis cows indicated more fitness of the model in kf crossbred cows as evident from low rmse value . 
 the latter confirms accuracy of gamma function model to predict milk yield of mastitis cows also [ 11 , 46 ] . 
 the similar value of r in normal and mastitis cows further indicated accuracy of model level . 
 the similar rmse values in mastitis and normal kf cows during different parities also support this fact . 
 the lactation curve parameters for fortnightly test - day milk yield exhibited similar trend in normal and mastitis infected cows by attaining peak milk yield in the 3rd and 4th td followed by a gradual decline in milk yield till the end of lactation . the steeper decline in descending slope ( c ) and 
 increase in initial ftdmy due to steeper rise in ascending slope from the 1st parity onwards could be used as a marker of persistency during different parities in cows . 
 further , higher r and lower rmse confirm the validity of gamma type function in predicting the milk yields in cows suffering from mastitis .","data pertaining to 11728 test - day daily milk yields of normal and mastitis karan fries cows were collected from the institute herd and divided as mastitis and nonmastitis and parity - wise . 
 the data of lactation curves of the normal and mastitis crossbred cows was analyzed using gamma type function . 
 ftdmy in normal and mastitis cows showed an increasing trend from td-1 to td-4 and a gradual decrease ( p < 0.01 ) thereafter until the end of lactation ( td-21 ) in different parities . 
 the ftdmy was maximum ( peak yield ) in the fourth parity . 
 parity - wise lactation curve revealed a decrease in persistency , steeper decline in descending slope ( c ) , and steeper increase in ascending slope ( b ) from 1st to 5th and above parity . 
 the higher coefficient of determination ( r2 ) and lower root mean square error ( rmse ) indicated goodness and accuracy of the model for the prediction of milk prediction performance under field conditions . 
 clinical mastitis resulted in a significantly higher loss of milk yield ( p < 0.05 ) . 
 the ftdmy was maximum ( p < 0.05 ) in the fourth parity in comparison to the rest of parity . 
 it is demonstrated that gamma type function can give the best fit lactation curve in normal and mastitis infected crossbred cows .",pubmed
"mouse lifespan , weight , and food consumption were monitored as described in detail previously ( 15 ) . 
 briefly , male b6c3f1 mice ( harlan breeders ; indianapolis ) were randomly assigned to treatment groups at 12 months of age . 
 two hundred ninety - seven control mice were shifted from ad libitum chow feeding ( diet # 5001 , purina mills , richmond , in ) to daily feeding with 13.3 kcal / day / mouse of control diet [ american institute of nutrition ( ain)-93 m diet ; diet no . 
 four groups of 18 mice each were shifted to daily feeding with an identical quantity of control diet supplemented with ndga at either 1.5 , 2.5 , 3.5 , or 4.5 g per kg diet ( approximately 1.59 , 2.65 , 3.71 , 4.77mg / kg body weight / day , respectively ) . 
 food consumption and health were monitored at the time of feeding , and any uneaten food noted . with few exceptions , 
 ndga was mixed with the powdered diet and cold - pressed into 1 g pellets by bio - serv . 
 the mice had ad libitum access to tap water , which was acidified ( ph 4.0 ) to reduce colonization by pseudomonas ( 16,17 ) . 
 the health of the mice was examined twice daily by laboratory staff and weekly by a veterinarian . 
 this study was approved by the institutional animal care and use committee at the university of california , riverside . 
 these lifespan studies utilized an unbalanced statistical design to minimize the number of mice per test group while maintaining statistical power ( 18 ) . 
 unbalanced designs have economic and logistic advantages for comparing multiple treatments to a common control ( 18 ) . 
 the group sizes in this study are similar to those required for a weibull survival analyses with a 75% probability of detecting an 10% increase in mean lifespan with a 1% probability of a false positive (   .01 ) . 
 the weibull analysis is more stringent than the comparison of kaplan  meier survival curves using the mantel 
 cox or gehan  breslow  wilcoxon tests , implemented in graphpad prism 5.01 , which are used here . 
 the significance of the differences in body weights between the treated and control groups was judged using a linear mixed effects model ( 19,20 ) as described previously ( 15 ) . in brief , we modeled the mean response by a set of fixed effects assumed to be shared by mice and a set of random effects that are unique to a particular mouse . additionally , our model imposed a common intercept since all mice were on the same diet at the time of the first measurement . to determine which group weights were significantly different than those of the controls , a bayesian information criterion ( bic ) 
 model selection criteria , a likelihood ratio test ( lrt ) , and an akaike s information criterion ( aic ) model selection criteria were used ( table 1 and results ) . 
 food consumption was determined by totaling the amount eaten by each treatment group during each time period , adjusted for the number of mice . 
 summary of the statistical analysis of mouse group weights ( figure 2 ) using bic model selection removing each diet individually 
 akaike s information criterion . 
 drosophila ( wild - type oregon - r - c ; bloomington drosophila stock center , department of biology , indiana university , bloomington , in ) lifespan determinations were performed as described in detail previously ( 21,22 ) , using 0 , 1 , or 3mg / ml ndga . 
 capillary feeder ( cafe ) assays ( 23 ) and fecal plaque assays ( fpas ( 24,25 ) ) were performed as described ( 26 ) . 
 the effect of ndga on fpa plaque size was determined as described ( 21,22 ) . 
 the effects of ndga on locomotor activity were determined as described previously ( 26 ) . 
 briefly , one day old male flies were exposed to 3 mm ndga in dmso or vehicle treatments in 9525 mm glass vials for 3 days ( at 25 c with 12 hour light / dark cycle ) . 
 after 3 days , movement of the flies was monitored for 72 hours using a trikinetics locomotor activity monitor ( lam 32 ) . 
 means  sem were calculated for 30 minutes time increments over the entire 72 hour trial ( 144 recordings ) . 
 an unpaired , two - sample t test was used to determine the significance of differences between experimental and control treatments using graphpad prism 5 . 
 mouse lifespan , weight , and food consumption were monitored as described in detail previously ( 15 ) . 
 briefly , male b6c3f1 mice ( harlan breeders ; indianapolis ) were randomly assigned to treatment groups at 12 months of age . 
 two hundred ninety - seven control mice were shifted from ad libitum chow feeding ( diet # 5001 , purina mills , richmond , in ) to daily feeding with 13.3 kcal / day / mouse of control diet [ american institute of nutrition ( ain)-93 m diet ; diet no . 
 four groups of 18 mice each were shifted to daily feeding with an identical quantity of control diet supplemented with ndga at either 1.5 , 2.5 , 3.5 , or 4.5 g per kg diet ( approximately 1.59 , 2.65 , 3.71 , 4.77mg / kg body weight / day , respectively ) . 
 food consumption and health were monitored at the time of feeding , and any uneaten food noted . with few exceptions , 
 ndga was mixed with the powdered diet and cold - pressed into 1 g pellets by bio - serv . 
 the mice had ad libitum access to tap water , which was acidified ( ph 4.0 ) to reduce colonization by pseudomonas ( 16,17 ) . 
 the health of the mice was examined twice daily by laboratory staff and weekly by a veterinarian . 
 this study was approved by the institutional animal care and use committee at the university of california , riverside . 
 these lifespan studies utilized an unbalanced statistical design to minimize the number of mice per test group while maintaining statistical power ( 18 ) . 
 unbalanced designs have economic and logistic advantages for comparing multiple treatments to a common control ( 18 ) . 
 the group sizes in this study are similar to those required for a weibull survival analyses with a 75% probability of detecting an 10% increase in mean lifespan with a 1% probability of a false positive (   .01 ) . 
 the weibull analysis is more stringent than the comparison of kaplan  meier survival curves using the mantel 
 cox or gehan  breslow  wilcoxon tests , implemented in graphpad prism 5.01 , which are used here . 
 the significance of the differences in body weights between the treated and control groups was judged using a linear mixed effects model ( 19,20 ) as described previously ( 15 ) . in brief , we modeled the mean response by a set of fixed effects assumed to be shared by mice and a set of random effects that are unique to a particular mouse . additionally , our model imposed a common intercept since all mice were on the same diet at the time of the first measurement . to determine which group weights were significantly different than those of the controls , a bayesian information criterion ( bic ) 
 model selection criteria , a likelihood ratio test ( lrt ) , and an akaike s information criterion ( aic ) model selection criteria were used ( table 1 and results ) . 
 food consumption was determined by totaling the amount eaten by each treatment group during each time period , adjusted for the number of mice . 
 summary of the statistical analysis of mouse group weights ( figure 2 ) using bic model selection removing each diet individually 
 akaike s information criterion . 
 drosophila ( wild - type oregon - r - c ; bloomington drosophila stock center , department of biology , indiana university , bloomington , in ) lifespan determinations were performed as described in detail previously ( 21,22 ) , using 0 , 1 , or 3mg / ml ndga . 
 capillary feeder ( cafe ) assays ( 23 ) and fecal plaque assays ( fpas ( 24,25 ) ) were performed as described ( 26 ) . 
 the effect of ndga on fpa plaque size was determined as described ( 21,22 ) . 
 the effects of ndga on locomotor activity were determined as described previously ( 26 ) . 
 briefly , one day old male flies were exposed to 3 mm ndga in dmso or vehicle treatments in 9525 mm glass vials for 3 days ( at 25 c with 12 hour light / dark cycle ) . 
 after 3 days , movement of the flies was monitored for 72 hours using a trikinetics locomotor activity monitor ( lam 32 ) . 
 means  sem were calculated for 30 minutes time increments over the entire 72 hour trial ( 144 recordings ) . 
 an unpaired , two - sample t test was used to determine the significance of differences between experimental and control treatments using graphpad prism 5 . 
 male mice were fed ndga in their food at 1.5 , 2.5 , 3.5 , or 4.5g / kg diet ( 160 , 267 , 373 , 480mg / kg bw / d ) , beginning at 12 months of age . 
 median lifespan was extended by 12% at 373mg / kg bw / d ( ~13.5mg / mouse / day ; mantel 
 cox p = .008 ; gehan  breslow  wilcoxon p = .009 ; figure 1 ) . 
 wilcoxon test gives more weight to deaths at early time points , while the mantel - cox test gives equal weight to all time points . despite its effects on median lifespan 
 , we found no effect of 373 mg of ndga / kg bw / d ( 3.5g / kg diet ) on maximum lifespan using the method of gao et al . 
 maximum lifespan is variously defined as the lifespan of the longest lived 10% or 20% of a cohort . here , we eliminated the lifespan data below a pooled quantile of 0.8 . the resulting mann 
 shown are the lifespans of mice consuming ain-93 m diet without additions ( filled circles ; median survival 983 days ) ; mice consuming 1.5 g of ndga / kg ain-93 m diet ( open circles ; median survival 969 days ; mantel 
 cox p = .769 ; gehan  breslow  wilcoxon p = .991 ) ; mice consuming 2.5 g of ndga / kg diet ( open squares ; median survival 1059 days ; mantel - cox p = .528 ; gehan  breslow  wilcoxon p = .490 ) ; mice consuming 3.5 g of ndga / kg diet ( open upward pointing triangles ; median survival 1099 days ; mantel - cox p = .008 ; gehan 
 breslow  wilcoxon p = .009 ) ; and mice consuming 4.5 g of ndga / kg diet ( open downward pointing triangles ; median survival 995 days ; mantel  cox p = .963 ; gehan  breslow  wilcoxon p = .647 ) . 
 the dose of ndga which extended median lifespan is within 12% of that initially reported to extend the lifespan of the nih - itp mice ( ~417mg / kg bw / d ( 12 ) ) . in our study , only this dose extended lifespan ( figure 1 ) . 
 recently , the nih - itp reported lifespan extension using censored data at higher and lower dosages . further study will be needed to resolve these differences . 
 the statistical design and animal husbandry used in these studies have been described in detail elsewhere ( 15,18,26,28 ) . 
 food consumption and body weight food consumption and body mass were monitored throughout the study ( figure 2 ) . 
 the mice ate essentially all their food each day , with the exception of a single excursion by the 1.5g / kg diet group late in life . 
 this is consistent with the instability of the weights in late life due to the smaller number of mice and the presence of more age - related pathologies . 
 shown is the body weight of the control ( closed black circles ) ; 40% cr fed ( closed orange circles ) ; 20% cr fed ( closed fuchsia circles ) ; 1.5 g ndga / kg diet ( closed gold circles ) ; 2.5 g ndga / kg diet ( closed green circles ) ; 3.5 g ndga / kg diet ( closed purple circles ) ; and 4.5 g ndga / kg diet ( closed blue circles ) . 
 food consumption as a percent of the kcal offered with respect to the kcals consumed during the preceding month is shown for control , ( open black circles ) ; 40% cr fed ( open orange circles ) ; 20% cr fed ( open fuchsia circles ) ; 1.5 g ndga / kg diet ( open gold circles ) ; 2.5 g ndga / kg diet ( open green circles ) ; 3.5 g ndga / kg diet ( open purple circles ) ; and 4.5 g ndga / kg diet ( open blue circles ) . 
 a preliminary report of these data from 365 to 1,063 days of age was published previously ( 14 ) . 
 the weights of the mice were analyzed using a mixed effects model with a bic model selection criterion , as described previously [ table 1 ; reference ( 15 ) ] . 
 these results suggest that the weights of the 2.5 and 4.5g / kg diet - treated mice , as well as the 20% and 40% cr mice were reduced significantly relative to those of the control group ( figure 2 ; table 1 ) . 
 the differences between the weights of the control and the 1.5 and 3.5g / kg diet - treated groups were close to significance by this test ( table 1 ) . 
 a lrt suggests that the weights of all the groups except the 1.5g / kg diet group differed significantly from those of the control group . 
 an aic model selection criteria suggests that all the diets groups differed significantly from the control group ( table 1 ) . 
 the group consuming the highest dose of ndga weighed less than the 20% calorically restricted ( 20% cr ) group at younger ages , when the data were more accurate due to sample sizes . 
 pathologies associated with mortality death - related pathology has not been reported previously ( 1012 ) . 
 the results of the necropsies from our study are summarized in tables 2 and 3 . 
 focusing on the aggregated results for the pathologies for all the groups , there was a highly or very highly significant increase in liver and lung tumors , enlarged thymuses ( indicative of thymomas or lymphomas ) , and hemorrhage into the peritoneum ( hemorrhagic diathesis ; table 2 ) . 
 response relationship for these pathologies , likely due to the relatively small group sizes . however , even the lowest dose of ndga produced significant pathologies ( table 2 ) . 
 ndga decreased the size of liver tumors by 24% relative to those found in the control mice , suggesting that it may decrease the rate of tumor growth . 
 however , the total mass of the tumors per mouse was unchanged , suggesting it may increase hepatic tumorigenesis ( tables 2 and 3 ; see discussion below ) . 
 necropsy results from the mouse longevity studies shown in figure 1 
  * number of necropsied mice in each treatment group . 
 significance of the differences from the control values were determined using fisher s exact test . for convenience , 
 liver tumor mass of the mice shown in table 2 and figure 1 * tumor mass was calculated as ( /6 )  l  w  h  1.0 g , where l is length , w is width , and h is the height of each tumor . 
 significant changes are in bold . in view of the clinical evidence of hepatotoxicity and nephrotoxicity in humans and rodents consuming ndga , we investigated whether ndga produced signs of toxicity at the concentrations used in these lifespan studies . 
 male b6c3f1 mice were treated with ndga or control diet for 9 weeks , and clinical chemistries performed . 
 alanine transaminase ( alt ) was significantly decreased by the higher doses of ndga , which is the opposite of what would be expected if the drug was toxic to the liver or kidneys . 
 aspartate transaminase ( ast ) , alkaline phosphatase , bilirubin , and creatinine levels , which can also be signs of toxicity if elevated , were unchanged . 
 con = control ; p = p - value . * eight male b6c3f1 mice were treated with ndga at each dosage or with control diet for 8 weeks . at 21 months of age 
 clinical chemistries were performed on the 68 mice in each group with no obvious signs of pathology at necropsy . 
 blood glucose levels were measured with the freestyle lite blood glucose monitoring system ( abbot laboratories ) . 
 the other blood tests were performed by the comparative pathology laboratory , university of california , davis . 
 all , results and p - values for all ndga treated groups considered together ; two lowest , the number of pathologies and the p - values for the 1.5 and 2.5g / kg diet fed mice considered together ; two highest , the number of pathologies and p - values for the 3.5 and 4.5g / kg diet fed mice considered together . 
 calculated using unpaired two - sample t tests for convenience , significant changes are indicated in bold . 
 however , the elevated triglyceride and glucose levels seem more likely to have resulted from the alterations in energy homeostasis evinced by the decrease in body weight in the absence of a change in caloric consumption ( figure 2 ; table 1 ) . 
 conservation of the lifespan response to further characterize the effects of the drug , we investigated its effects on the lifespan of drosophila . 
 optimum oral treatment with ndga at 3.0mg / ml medium increased the median lifespan of the flies by approximately 23% ( figure 3 ) . 
 these results suggest that at least some of the target(s ) of ndga action are phylogenically conserved between mice and flies . 
 the relatively short lifespans of the control flies are the result of using large flybottles , which induce more flight time ( 29 ) , male flies only ( 30 ) , mildly elevated incubation temperatures ( 31 ) , and food with a high protein concentration ( 3234 ) . 
 each of these parameters somewhat shortens lifespan , although the flies remain responsive to compounds that lengthen lifespan [ e.g. references ( 21,22,26 ) ] . 
 shown is the survival of drosophila fed diets containing either vehicle alone ( closed circles ; median survival 20 days ) , vehicle containing 1.0mg / ml ndga ( upward pointing triangles ) , or vehicle containing 3.0mg / ml ndga ( downward pointing triangles ; median survival 26 days ; both mantel  cox and gehan  breslow 
 because drug induced cr or reduced locomotor activity might increase drosophila lifespan ( 29,35 ) , we investigated these parameters in ndga - treated flies . 
 we found no effect of ndga on food consumption ( tables 5 and 6 ) , body weight ( table 7 ) , or locomotor activity ( table 8) . food consumption was quantified in two ways . 
 we used our modifications of the cafe assay ( table 5 ) and the fecal plaque assay [ fpa ; table 6 ; references ( 21,22 ) ] . no effect on food consumption 
 long - term ndga treatment also did not change body weight ( table 7 ) . 
 locomotor activity was quantified using a lam 32 activity monitor ( trikinetics ) as described previously [ table 8 ; reference ( 26 ) ] . 
 there was no significant difference between the activity of flies consuming food containing ndga or an equivalent volume of vehicle . 
 together , these results indicate that the effects of ndga on drosophila lifespan were not due to cr or altered locomotor activity . 
 further , we did not detect a disequilibrium between food intake and weight in this species as we did in the mice . 
 ndga does not alter food consumption in drosophila as measured using cafe assays * one day old male flies were starved for 2 hours and exposed to capillary tubes containing a solution of 5% sucrose and 5% yeast extract with the addition 1% by volume of either 3 mm ndga dissolved in dmso , or dmso alone . a small volume of red food coloring was added to the food to aid in the visualization of the liquid . for a complete description see methods . 
 consumption per fly is the mean  sem of the difference between the initial volume in the capillary pipets at the start and completion of assay conducted with flies present less the average amount of liquid loss to evaporation in control assays conducted in the absence of flies , divided by the number of flies in the assays . 
 four bottles per condition ( 50 flies per bottle ) . each bottle had four capillary tubes inserted into its sides . 
 nul control bottles were identical except for the absence of flies . 
 the significance of the difference in food consumption between the treated and control flies was determined using an unpaired , two - sample t test . 
 two day old flies were starved for 2 hours and exposed to capillary tubes containing a solution of 5% sucrose and 5% yeast extract with the addition 1% by volume of either 3 mm ndga dissolved in dmso , or dmso alone . a small volume of red food coloring was added to the food to aid in the visualization of the fecal plaques . 
 the significance of the differences between the treated and control groups was determined using an unpaired two - sample t test . 
 * one day old flies were fed for 7 days with food containing either 3 mm ndga or an equal volume of vehicle ( dmso ) . 
 a total of 7 groups ( n ) of 25 flies were weighed for each treatment condition . 
 mean weight  sem was calculated by dividing the total weight in milligrams of each group of flies by the number of individuals in the group . 
 the significance of the differences in weight was determined using unpaired two - sample t tests . 
 * flies were treated with either 3 mm ndga , dissolved in dmso , or medium containing an equal volume of dmso alone for 3 days , followed by 72 hours of monitoring at 25 c in a lam 32 activity monitor ( trikinetics ) . 
 ten flies in each of 10 vials were monitored simultaneously for each condition . 
 the average number of infrared beam disruptions per 72-hour period by the groups of 10 flies . 
 the significance of the differences in the mean number of beam breaks for treated and control flies determined using unpaired two - sample t tests . 
 the results presented here show that ndga can extend murine and drosophila lifespan . in our study , 
 ndga consumption , even at doses below its therapeutic dose , was associated with an increase in multiple tumor types and hemorrhagic diathesis . 
 the drug also increased the lifespan of drosophila , suggesting its target(s ) and mechanism(s ) of action may be phylogenetically conserved . 
 however , aspects of the response in flies were unlike those in mice . in flies 
 , ndga did not appear to affect energy uptake or utilization . in our studies , 
 3.5g / kg diet ( 373 mg ndga / kg bw / d ) extended lifespan , but higher and lower dosages were ineffective . 
 the nih - itp found that ndga extended male mouse lifespan at 2.5 g ndga / kg diet , which they estimated supplied approximately 417mg / kg bw / d ( 10,11 ) . 
 however , the actual dose was uncertain because food intake was not continuously measured in this study . 
 more recently , they report that ndga at 0.8 or 5.0g / kg food produced lifespan effects similar to that of 2.5g / kg diet . 
 these data were censored at 30% survival , and thus further study will be required to determine the dose response range of the lifespan effect in mice ( 12 ) . 
 the nih - itp did not report necropsy or toxicity studies for their ndga - treated mice ( 10,12 ) . 
 based on the mortality - related pathologies we found , there was an association between ndga consumption and an increased incidence of liver , lung , and thymus tumors , and increased hemorrhagic diathesis . 
 there was little evidence for acute liver or kidney toxicity as measured by serum chemistries . 
 however , others reported that treatment of mice with 5.0 g ndga / kg diet , a concentration reported to extend lifespan by the nih - itp , stunted the growth of male and female mice , and induced inflammatory cecal lesions , hemorrhages , and cysts in rats [ reviewed in ( 6 ) ] . 
 thus , the doses of ndga which extend murine lifespan appear to overlap the dosages which produce serious pathologies . in humans , 
 the consumption of dietary supplements containing creosote leaf and stem extracts resulted in toxic hepatitis , cirrhosis , and fulminant liver failure ( 4,7,36,37 ) . 
 however , ndga also was found to suppress signaling through the igfir and to suppress the androgen - stimulated growth of human prostate cancer cells in culture ( 3840 ) . 
 these studies led to clinical investigation of its safety and efficacy for the treatment of prostate cancer ( 41,42 ) . in humans , continuous oral dosing at up to 2.5g / d ( ~31mg / kg bw / d ) , one - tenth of the dose which extended lifespan in this study in mice , was tolerated , except for the development of elevated ast or alt ( transaminitis ) in some patients ( 42 ) . 
 drug dosages in rodents are often scaled down approximately 10-fold in mg per kg body weight for investigational studies in humans ( 14 ) . in a phase ii study of men with non - metastatic hormone - sensitive prostate cancer , oral doses of 2.0 g 
 ndga / d in 28 day cycles produced transaminitis in 8 patients ( 67% ) . 
 the development of less toxic derivatives of ndga , such as tetra - o - methyl nordihydroguaiaretic acid , are underway for potential use as cancer therapeutics ( 43,44 ) . 
 possible mechanisms for the longevity effects of ndga a large number of molecular mechanisms have been proposed to explain the presumed , but as yet largely undocumented , health benefits of ndga [ reviewed in ( 4 ) ] . 
 perhaps the most probable mechanism for the longevity effects of ndga is its inhibitory effects on the activity of mammalian target of rapamycin complex 1 ( mtorc1 ( 45 ) ) . 
 mtorc1 inhibition is a well - established mechanism for the extension of mouse and drosophila lifespan ( 22,4649 ) . 
 a recent review of the literature argues that rapamycin inhibition of mtorc1 extends mouse lifespan by suppressing cancer formation and growth ( 50 ) . 
 however , it will be important to discover whether the dosages of ndga that extend lifespan actually inhibit mtorc1 activity in tissues shown to be important to its longevity effects . in our studies , ndga decreased the mass of liver tumors by 24% relative to those found in control mice , suggesting that it decreased the rate of liver tumor growth ( table 3 ) . 
 these observations are consistent with in vitro and in vivo studies showing that ndga inhibits the growth and proliferation , and induces the apoptosis of multiple cancer cell types ( 5154 ) . 
 these effects likely result from the inhibitory effects of ndga on growth factor signaling ( 3840 ) . in mammals 
 , ndga inhibits igfir activation , igfir and her2 tyrosine kinase activity , androgen dependent growth of cultured prostate tumor cells , and growth of cultured her2-overexpressing human breast cancer cells ( 3840 ) . in our studies 
 , ndga appeared to increased hepatic tumor number ( tables 2 and 3 ) , and to increase the prevalence of lung and thymus tumors ( table 2 ) . 
 taken together , the data suggest that while ndga reduces tumor growth rates , it increases tumor formation . 
 inhibition of igfi receptor signaling can enhance rodent lifespan , although these effects are sex and background dependent ( 55,56 ) . 
 there is less of an effect on lifespan on the c57bl/6j background than on the 129/svpas background ( 56 ) . 
 female mice heterozygous null for the igf1r exhibit extended longevity , while their male counterparts do not ( 56,57 ) . 
 in other studies , neither male nor female mice with reduced igfi levels experience an increase in mean lifespan ( 55 ) . 
 together , these results suggest that if a lifespan effect is observed when igfr activity is inhibited , females , rather than males respond . 
 thus , this response is the opposite of that found with ndga , where males rather than females respond . 
 thus , reduced igfir activity alone is unlikely to be the major mechanism by which ndga extends murine lifespan . 
 hormetic stimuli are thought to produce molecular damage at low doses , which stimulate increased maintenance and repair , and thereby increased longevity ( 58 ) . at high doses 
 masoro and others have proposed that cr is a hormetic agent ( 35,59,60 ) . in one example , the genic profiles produced by a group of progeroid mutations recapitulate the profiles produced by the prolongevity treatments of cr and gh deficiency in mice ( 60 ) . 
 the data regarding ndga are not sufficiently developed to indicate whether it is a hormetic longevity agent . 
 it appears to produce molecular damage at its optimum dosage for increasing lifespan , as evinced by the induction of cancers ( table 2 ) and modest liver and kidney toxicity in humans ( 41,42 ) and mice ( table 4 ) . 
 an unanswered question is whether the less toxic derivatives of ndga such as tetra - o - methyl nordihydroguaiaretic acid will also be capable of extending murine and drosophila lifespan ( 43,44 ) . 
 one theory proposes that ndga exerts positive effects on health because it is a potent scavenger of activated oxygen and nitrogen species in vitro and in vivo ( 4 ) . 
 however , exogenous antioxidants have not been shown to reproducibly extend mammalian lifespan , and they can shorten it ( 15,28,6164 ) . 
 further , the consumption of agents that increase oxidative stress , rather than reduce it , appear to be associated with enhanced longevity in mammals and other species ( 65 ) . 
 ndga reportedly suppresses proinflammatory gene expression and prostaglandin e 2 production and thereby inhibits arachidonic acid 5-lipoxygenase and cytokine - stimulated activation of microglia and macrophages ( 66,67 ) . 
 some data support an inverse correlation between inflammation and lifespan ( 68 ) . however , correlative evidence has been misleading in the context of oxidative stress ( 61 ) . in the absence of direct evidence for an association between reduced inflammation and extended lifespan , 
 inhibition of oxidative phosphorylation can increase rodent and fly lifespan ( 69,70 ) . however , decreased rates of oxidative phosphorylation are not consistent with the dose responsive loss of body weight observed in our mice ( figure 2 ; table 1 ) . 
 inhibition of oxidative phosphorylation should preserve body weight , since it reduces the utilization of calories . in flies 
 , ndga had no effect on body weight , physical activity , or food consumption ( tables 58 ) . 
 thus , our data are inconsistent with the extension of lifespan through inhibition of oxidative phosphorylation . 
 the funders had no role in study design , data collection or analysis , decision to publish , or preparation of the manuscript .","mesonordihydroguaiaretic acid ( ndga ) extends murine lifespan . 
 the studies reported here describe its dose dependence , effects on body weight , toxicity - related clinical chemistries , and mortality - related pathologies . in flies 
 , we characterized its effects on lifespan , food consumption , body weight , and locomotion . 
 b6c3f1 mice were fed ain-93 m diet supplemented with 1.5 , 2.5 , 3.5 , or 4.5 g ndga / kg diet ( 1.59 , 2.65 , 3.71 and 4.77mg / kg body weight / day ) beginning at 12 months of age . only the 3.5mg / kg diet produced a highly significant increase in lifespan , as judged by either the mantel 
 cox log - rank test ( p = .008 ) or the gehan  breslow 
 wilcoxon test ( p = .009 ) . 
 ndga did not alter food intake , but dose - responsively reduced weight , suggesting it decreased the absorption or increased the utilization of calories . 
 ndga significantly increased the incidence of liver , lung , and thymus tumors , and peritoneal hemorrhagic diathesis found at necropsy . 
 however , clinical chemistries found little evidence for overt toxicity . 
 while ndga was not overtly toxic at its therapeutic dosage , its association with severe end of life pathologies does not support the idea that ndga consumption will increase human lifespan or health - span . 
 the less toxic derivatives of ndga which are under development should be explored as anti - aging therapeutics .",pubmed
"genetic variation contributes to both disease susceptibility and treatment response . genome - wide association studies ( gwas ) have enabled rapid discovery of genetic variants contributing to the pathogenesis of complex genetic diseases ( manolio 2010 ) , as well as detection of many pharmacogenetic markers ( link et al . 
 the driving hope of these major advances in genetic epidemiology is that promotion of personalized medicine will improve medical decision - making . 
 although use of the term personalized medicine is often limited to the identification of the optimal drug and the optimal dosage for a subgroup of patients , current personalized medicine applications are far more broad , and might include situations of withholding treatment , preventive interventions , or targeted treatment options for individual patients . in prostate cancer , for example , dna biomarker tests may be used to determine whether treatment may be safely delayed for a period of watchful waiting . if the tumor is demonstrated due to lack of genes causing an aggressive form of the cancer , it may remain stable for decades , and the need for radical surgical resection with subsequent radio- or chemotherapy may be obviated ( kroll 2008 ) . 
 in contrast , in other instances , genetic profiles may be used to determine preventive interventions . 
 this approach is already used for some forms of hereditary cancer , in which individual genetic testing is the basis for deciding upon specific , sometimes very radical interventions such as preventive surgery ( kroll 2008 ) . beyond treatment schemes that are applied identically across large subgroups of patients  to which some authors have applied the distinct term stratified medicine ( trusheim et al . 
 2007)other personalized medicine applications offer targeted treatment options for individual patients . anti - inflammatory therapies , for example , such as anti tnf , anti il-6 , or anti il-1 , 
 are thought to be effective in inflammatory diseases , such as crohn s disease ( buchner et al . 
 other molecules seem suitable for use as anti - inflammatory therapy as well . to choose one or a combination of different anti - inflammatory therapies , the physician might first obtain the genetic profile of a patient by sequencing . depending on the individual dna profile , the physician might select the anti - inflammatory combination therapy , of which there are many . 
 though foundational to personalized medicine , biological markers , biomarkers for short , are diversely defined in the literature . 
 gallo et al . ( 2011 ) summarize some of the definitions , and observe that the most commonly adopted definition states that  a biomarker is any substance or biological structure that can be measured in the human body and may influence , explain or predict the incidence or outcome of disease  . 
 however , it is a matter of debate whether the qualification that biomarkers must be measured in the human body is a reasonable limitation . 
 ( 2011 ) who define  biomarkers as any substance , structure or process that can be measured in biospecimen and which may be associated with health - related outcomes  . from our perspective , this definition is too general and should include a specific association with a health or clinical outcome . 
 our preference , therefore , is for the definition developed by the biomarkers definitions working group ( 2001 ) :  a biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes , pathogenic processes or pharmacologic responses to a therapeutic intervention . 
 although more than a decade old , this definition is comprehensive and sufficiently broad to capture the full range of current biomarker applications , described in detail in the table 1.table  1important terms in biomarker studiestermexplanationbiomarkera characteristic that is objectively measured and evaluated as an indicator of normal biological processes , pathogenic processes , or pharmacologic responses to a therapeutic intervention ( biomarkers definitions working group 2001).cancer biomarkera biomarker that is present in tumor tissue or serum and includes many different molecules , such as dna , mrna , or proteins . 
 tumor biomarkers are measured in tumor tissue , and tumor dna biomarkers are measured from tumor tissue.clinical endpointa characteristic or variable that reflects how a patient feels , functions , or survives ( biomarkers definitions working group 2001).companion endpointa biomarker that is essential to the efficacy and safety of a corresponding therapeutic product ( food and drug administration 2011).copy number variant ( cnv ) biomarkera biomarker of genomic variation in which blocks of dna are missing or for which multiple copies exist.diagnostic biomarkera biomarker that relates to the diagnosis or severity of disease . 
 the most important diagnostic biomarkers are screening biomarkers.disease biomarkera biomarker that relates to a clinical endpoint or measure of disease ( kroll 2008).dna biomarkera germline biomarker , such as snps , strs , deletions , insertions , or other variation on the dna sequence level.efficacy biomarkera biomarker that predicts a beneficial effect of a given treatment ( kroll 2008).epigenetic biomarkera biomarker that measures epigenetic alterations , such as dna methylation , histone methylation , histone acetylation , micrornas , or other non - coding rna ( bock 2009).monitoring biomarkera biomarker to monitor efficacy or side effects of a drug treatment.prognostic biomarkera biomarker that predicts the likely course of disease in a defined clinical population under standard treatment conditions.prediction modela predictive test including multiple markers.predictive biomarkera biomarker that forecasts the likely response to treatment ( buyse et al . 
 2011 ) . treatment response may be measured either as efficacy or as safety.predictive testtwo definitions exist in the literature a test of test of probability for an individual to develop a disease ; alternatively , a test which discriminates between individuals who will develop a disease and those who will not ( janssens and van duijn 2008).risk predictionthe generation or validation of models which make a prognosis for developing a disease or the prognosis for attaining a clinical endpoint.safety biomarkera biomarker that indicates adverse response to a treatment ( sistare et al . 
 toxicity biomarkers are special cases of safety biomarkers.screening biomarkera biomarker to discriminate between healthy individuals and those in an early stage of the disease ( kroll 2008 ) , ideally while subjects are asymptomatic.staging biomarkera biomarker that distinguishes between different stages of chronic disease ( kroll 2008).stratification biomarkersee predictive biomarker.surrogate biomarkera biomarker that is regarded as a valid substitute for a clinical endpoint . 
 a surrogate endpoint is expected to predict clinical benefit ( or harm or lack of benefit or harm ) ( biomarkers definitions working group 2001 ; kroll 2008).target biomarkera biomarker that reports interaction of the drug with its target ( kroll 2008).toxicity biomarkera biomarker that reports to the toxic effect of a drug on an in vitro or in vivo system ( kroll 2008 ) . 
 important terms in biomarker studies in personalized medicine , it is necessary to distinguish between prognostic and predictive biomarkers . following buyse et al . 
 ( 2011 ) , the difference is that prognostic biomarkers help in predicting the progress of the disease , while predictive biomarkers are connected with the response to a treatment . 
 having clarified fundamental terms , we will proceed to a discussion of similarities and differences of biomarkers , illustrate current uses of some of these biomarkers , and outline specific aspects of diagnostic , prognostic , and predictive biomarker studies . 
 finally , we will discuss various study designs for the validation of predictive biomarkers in detail . 
 genetic information , coded within dna , requires stability because dna directs the production of proteins required for the cell structure and function of cells over a lifetime . 
 some authors state that dna is stable over an individual s lifetime ( hicks and coquoz 2009 ) , and biomarkers explicitly representing this stability are termed  dna biomarkers  in the discussion which follows . 
 single nucleotide polymorphisms ( snps ) , short tandem repeats ( strs ) , deletions , insertions , or other variation on the dna sequence level are among this group . due to the availability of high - throughput molecular biological facilities , snps are the most commonly used type of dna variation . in most applications snps 
 cancer is a disease that involves changes to the dna at the cellular level , and these changes can be measured in the tumor . 
 distinct from dna biomarkers outlined above , we will use the term  dna tumor biomarkers  to indicate biomarkers specific to cancerous tumors . 
 typically , only the presence or absence of a mutation in a gene is determined . 
 finally , we use the term  general biomarkers  for all other forms of biomarkers , such as rna , protein , or metabolite measurements which can be measured in biofluid , tissue , or even cell lines . 
 while most general biomarkers share the property of being quantitative with positive measurement values , both dna biomarkers and dna tumor markers are discrete in nature ( table  2 ) . nevertheless , 
 when used in the diagnostic process , thresholds need to be introduced for all types of biomarkers to relate biomarker measurements to clinical decision-making.table  2differences between dna biomarkers , dna tumor biomarkers , and general biomarkerscharacteristicdna biomarkerdna tumor biomarkergeneral biomarkerlevel of measurementdiscrete . in snps , one of three different genotypes 
 is observed per subject , in generaldiscrete . in general , the measurement is whether a specific gene is mutated or notcontinuous . 
 rna , protein , and metabolite concentrations may take almost any continuous positive valuestability , reproducibilityyesnot necessarily as different mutations may be present in different parts of the tumoronly at one specific time pointsuitable for therapy monitoringnoyesyessuitable for pharmacodynamicsnoyesyessuitable as surrogate markernono , in generalyescomplexity of measurementlowhighhightime required for measurement ( includes drawing and preparation of sample)lowhighhightime of measurementdoes not have to be specifiedneeds to be specified in advanceneeds to be specified in advanceretrospective validation of biomarkeryesnonodurability  of the final biomarker testshort- to long - term ; multimarker sets may be already obsolete at start of prospective studymid - term to long - termmid - term to long - termstudy designretrospective or prospectiveprospective onlyprospective onlydna biomarkers are generally measured in the blood , tumor dna biomarkers are measured in tumor tissue , general biomarkers may be measured in biofluid , tissue , or cell lines differences between dna biomarkers , dna tumor biomarkers , and general biomarkers dna biomarkers are generally measured in the blood , tumor dna biomarkers are measured in tumor tissue , general biomarkers may be measured in biofluid , tissue , or cell lines important differences between dna biomarkers and dna tumor or general biomarkers stem from the fact that dna is stable over the entire lifetime ( table  2 ) . 
 dna biomarkers are reproducible , can be measured at any point in time , and may be used in both prospective and retrospective studies . 
 specifically , dna biomarkers can be prospectively validated in biobanks , i.e. , in studies , where clinical information has already been collected . 
 the authors stress that they would not call such kind of study a prospective one . in general , 
 sample drawing , handling and storage protocols are generally also simpler for dna biomarkers , and laboratory time and cost for measurement is lower . 
 first , as they do not vary over lifetime , they can not be used for therapy monitoring , pharmacodynamics , or as surrogate markers . 
 a second general problem is  durability  , as the rate of discovery of new dna biomarkers is frequently more rapid than their product cycles . by the time 
 , a dna multimarker test has successfully passed all steps for marketing approval , including refunding by health insurance companies , it may already be rendered scientifically obsolete by newly discovered dna biomarkers with seemingly better performance . 
 pertinent differences between dna tumor biomarkers and general biomarkers ( table  2 ) include the fact that dna tumor biomarkers generally can not serve as surrogate markers  while general biomarkers often can  and the fact that dna tumor biomarkers can show greater variation , depending on how they are measured . 
 if a dna tumor biomarker is measured through biopsy , for example , it is possible for one biopsy probe to be tumor free , while another contains tumor cells , and this difference may result in different dna tumor biomarker results . 
 in contrast , if a general biomarker is measured , for example , from plasma , it is generally stable for the time point of measurement . 
 in this section , we illustrate current applications of biomarkers for diagnosis , prognosis , and prediction in personalized medicine . 
 details of the examples outlined in this section are provided in table  3 , together with additional examples of general biomarkers , and in particular , dna biomarkers and dna tumor biomarkers , in current use.table  3examples for biomarkers in current usenametyperange of applicationcommercial useindicationtime of measureoutcomereferenceblueprintdna tumorpredictiveyesbreast cancerknown diagnosis , after surgeryreaction of individual therapieskrijgsman et al . 
 ( 2012)egfrdna tumorpredictivepcoadvanced non - small - cell lung cancerknown diagnosis , before first - line therapyegfr tki or chemotherapykeedy et al . 
 ( 2011)il28bdnapredictive  hepatitis c virus 1 ( hcv-1)known diagnosis , before treatmentresponse to treatment with pegylated interferon ( peg - ifn ) combined with ribavirin ( rbv)holmes et al . 
 ( 2011)k - rasdna tumorprognosticpcoadvanced colorectal cancerknown diagnosis , before chemotherapytreatment with cetuximab yes or nokarapetis et al . 
 ( 2008)mammaprintdna tumorprognosticyesbreast cancerknown diagnosis , after operationprecise stage of tumor , aggressivity of tumorbuyse et al . 
 ( 2011)oncotypdxdna tumorpredictive / prognosticyeser - positive , her2-negative breast cancer , colon cancerknown diagnosis , after operationchemotherapy recommended yes / nobuyse et al . 
 ( 2011)point - of - care tests : rheumachecccpoint assaygeneraldiagnostic ( screening)yesrheumatoid arthritis ( ra)before first symptomaticearlier therapy for raegerer et al . 
 ( 2009)slco1b1dnapredictive  myocardial infarctionknown diagnosis , before treatmentreduction of statin doses cause of statin - induced myopathy , security monitoringlink et al . 
 ( 2008)egfr epidermal growth factor receptor , egfr tki egfr tyrosine kinase inhibitor , er estrogen receptor , her2 hormone receptor , k - ras kirsten rat sarcoma viral oncogene homolog , pco provisional clinical opinion of the american society of clinical oncology examples for biomarkers in current use egfr epidermal growth factor receptor , egfr tki egfr tyrosine kinase inhibitor , er estrogen receptor , her2 hormone receptor , k - ras kirsten rat sarcoma viral oncogene homolog , pco provisional clinical opinion of the american society of clinical oncology diagnostic biomarkers are biomarkers used to determine the diagnosis or severity of a disease . 
 the most important within this group are screening biomarkers ( table 1 ) , which are used to discriminate between healthy individuals and those in an early stage of a disease . 
 for example , the commercially available point - of - care tests ( poct ) rheuma - chec and ccpoint , test serum for antibodies to mutated citrullinated vitmentin ( mcv ) or citrullinated peptides / proteins ( anti - ccp antibodies ) , in order to screen for rheumatoid arthritis in non - symptomatic , healthy persons ( egerer et al . 2009 ) . if a diagnosis is known , prognostic biomarkers help to predict the likely course of disease in a defined clinical population under standard treatment conditions . for example , mammaprint , a dna tumor biomarker for breast cancer prognosis is used following surgery to indicate whether risk of metastasis is low or high , and guide physicians to determine the best kind of treatment for the individual patient . 
 in fact , in the united states , mammaprint is cleared by the food and drug administration ( fda ) as an in vitro diagnostic multivariate index assay ( ivdmia ) ( slodkowska and ross 2009 ) . in germany , as of 2012 , some health insurance companies will pay the cost of the test for particular cases . 
 predictive biomarkers predict the likely response of a patient to a special treatment in terms of efficacy and/or safety and thus support clinical decision - making ( table 1 ) . 
 for example , gwas conducted by three independent groups from north america , australia / northern europe , and japan ( holmes et al . 
 2011 ) demonstrated that the il28b gene is a strong indicator for response to standard treatment in patients with hepatitis c virus-1 ( hcv-1 ) infection . 
 further , there is evidence for population stratification in the il28b gene , such that treatment response varied across different ethnic groups . 
 specifically , caucasians with the  good response  genotype were more likely to benefit from treatment . 
 this fact is also mentioned in a provisional guidance for the treatment of hcv ( thomas et al . 
 2012 ) , which notes that treatment with special inhibitors is expected to be less efficacious in persons with the  non - response  genotype , or african ancestry . 
 while some biomarkers have already been approved by the fda , the use of others has been recommended in clinical guidelines such as the provisional clinical opinion from the american society of clinical oncology ( asco ) , which publishes clinical direction based on potentially practice - changing data from major studies . 
 a recent example of this is a test for epidermal growth factor receptor ( egfr ) mutation in patients with advanced non - small - cell lung cancer , which determines whether or not the first - line egr tyrosine kinase inhibitor therapy is indicated ( keedy et al . 
 regulatory approval is required before a new drug can enter the market . in the preclinical program , safety , pharmacology and pharmacodynamics 
 the first three clinical phases establish pharmacokinetics , pharmacodynamics , the required dose , clinical efficacy and benefit / risk . following marketing approval , 
 effects on morbidity and mortality endpoints , or relative effectiveness are investigated in the fourth phase ( european medicines agency 1998b ) . 
 development programs of diagnostic tests undergo a similar process ( european medicines agency 2009 ) . in phase 
 i , it must be demonstrated that the diagnostic test may be safely applied to humans , and that the technical properties are well understood , such as mode of application , reproducibility , etc . in phase ii 
 phase iii studies are the so - called validation studies , in which the diagnostic test and subsequent diagnostic workup is carried out in the same manner and setting as it would be in later clinical and diagnostic practice . 
 finally , phase iv diagnostic studies are performed to investigate whether the application of the test leads to a measurable improvement of the clinical outcome in a broader population . 
 this phase concept is oriented towards marketing approval of diagnostic agents that are intended for in vivo administration , such as radiopharmaceuticals or contrast agents for use in imaging techniques , such as magnetic resonance imaging . in our own projects 
 , we have found an extended phase approach , summarized in table  4 , to be more helpful from a developmental perspective for diagnostic or prognostic biomarkers ( compare the phase models of pepe 2003 , p. 215 
 2009).table  4phases of diagnostic or prognostic biomarker studiesphasediagnostic and prognostic biomarkerstypical sample sizesdescriptionaim of studyiadiscoveryidentification of promising biomarkers10100ibassay development , assay validationdefine and optimize analytical process into robust , reproducible , and valid device10100icretrospective validationclinical assay detects disease ; development of first algorithm for combination test50500iiretrospective refinementvalidation of early detection properties of biomarker ( set ) ; development and/or refinement of algorithm(s ) for combination tests1001,000iiiprospective investigationdetermination of diagnostic accuracy ( sensitivity , specificity ) in the situation of clinical routine2001,000ivarandomized controlled trialquantification of effect of making the biomarker information available to the doctor to reduce disease burden2001,000ivbhealth economics studyquantification of cost - effectivenessstrongly depends on clinical consideration of clinical risk phases of diagnostic or prognostic biomarker studies in phase i of this extended approach , we delineate three distinct sub - phases : discovery ( ia ) , assay development and validation ( ib ) , and retrospective validation ( ic ) . the discovery study ( phase ia ) 
 the discovery phase might be a gwas , a genome - wide meta - analysis or even a whole genome sequencing study . in proteomics , 
 this discovery phase might consist of expression clone arrays containing tens of thousands of recombinant human proteins ( for a recent application to prostate cancer , see massoner et al . 
 2012 ) or multiplex protein antigen analysis on a luminex platform ( linkov et al . 
 the high - throughput arrays or whole genome sequences will not be the final product for the diagnostic test to be released on the market . 
 therefore , in phase ib the identified biomarkers are adapted to a laboratory setting which might be integrated into clinical routine . 
 the complementary technology introduced in this phase is generally more reliable and precise , i.e. , its coefficient of variation is lower and typically has reduced bias . for dna biomarkers , this phase combines assay transfer with a better - suited laboratory platform and the choice of the dna marker(s ) to be typed on this platform . in proteomics , this might represent the development of an elisa , such as the elisa for agr2 in voided urine for detecting secreted prostate cancer ( wayner et al . 
 2011 ) . following the discovery phase and the change in the specific laboratory technology , a first retrospective validation is generally performed ( phase ic ) to determine whether the results from the imprecise high - throughput technology still hold true . 
 it is important to note that the sample size in this first retrospective validation is higher than in the initial high - throughput search ( table  4 , last column ) . 
 subsequent to retrospective validation , multimarker models are developed , either as part of phase ic , with the initial cohort of patients , or as part of phase ii , in a retrospective study using patients different from those of phase i. whether initially developed in phase i or ii , this model will always undergo refinement with the second group of patients . 
 nevertheless , a prospective investigation in phase iii may also often be necessary to obtain reliable estimators for sensitivity and specificity of dna biomarker tests , or , for prognostic biomarkers , to attain reliable estimates of the prognosis , e.g. , through the use of brier scores . to assess the impact of the diagnostic dna biomarker on patient management , a randomized controlled trial is conducted ( in phase iva ) , following marketing approval demonstrating that treating doctors knowledge of the test result improves patient outcome . 
 finally , health economics studies may be carried out in order to assess cost - effectiveness of the diagnostic test ( phase ivb ) . a challenging element of this extended phase model for diagnostic and prognostic biomarkers is the term  prospective validation  for phase iii . because germline dna is assumed to be stable , the prospective investigation of an assay in an already ascertained biobank may be considered to be prospective . however , it must be definitively demonstrated that the biobank has not been used before , because multiple testing will arise if a biobank is used for biomarker validation more than once . proper definition of standards as to when biobanks may be used for prospective validation of dna biomarkers is complex . 
 the situation becomes even more complex when considering dna tumor biomarkers or general biomarkers , as these biomarkers may change over time such that the time point of sample drawing may also be crucial . 
 furthermore , different sample handling and storage protocols as well as age of samples may have an effect not only on biomarker quality but also on biomarker levels ( table  2 ) . 
 irrespective of the phase model used ( for other alternatives , see , e.g. , zhou et al . 
 2001 , p. 61 ) , assay sensitivity  the technical and analytical validity of the biomarkers  is a mandatory requirement . given high assay sensitivity , the problem of missing values can be ignored because they are assumed to occur only due to random technical failure and not any systematic process . 
 examples for current use of biomarkers  , above , and detailed in table  3 , a number of biomarkers have recently been identified to predict treatment outcome , and some have proven particularly effective in resolving unconvincing or ambiguous clinical trial results . 
 for example , in the multicenter ipass ( iressa pan - asia study ) trial of advanced non - small cell lung cancer ( nsclc ) , patients treated with either gefitinib or carboplatin plus paclitaxel , progression - free survival curves of both treatment groups crossed in the general population of patients ( 1,200 previously untreated east - asian non - smokers or former light smokers ) . 
 when treatment groups were stratified for tumors bearing epidermal growth factor receptor gene ( efgr ) mutation , however , important differences emerged : egfr - positive patients progression - free survival was consistently longer in the gefinitab treatment group , while egfr - negative patients progression - free survival was longer in the carboplatin 
 paclitaxel group ( mok et al . 2009 ) . as noted in sect . 
 examples for current use of biomarkers  , the strength of the relationship was sufficiently compelling that the american society for clinical oncology has since recommended that patients with nsclc should have their tumors tested for egfr mutations to determine the most appropriate first - line therapy ( keedy et al . 
 2011 ) . in most instances these biomarkers have been identified after the conduct of phase iii trials that were intended to provide the pivotal evidence about efficacy and positive benefit / risk of an experimental treatment required to justify market approval . 
 an important consequence of the retrospective nature of these investigations was that the appropriateness of the biomarker validation had to be assessed on a case - by - case basis . in july 2011 , 
 the fda issued a draft guidance for industry for  in vitro companion diagnostic devices 
 , which are predictive biomarkers essential for safe and effective use of a corresponding therapeutic product ( food and drug administration 2011 ) . 
 the draft guidance anticipated three uses of companion diagnostics : identification of patients most likely to benefit from a particular therapeutic product;identification of patients likely to be at increased risk for adverse reactions to a therapeutic product , and;monitoring treatment response to adjust treatment in order to improve efficacy and safety . 
 identification of patients most likely to benefit from a particular therapeutic product ; identification of patients likely to be at increased risk for adverse reactions to a therapeutic product , and ; monitoring treatment response to adjust treatment in order to improve efficacy and safety . 
 if either treatment effect or tolerability differs with respect to the companion diagnostic , the diagnostic test can be used to refine the patient population . 
 dna biomarkers and dna tumor biomarkers can not be used for treatment monitoring for obvious reasons . 
 the fda guideline suggests that because companion diagnostics provide critical information for the appropriate use of drugs , they require validation as part of the evaluation of efficacy of the experimental treatments , and information about the diagnostic is reflected in the drugs labeling . 
 the use of biobanks for prospective validation , therefore , might play a role for already approved drugs , if biomaterial allows systematic investigations into improved efficacy or safety in biomarker - defined subgroups . within the pharmaceutical industry 
 , such research will become increasingly important to pharmacovigilance and post - marketing surveillance of drug use . 
 valid conclusions are , however , not possible if material for genotyping is available for only so - called convenience samples ( wang et al . 
 , clinical data should substantiate that efficacy and/or benefit / risk of the experimental treatment as compared to control is substantially larger in patients who test positive with the companion diagnostic test than in those who test negative . 
 if the experimental treatment shows comparable positive effects in test - negative patients and in test - positive patients , the diagnostic test would not be required , and the new treatment might simply be provided to all patients . 
 thus , diagnostic tests are only of value if clinical data or ethical reasons support that treatment be withheld in test - negative patients . in the context of clarifying specific circumstances in which a new drug might be used appropriately , information from test - negative patients 
 must be available in order to justify population refinement , and the costs that use of the biomarker prior to treatment will incur on the health insurance system . 
 it may be a matter of debate , or even discriminative power of the companion biomarker , whether this information might be obtained from a phase ii clinical trial , or whether it should be obtained from the same trial used to demonstrate efficacy of the experimental treatment . 
 obviously , there is no need to demonstrate in statistical terms that the experimental treatment does not have advantages over control in test - negative patients . 
 however , if in the same trial there is no clear trend towards superior efficacy or improved benefit / risk , especially in a phase ii trial of limited size , the usefulness of the companion biomarker might be questioned . 
 official regulatory guidance about appropriate study designs to validate companion diagnostics together with new drugs is still sparse , although both the fda and european medicines agency ( ema ) have established procedures , in which applicants may negotiate the amount of evidence required for marketing authorization in this setting ( european medicines agency 2012 ) . 
 pioneering work on clinical trial designs for predictive biomarker validation has been performed by sargent et al . 
 2011 ) , in their seminal review , discuss 10 different study designs for co - development of an experimental drug and a companion diagnostic test . in selection designs , patients are first tested with the companion test . 
 only test - positive patients are then randomized to treatment or control ( fig . 
 ( 2011 ) observe , although these selection designs provide clear information about treatment efficacy , they fall short in substantiating the usefulness of the companion test , as they do not demonstrate lack of benefit in marker - negative patients . 
 as soon as a test incurs costs , its usefulness for application should be quantified , and for this a selection design is only of limited value.fig . 
 a traditional randomized - control design where the randomization ( r ) to standard ( std ) or experimental ( exp ) therapy is independent from the results of the biomarker test . 
 randomization to std or exp is performed in the total patient population stratified by the result of the biomarker results . 
 c restricted design . to reduce effort , only biomarker - positive patients are randomized to std or exp . claims about the utility of a biomarker can not be made from this trial alone . 
 d patients are randomized according to treatment based on biomarker or non - biomarker - dependent strategy . 
 this is the most flexible design that provides information about specific individualized treatment rules according to , e.g. , a dna profile common study designs for biomarker studies . a traditional randomized - control design where the randomization ( r ) to standard ( std ) or experimental ( exp ) therapy is independent from the results of the biomarker test . 
 randomization to std or exp is performed in the total patient population stratified by the result of the biomarker results . 
 c restricted design . to reduce effort , only biomarker - positive patients are randomized to std or exp . 
 claims about the utility of a biomarker can not be made from this trial alone . 
 d patients are randomized according to treatment based on biomarker or non - biomarker - dependent strategy . 
 this is the most flexible design that provides information about specific individualized treatment rules according to , e.g. , a dna profile in the interaction or biomarker - stratified design ( fig . 
 1b ) , patients are first tested for the biomarker , and then randomized to treatment or control with stratification by the companion biomarker s test result . 
 this study design might be considered the gold - standard design for providing a sound basis for decision - making about the efficacy and benefit / risk of the experimental drug and the ability of the companion diagnostic to identify the appropriate patient population to be treated . 
 the significance of the interaction test for the treatment effect in the test - positive and test - negative stratum may serve as a yardstick for the usefulness of the companion biomarker . 
 if the interaction effect is significant , treatment effects in test - positive and test - negative patients differ ; this difference alone does , however , not in itself assure the utility of a companion diagnostic . 
 a small but significant treatment effect in test negatives , even if less marked than the effect in the biomarker - positive population , might still be of clinical importance . 
 similarly , if the treatment effect in the larger population is small and irrelevant , the subgroup of biomarker - positive patients might be too small to achieve sufficient power in the interaction test . 
 evaluation of efficacy and benefit / risk should always consider the size of the estimated treatment effect as well . 
 adaptive study designs offer a promising means to stop recruitment for futility in test - negative patients as soon as sufficient evidence about lack of efficacy in this subgroup is available . 
 strategies based on conditional power may be used to formalize adaptive study designs to some extent ( lachin 2005 ; proschan and hunsberger 1995 ; schfer et al . 
 however , in the end , the assessment of the treatment effect size and the extent of benefit / risk must be set into perspective . in instances 
 where no gold standard are available , the utility of a diagnostic test may be evaluated by comparing biomarker - guided treatment with the standard , non - biomarker - based treatment selection . a highly simplified version of this is depicted in fig . 
 1c with only two profiles and two treatments in the biomarker - guided treatment group . 
 again , significant differences between the outcome in the biomarker - guided and the conventional treatment group may be difficult to achieve and may fall short in demonstrating the efficacy of the experimental biomarker 
 . the individual profile design , which includes a large number of different profiles leading to the selection of one out of a large number of different treatments , is easily validated if it is planned and understood as a strategic trial comparing conventional treatment selection to an individualized decision rule ( fig . 
 for example , an individualized therapy might combine several monotherapies , each selected based on the presence or absence of a specific dna variant . 
 however , they pose new challenges to the regulatory system , requiring that the precise particularities of the application of drugs as mono - therapy , or in combination are well understood and substantiated with clinical trials data . obviously , there is a gap between the validation of the biomarker - based treatment selection rule and the efficacy of the individual treatments to be applied based on a complex decision rule . 
 consideration needs to be given to the question of how this gap can be filled . 
 for example , the global statistics may demonstrate superiority of the individualized treatment selection when compared to the current standard . at the same time 
 , some of the individual treatment combinations may seem to be inferior to the current standard . here 
 , it is clear that an upfront discussion is required to formulate the circumstances under which such inconsistencies can be ignored and under which the overall validity of conclusions has to be questioned . 
 similarly , none of the many investigated treatments or treatment combinations may provide sufficient clinical information enabling the assessment of the safety of the suggested combination therapy in the way this is done in the standard drug development process , and we acknowledge that this standard process is justified by historical fallacies . unless clear evidence is available that all drugs under investigation are safe and can be combined freely , a substantial amount of clinical data needs to be provided for each of the recommended treatments and treatment combinations . 
 at least a basic assessment of safety is required because safety or benefit / risk may be different for different subpopulations identified with the biomarker rule . 
 along the line of the fda definition of a companion test such information is a pre - requisite to use a complex decision rule in patient management . 
 of course , sample - size requirements will substantially increase with the number of treatments and treatment combinations under investigation , and the ipass trial can be considered as a model for this . in some instances 
 , biomarkers may be investigated that are closely related to the mechanism of action of the companion drug , and in these instances it may be possible to provide the required information about the usefulness of the companion test as early as phase ii drug development . in most instances , 
 however , the traditional design  randomizing patients to treatment and control irrespective of biomarker outcome  may still be the optimal approach , such that overall superiority of a drug should first be demonstrated , subgroups defined by the companion test excluded from the labeling in retrospect , following the precautionary principle that harm does not need to be proven ( fig . 
 the credibility of this concept depends on the degree of independent validation that can be found in the different studies during the development program and the completeness of the sampling in every study . 
 convenience sampling in subpopulations of poorly defined origin should be avoided ( wang et al . 2010 ) . 
 the three pillars of a physician s work are diagnosis , therapy , and prognosis . 
 biomarkers form the basis for all aspects of personalized medicine , from  what does the patient have ?  ( diagnosis ) to  what can the patient do about it ?  ( therapy ) to  how bad is it ?  ( prognosis ) . 
 although the characteristics and applications of dna biomarkers , dna tumor biomarkers and general biomarkers differ substantially , the underlying methodological principles to validate each for use in clinical routine are identical . 
 dna biomarkers are distinct from general biomarkers in several ways , such as protein expression levels , gene expression levels or even epigenetic biomarkers . 
 further , since germline dna does not change over time , some studies utilizing biobanks may be interpreted as prospective biomarker studies under very special conditions . 
 however , the critical aspect prior to marketing approval of a biomarker is how  convincing evidence  may be achieved and when it has been achieved . 
 novel strategies must be developed so that a biomarker assessed in biobanks can be considered validated , in particular if biobanks are to be used multiple times . 
 an important aspect of biobank biomarker studies is the transparency of the approach , which directly relates to the quality of reporting . in general , 
 to date , reporting of biomarker studies has been weak . in a systematic review of studies published between 2004 and 2006 , fontela et al . 
 ( 2009 ) evaluated diagnostic studies that used commercially available test kits for tuberculosis , hiv , and malaria , and found that all of the 238 articles fulfilling the study inclusion criteria had design issues . in only 10  % of the studies the reference standard was adequately described . 
 nine of the 25 indicators of the standards for reporting of diagnostic accuracy ( stard ) ( bossuyt et al . 
 2004 ) were reported in fewer than 25  % of the studies , even though the studies reviewed were published immediately following publication of the stard statement . 
 ( 2006 ) have effectively demonstrated for therapeutic studies , if reporting guidelines are adopted by journals , and in consequence by authors , the quality of reporting is measurably improved . in addition , it is substantially simpler to read articles if authors follow reporting statements . 
 therefore , authors of biomarker studies are strongly encouraged to report their work using appropriate reporting guidelines , such as stard , strengthening the reporting of observational studies in epidemiology  molecular epidemiology ( strobe - me ; gallo et al . 2011 ) , reporting recommendations for tumor marker prognostic studies ( remark ; mcshane et al . 
 2010 ) or the preferred reporting items for systematic reviews and meta - analyses ( prisma , formerly quorum ; moher et al . 2009 ) ; for a complete list , the reader may refer to the equator network at http://equator-network.org . 
 while validation of diagnostic and prognostic biomarkers does not need to be linked to a specific therapy but rather to standard of care , validation of predictive biomarkers must be closely linked to the specific therapy under consideration . in order to gain marketing approval , pivotal drug trials , 
 i.e. , randomized controlled phase iii clinical trials generally operate from the paradigm recruiting a broad spectrum of patients , not only to achieve higher enrollment numbers , but also to investigate the generalizability of findings . 
 if , however , a drug has different levels of efficacy and safety in different ethnicities , as in for example gemcitabine , tamoxifen ( sai and saito 2011 ) or warfarin ( el rouby et al . 
 2004 ) , it might be possible to leverage the power to detect efficacy and demonstrate safety of a drug as a rationale to conduct trials in ethnically homogenous populations . 
 although the ich e5 guideline ( european medicines agency 1998a ) clearly outlines a framework to evaluate the impact of ethnic factors , the use of an ethnically homogeneous population to evaluate the effect of a biomarker might be more promising if the aim is detection of different effects between biomarkers ( see the study of link et al . 
 if recruitment is done on a global level in a phase iii clinical trial , one has to expect substantial population stratification , i.e. , heterogeneity in the patient population , and as sample sizes per ethnic group are eventually small , identification of biomarkers might be hindered . even if a biomarker or a set of biomarkers has been identified to be a good diagnostic , prognostic , or predictive marker , application to clinical routine 
 although predictive biomarkers may be readily applied , as specific therapies rely on biomarker results , clinical applications for diagnostic or prognostic biomarkers are more complex . here , use in clinical routine will depend on the ease of applicability , including the media for biomarker measurement , and whether clinicians can measure the biomarker in their own laboratory , a local laboratory , or a specialized laboratory at some distance from their practices . also important 
 if clinicians are able to perform calculations by hand and the rules are easy to interpret , acceptance of the biomarker(s ) is more likely than if some kind of  black box  is required . 
 therefore , classifications and probabilities estimated by a logistic regression model are more likely to be accepted by clinicians than results obtained by machine learning methods , such as artificial neural networks or support vector machines 
 we conclude that a priori planning of research strategies is vital for the identification and validation of biomarkers .","biomarkers are of increasing importance for personalized medicine , with applications including diagnosis , prognosis , and selection of targeted therapies . 
 their use is extremely diverse , ranging from pharmacodynamics to treatment monitoring . 
 following a concise review of terminology , we provide examples and current applications of three broad categories of biomarkers  dna biomarkers , dna tumor biomarkers , and other general biomarkers . 
 we outline clinical trial phases for identifying and validating diagnostic and prognostic biomarkers . 
 predictive biomarkers , more generally termed companion diagnostic tests predict treatment response in terms of efficacy and/or safety . 
 we consider suitability of clinical trial designs for predictive biomarkers , including a detailed discussion of validation study designs , with emphasis on interpretation of study results . 
 we specifically discuss the interpretability of treatment effects if a large set of dna biomarker profiles is available and the number of therapies is identical to the number of different profiles .",pubmed
"myosin va ( myova ) is a processive molecular motor   involved in intracellular cargo transport along the actin cytoskeleton [ 2 , 3 ] . 
 each dimerized molecule consists of a pair of n - terminal motor domains ( i.e. , heads ) connected to lever arms , followed by a coiled - coil rod domain , ending in a globular c - terminal cargo - binding domain . 
 myova 's directed movement is accomplished by a conformational change in the motor domain following atp hydrolysis [ 5 , 6 ] . 
 this motion is amplified by the lever arms , resulting in the motor 's processive motion as each head takes 72  nm steps in a hand - over - hand fashion [ 79 ] . 
 each lever arm consists of six tandem -helix motifs , stabilized by calmodulin or calmodulin - like light chains . 
 calcium is required to activate the atpase activity of myova ; however in the presence of high calcium ( > 1  m ) , motor processivity is reduced   and one or more calmodulins may dissociate from each lever arm . 
 calmodulin dissociation may render the lever arm highly compliant , which could reduce the efficiency of both the lever arm displacement amplification and the strain - dependent gating between the two heads , which are necessary for the motor 's processivity . 
 single myova are adept at maneuvering through actin intersections and traveling along actin bundles in vitro [ 1417 ] . 
 myova must have sufficient structural flexibility to permit the unbound leading head to undergo a broad diffusive search [ 7 , 1820 ] , allowing myova to switch actin tracks even when the angle between intersecting actin filaments is ~150 . 
 the structural domains that are critical to myova 's maneuverability have yet to be determined but may reside within the lever arms or at the lever arm / coiled - coil junction . 
 here we used tethered particle microscopy ( tpm ) to directly measure the flexural stiffness of individual myova motors and their subdomains . 
 tpm has been used extensively to characterize the contour length of dna tethers [ 2225 ] . 
 tpm has additionally been coupled with monte carlo simulations of nanoparticles attached to wormlike chain tethers to estimate , with high precision , the persistence length ( a measure of flexural stiffness ) of double - stranded dna . 
 our utilization of tpm involved anchoring a myova - actin complex to a microscope slide and then attaching a visible particle ( i.e. , fluorescent quantum dot ( qdot ) ) to a desired location along the motor . 
 thermal energy caused the qdot to be randomly displaced within bounds defined by the length and stiffness of the effective tether ( i.e. , the myova ) . 
 the resulting motion was measured by comparing the difference in apparent image size of the tethered qdot to a stationary , reference qdot , both of which are viewed over a long ( ~2.5  s ) exposure time [ 24 , 25 ] . while a qdot held in a fixed location has an apparent image size ( sr ) which is dependent on its diffraction limited point spread function , tethered qdots will appear larger ( st ) depending on the length and flexibility of the tether ( figure 1 ) . by attaching qdots to expressed myova constructs of different lengths ( figure 2 ) , we isolated the contributions of individual structural domains , for example , the lever arm and rod domain , to the overall flexibility and flexural stiffness of the myova molecule . 
 in addition , we hypothesized that calmodulin disassociation from the myova lever arm decreases the lever arm 's flexural stiffness , thus contributing to the motor 's compromised processivity in the presence of calcium . 
 various myova constructs , listed as follows , were expressed in the baculovirus / sf9 cell system and purified as described previously . 
 these constructs allowed the flexural stiffness of myova subdomains to be defined by tpm ( figure 2 ) . 
 full - length murine myova ( myova - fl ) was expressed as was a double - headed short construct ( myova - hmm ) , engineered by truncating the myova - fl heavy chain at residue 1098 , followed by a biotin tag and a flag epitope tag at the c - terminus to facilitate purification [ 14 , 28 ] . 
 double - headed myova constructs were coexpressed with calcium - insensitive calmodulin ( camall ) as described previously . 
 single - headed myova ( myova - s1 ) was truncated at amino acid 908 with a biotin tag at the c - terminal end and coexpressed with wild - type calmodulin and purified as described . 
 the myova - s1 and its wild - type calmodulins are sensitive to calcium in that calmodulin(s ) will dissociate from the lever arm in the presence of calcium in the solution . 
 the biotin tag is an 88-amino - acid segment from the escherichia coli biotin carboxyl carrier protein , which was biotinylated at a single lysine residue ( located 35 amino acids from the c - terminus ) during expression in sf9 cells . 
 n - ethylmaleimide- ( nem- ) modified skeletal muscle myosin was prepared as previously described   and was used to strongly adhere actin filaments to the glass surface . 
 actin filaments were isolated from chicken pectoralis ; labeled with tetramethyl rhodamine isothiocyanate ( tritc ) phalloidin overnight in buffer a ( 25  mm imidazole , ph 7.4 , 4  mm mgcl2 , 1  mm egta , 25  mm kcl , and 10  mm dtt ) prior to experiments . carboxylated qdots , emitting at 655  nm ( invitrogen - molecular probe , eugene , or ) , were attached to the myova - fl cargo - binding domain by incubating a 4  :  1 mixture of qdots to myova - fl for 15  min on ice in buffer b ( 25  mm imidazole , ph 7.4 , 4  mm mgcl2 , 1  mm egta , 25  mm kcl , 10  mm dtt , oxygen scavenger composed of 0.1  mg / ml glucose oxidase , 0.020.18  mg / ml catalase , and 3.0  mg / ml glucose ) and 0.1  mg / ml bsa . 
 streptavidin - conjugated qdots , also emitting at 655  nm ( invitrogen - molecular probe , eugene , or ) , were attached to the c - termini of the myova - hmm and myova - s1 constructs by mixing qdots and construct ( 4  :  1 ratio ) and incubating for 15 minutes on ice in buffer b plus 0.5  mg / ml bsa . 
 the mixture was further diluted to a final myova construct concentration of ~0.1  nm before imaging . 
 to attach streptavidin - conjugated qdots to actin filaments , biotinylated actin filaments were prepared by mixing 1  m actin filament , 500  nm tritc - phalloidin , and 500  nm biotin - phalloidin ( invitrogen ) in buffer a and then incubating overnight at 4c . 
 biotinylated actin filaments ( 50  nm ) were attached to the glass surface through nem - modified myosin ( see section 2.3 for details ) . after washing the flow cell with buffer 
 then 24  nm streptavidin - conjugated qdots ( emitting at 655  nm ) were introduced into the flow cell . to remove the unbound qdots , 
 the flow cell was washed by buffer a. a 20  l flow cell was constructed as described . 
 first , 20  l of nem - modified myosin   at 0.5  mg / ml in buffer c ( 25  mm imidazole , ph 7.4 , 4  mm mgcl2 , 1  mm egta , 300  mm kcl , and 10  mm dtt ) was introduced and incubated for 2 minutes at room temperature . 
 nem - modified myosin was used to fix the actin filaments to the glass surface , as it has strong binding affinity to actin . after washing the flow cell using buffer b , 20  l of 0.5  mg 
 / ml bsa in buffer b was infused and incubated for 2  min , followed by a buffer b wash . 
 tritc - phalloidin labeled actin in buffer b was then introduced into the flow - cell chamber and incubated for 2  min , followed by a buffer b wash containing a myova : qdot complex . 
 all myova constructs were strongly bound to actin in the absence of mgatp , that is , in rigor . 
 it is assumed that myova constructs bound to the top surface of the actin filaments due to the high entropy required to approach the filaments from the sides . to measure the calcium - dependent flexibility of the myova - s1 construct , 
 calcium was added to the myova - s1:qdot complex in buffer b , to a final concentration of 1  m , and then introduced into the flow cell . 
 single molecule imaging was performed at room temperature ( 23  1c ) using a nikon eclipse ti - u inverted microscope equipped with a planapo objective lens ( 100x , 1.49  n.a . ) through the objective tirf microscopy . 
 images were acquired using piper software ( piper control v2.3.14 software , stanford photonics , stanford , ca ) and a high resolution ( 95  nm per pixel ) digital camera ( standard photonics , turbo - z , stanford , ca ) . 
 qdots were excited with a 473  nm argon laser line and images obtained using a 655  20  nm emission filter ( chroma technologies , rockingham , vt ) at 8.3  ms integration time for 300 frames . following imaging of the qdots , a single image of the actin filaments in the same visual field 
 was obtained by exciting the tritc - phalloidin labeled actin with a 532  nm laser line and the emission acquired through a 605  35  nm filter ( chroma technologies , rockingham , vt ) and a 67  ms exposure time . 
 first , each series of frames was converted to double precision and summed for an effective integration time of 2.49  s , which was determined to be suitably long to sample the full extent of particle motion ( see results and discussion ; figure 3 ) . 
 summation of short exposures was chosen for this study rather than a single long exposure in order to reduce noise and confirm that the microscope stage did not drift during the experimental protocol . 
 qdots were automatically identified ( figures 4(a ) and 4(b ) ) by thresholding the summed images to binary and recording the centroids of regions which fit three different selection criteria . 
 regions were included based on size ( < 144 pixels ) and elliptical eccentricity ( < 0.86 , which is an aspect ratio of approximately 2  :  1 ) to reject regions likely representing multiple qdots in close proximity to each other . additionally , distance from edges of the image 
 qdot apparent image sizes were then measured by extracting a twelve - pixel square subwindow around each qdot ( figures 4(c ) and 4(d ) ) . 
 this window size was suitably large to fully encompass the qdot image that on average was approximately seven pixels in diameter . 
 each window was then fit , using the least squares method , by a two - dimensional gaussian function ( figure 4(e ) ) , in local coordinates x - y of the form(1)g = a12x2ex+x2/2x212y2ey+y2/2y2+b , where a is the intensity of the qdot , b is the mean intensity of the background , x and y are the x - axis and y - axis standard deviations of the gaussian function , and x and y are offsets between the center of the two - dimensional gaussian function and the center of the window . 
 an image size parameter , s , for each qdot was then defined as the average of x and y multiplied by a microscope image calibration factor ( 95  nm per pixel ) . 
 surface bound qdots ( i.e. , stationary and fixed ) were used as an internal reference and control for the tethered qdot image size parameters in the same field of view , whether they be on an actin filament or a myova construct . the reference and 
 tethered qdots were differentiated by merging images of qdots and actin filaments and manually selecting the qdots not colocalized with actin as reference qdots ( figure 4(a ) ) . 
 when two orthogonal ( i.e. , not correlated ) signals oscillating around a central point are summed , the resulting variance is the sum of the variances of the two signals . given two signals with standard deviations 1 and 2 , the sum of the two signals will thus have a deviation , 3 , given by the square root of the sum of squares:(2)3=12+22 . 
 both the reference image size parameter , sr( ) , and the tethered particle motion , t( ) ( where  may refer to images with qdots bound to actin filaments , a , or myova constructs , m ) , are defined as the deviations of normally distributed random signals , which , added together , result in an apparent image size parameter for the tethered qdot ( i.e. , st( ) ) . 
 therefore , st( ) results from letting 1 = sr( ) and 2 = t( ) in ( 2 ) . 
 this then allows the motion of the tethered particle to be determined when median values of reference ( sr( ) ) and tethered qdot ( st( ) ) image size parameters are known as in ( 3 ) . due to nonnormality of image size parameter frequency distributions ( figure 5 ) , 
 equation ( 2 ) may also be used to relate the deviations of any two points on a randomly oscillating structure that is fixed at one end to a rigid structure . 
 this is particularly useful for canceling out the compliance of the nem - modified myosin , which results in motion of the actin filaments , t(a ) . 
 the motion of a qdot tethered by a myova construct , corrected for actin filament motion , ma , may then be determined knowing the myosin tethered qdot motion , t(m ) , and the baseline actin filament motion , t(a):(4)ma=tm2ta2 . by substituting ( 3 ) into ( 4 ) , the motion of a particle tethered by a myova construct relative to actin , ma , 
 was related to the various median measured image size parameters:(5)ma = stm2srm2sta2+sra2 . for median values of st( ) and sr( ) measured for each construct , the standard error of the median ( sem ) was calculated from the standard deviation ( sd ) and sample size ( n ) assuming an approximately gaussian distribution:(6)sem=1.25sdn . 
 error propagation through ( 5 ) ( ema ) was calculated from the median image size parameters of tethered and actin - bound qdots ( st(m ) and st(a ) ) along with the sizes of their associated reference qdots ( sr(m ) and sr(a ) ) and their respective standard errors ( et(m ) , et(a ) , er(m ) , and er(a)):(7)ematastm2etm2+tasrm2erm2+tasta2eta2+tasra2era2=stm2etm2+srm2erm2sta2eta2+sra2era2stm2srm2sta2+sra2 . 
 the flexural stiffness for the various myova constructs , k , was determined by equipartition due to thermal energy , resulting in brownian motion of the tethered qdot relative to the actin filament , ma , where kb is boltzmann 's constant and t is temperature in degrees kelvin:(8)k = kbtma2 . 
 the persistence length of the tether , lp , is defined as ei / kbt , where ei is the flexural rigidity of a prismatic rod . 
 assuming that the tether acts as a cantilevered beam , with load imparted primarily through the qdot at the end , the effective spring constant , k , can be related to flexural rigidity by k = 3ei / l . combining these two expressions and substituting into ( 8) allow lp to be calculated directly from the motion of the tethered qdot relative to actin ( ma ) and the length of the molecule , l ( see figure 2):(9)lp = l33ma2 . 
 in addition to being used to measure the flexural stiffness of myova constructs , the effective motions of the distal rod domain were isolated by using the procedure above with the motion of qdots bound to myova - hmm constructs rather than actin filaments as the baseline of comparison for myova - fl . 
 the validity of the tethered particle motion approach was confirmed by defining the relationship between myosin tethered qdot motion relative to the actin filament ( ma ) ( see ( 5 ) ) and the assumed tether length , l , for the various myova constructs , which was estimated to span from the actin filament surface to the center of the attached 20  nm qdot , with the length of the myova construct based on its structure   ( see figure 2 ) . to test the significance or the trend in ma with l 
 , a linear regression was performed using prism 6 ( graphpad inc . ) . in general 
 , ma was expected to increase with increasing l , which in fact was the case ( see figure 6 ) . 
 a pilot study showed no significant effect of up to 100  nm of displacement out of the focal plane on qdot image size parameters ( see figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2015/465693 ) , confirming that the trend in increasing ma with l was , in fact , the result of tether compliance and not focal separation   between tethered and reference qdots . 
 to estimate the flexural stiffness of myova - fl and its subdomains , we utilized long exposure time tpm imaging of qdots attached to various myova constructs that served as flexible tethers ( figure 2 ) . 
 the long exposure times were necessary since the brownian excursion time for a qdot tethered to a myova construct should have been on the order of microseconds . based on our experience , imaging qdots with acceptable signal  : 
 thus , accurately assigning the tethered qdot 's location at a single point in time was effectively impossible . 
 however , at the other extreme , millisecond exposures do not fully sample the qdot 's excursion overall times . 
 therefore , to accurately estimate myova 's flexural stiffness , the full range of tethered qdot motion is needed ; otherwise the ambiguity in tracking the qdot 's position results in a significant underestimate of motion . 
 imaging for relatively long exposure times ( 100  ms ) by summing sequentially acquired images ( see section 2 ) ( figure 3 ) circumvents this issue by sampling the full distribution of particle locations and becomes insensitive to exact exposure times . 
 a further advantage of tpm is the ability to efficiently estimate the flexural stiffness of a large number of myova constructs simultaneously within each visual field . 
 we measured image size parameters for qdots bound to actin and tethered by various myova constructs . before relating these parameters to estimates of tether motion and thus flexural stiffness 
 , it was important to control for the effective motion ( i.e. , noise ) in the imaging system , the mode of qdot attachment , and the actin filament movement to which the various myova constructs were bound ( figure 2 ) . to control for the imaging system noise 
 , image size parameters were obtained for qdots bound to the microscope slide and the nem - modified myosin layer under identical imaging conditions ( figure 2 ) . 
 these image size parameters were no different ( 185.8  2.8  nm and 184.3  2.3  nm mean  sem , resp . ) , indicating that image size parameters for qdots in the visual field that were not bound to actin and are suitable for use as stationary , surface - bound qdot references , sr . 
 however , qdots bound directly to actin filaments showed 17  nm of motion , t(a ) , relative to reference qdots , r(a ) ( table 1 ) . this amount of motion indicates substantial compliance in the nem - modified myosin attachment to the actin filaments , requiring the use of actin - bound qdots and their motion as a corrective baseline for myova stiffness estimates ( table 1 ) . 
 using a range of different length myova constructs ( figure 2 ) , we observed a proportional increase in tethered qdot motion relative to actin , ma , with increasing tether length , that is , the length of the myova construct , l ( table 1 ; figure 6 ) . 
 the linear regression of ma with respect to l was statistically significant ( slope = 0.341 , p < 0.01 ) , with all points falling between the theoretical bounds of 1 ( which assumes a tether with zero stiffness ) and 0 ( perfectly rigid tether ) . 
 in addition , to the flexural stiffness of the various myova constructs , we could estimate the flexural stiffness of myova subdomains , that is , the lever arms , lever arm / coiled - coil junction , and distal rod ( table 1 , figure 7 ) as presented below . 
 myova 's lever arms and their elasticity   are critical to the motor 's processive movement . 
 differential strain in the motor 's lever arms while undergoing processive movement is apparent in electron   and atomic force   microscopic images in which bending of the leading head 's lever arm is observed as it attempts its power stroke against the resistive load of the strongly bound trailing head . with the kinetics of the chemomechanical cycle of the individual heads being load - dependent , 
 the difference in strain experienced by the leading and trailing heads is believed to underlie the gating mechanism that assures forward displacement of the motor along actin [ 21 , 36 ] . 
 therefore , the lever arm flexural stiffness may be tuned via calcium signaling to effectively allow or inhibit this necessary intramolecular communication between heads . using tpm , 
 the motion of qdots attached to the c - termini of single - headed myova - s1 constructs relative to actin - bound baseline qdots was used to estimate the lever arm stiffness ( see ( 5 ) ) . 
 based on electron microscopic images , the position of a strongly bound myova head domain appears fixed on the actin filament relative to the observed swinging motion of the lever arm during the power stroke . 
 therefore , we assumed that the tethered qdot motion predominantly reflects the 0.0346  pn / nm flexural stiffness of the lever arm ( figure 7 ; table 1 ) with a corresponding persistence length of 242  nm . a significantly higher stiffness value of 0.21  pn / nm for the myova - s1 was estimated using oscillatory length perturbations in the laser trap assay . 
 however , this approach most likely imposed a combination of bending and axial tension , which would be expected to lead to higher stiffness values . 
 the tpm method more closely approximates pure bending , so that the tpm estimate for persistence length agrees with our previous estimate of 310  nm , based on myova - s1 step size measurements in a laser trap assay . in these earlier studies , 
 the motor itself imposed an internal bending strain on the lever arm due to the power stroke working against the stiffness of the laser trap and thus would have been dominated by bending rather than tensile forces . following the addition of calcium , the average motion of qdots tethered to the myova - s1 c - terminus increased by 10% , indicating a 40% reduction in lever arm flexural stiffness ( figure 7 ; table 1 ) . a substantial decrease in stiffness 
 is expected , given that the addition of calcium can disassociate as many as three calmodulins from each lever arm , exposing the underlying alpha - helix , which by itself is assumed to be unstable [ 12 , 13 , 37 ] . 
 it is important to note that although calmodulins could dissociate from the lever arm in the absence of calcium without excess calmodulin in the assay buffer , as in our experimental conditions , we do not believe this was the case . 
 it has been shown that myova - hmm processivity , as defined by the motor 's run length , is a sensitive measure of interhead communication that relies on rigid lever arms [ 12 , 37 ] . 
 thus , the addition of calcium leads to an abrupt decline in processivity as calmodulins dissociate and the lever arms become compliant [ 12 , 37 ] . however , in control experiments ( figure s4 in supplemental materials ) , run lengths for myova - hmm with and without excess calmodulin in the assay buffer were unchanged , confirming that the lever arms ' calmodulin occupancy under our zero calcium conditions was fully intact . 
 interestingly , in addition to an increase in the average tethered qdot motion with calcium , there was a large increase in the variance of the qdot motion , with the sem increasing from 3  nm to 21  nm ( figure 6 ) . 
 the number of calmodulins dissociated from each individual lever arm is variable , with an average of 2.2 per lever [ 13 , 37 ] , which may account for the broad distribution of observed tethered qdot motions for the myova - s1 construct in the presence of calcium ( figure 6 ; table 1 ) . 
 the observed decrease in lever arm flexural stiffness is likely large enough to negatively impact the interhead communication and gating   necessary for force production and processivity . 
 the myova rod domain lies between the lever arms and the globular tail domain ( figure 7 ) . 
 this rod is predominantly -helical coiled - coil and contains two pest sites , with the -helical coiled - coil in the proximal rod prior to the first pest site necessary for myova dimerization   ( figure 7 ) . to estimate the flexural stiffness of the distal rod domain , we compared the tethered qdot motion of the myova - fl construct to that of the myova - hmm construct , which was truncated just before the first pest site ( figures 2 and 7 ) . 
 our measurements suggest that the distal rod segment , itself , has a flexural stiffness of 0.0326  pn / nm . unlike the lever arm , however , it is difficult to apply ( 9 ) and calculate a persistence length for the rod segment , as the exact length and structure of the distal rod are unknown , with estimates of the distal rod ranging up to 38  nm not including the 7  nm globular tail domain [ 4 , 27 ] . 
 the observed increase in tethered particle excursion requires the distal rod to have some effective length , though the measured stiffness requires a structure composed of either a long ( ~35  nm ) segment of coiled - coil ( lp = 100  nm ) or a series of coiled - coil segments interspersed with highly compliant disordered segments . 
 both the regulatory interaction between the globular tail and motor domains [ 27 , 40 , 41 ] and the variation in apparent rod lengths in electron micrographs strongly suggest the latter . 
 interestingly , the flexural stiffness for the myova - hmm and myova - fl constructs is an order of magnitude lower than both the distal coiled - coil rod domain and lever arms ( table 1 , figure 7 ) . with the tpm measurements for both of the myova - hmm and myova - fl constructs obtained in rigor 
 , we assume that for either construct both heads are strongly bound to the actin filament . 
 the two - headed bound state with the two lever arms in a triangular arrangement   should give a combined stiffness for the two levers , which is greater than a single lever arm and greater than the flexural stiffness of the rod . 
 thus , the extremely low myova - hmm and myova - fl flexural stiffness require that the lever arm / rod junction must be highly compliant . 
 this compliance must contribute to the free diffusive search of the unbound leading head [ 18 , 19 ] , allowing myova to maneuver through actin - actin intersections   and to switch tracks on actin bundles . 
 we have developed a unique system , utilizing long exposure tpm , to measure the flexural stiffness of single molecules with lengths less than 100  nm . 
 the estimates of flexural stiffness for various myova constructs and subdomains by tpm offer direct insight into the structural components of the myova motor that are critical to its functional capacities . 
 specifically , the lever arms must be rigid enough to allow intramolecular strains to be communicated between heads for proper gating and control of the motor 's processivity . 
 the compliance of the lever arm / rod junction must have sufficient flexibility to allow myova to avoid intracellular obstacles and deliver cargo efficiently to its destination in vivo through the dense actin meshwork . 
 in addition , the potential for alterations in the lever arm stiffness with changes in intracellular calcium may provide a measure of tunability in regulating cargo transport and delivery linked to changes in the cell 's ionic environment .","myosin va ( myova ) is a processive molecular motor involved in intracellular cargo transport on the actin cytoskeleton . 
 the motor 's processivity and ability to navigate actin intersections are believed to be governed by the stiffness of various parts of the motor 's structure . 
 specifically , changes in calcium may regulate motor processivity by altering the motor 's lever arm stiffness and thus its interhead communication . in order to measure the flexural stiffness of myova subdomains , 
 we use tethered particle microscopy , which relates the brownian motion of fluorescent quantum dots , which are attached to various single- and double - headed myova constructs bound to actin in rigor , to the motor 's flexural stiffness . based on these measurements , the myova lever arm and coiled - coil rod domain 
 have comparable flexural stiffness ( 0.034  pn / nm ) . 
 upon addition of calcium , the lever arm stiffness is reduced 40% as a result of calmodulins potentially dissociating from the lever arm . 
 in addition , the flexural stiffness of the full - length myova construct is an order of magnitude less stiff than both a single lever arm and the coiled - coil rod . 
 this suggests that the myova lever arm - rod junction provides a flexible hinge that would allow the motor to maneuver cargo through the complex intracellular actin network .",pubmed
"the aim of this study was to determine whether clinical outcome after surgical resection of esophageal adenocarcinoma ( eac ) or esophageal squamous cell carcinoma ( escc ) could be predicted by functional polymorphisms in different proto - oncogenes and tumor suppressor genes . 
 six single nucleotide polymorphisms ( snps ) in the aurka ( rs2273535 ) , erbb2 ( rs1136201 ) , mdm2 ( rs2279744 ) , cdh1 ( rs5030625 ) , cdkn2a ( rs11515 ) , and tp73 ( rs2273953 ) genes were genotyped in a consecutive cohort of 346 esophageal cancer patients , who had underwent surgical resection with curative intent . 
 associations with disease - free survival ( dfs ) were analyzed with kaplan  meier curves and cox regression , adjusting for potential confounders . 
 univariate analysis showed no significant associations between the tested polymorphisms and dfs in patients with eac or escc . 
 however , in a multivariate analysis , patients with eac carrying the heterozygous mdm2 ( rs2279744 ) t / g genotype had significantly improved dfs compared with patients carrying the wild - type genotype ( adjusted hazard ratio ( ahr ) , 0.63 ; 95% confidence interval ( ci ) [ 0.450.88 ] ) . 
 patients with eac harboring the homozygous cdh1 ( rs5030625 ) ga / ga genotype had a significantly reduced survival as compared with patients carrying the wild - type genotype ahr 4.0 , 95% ci [ 1.411 ] . 
 in a large cohort of esophageal cancer patients , the mdm2 t / g and cdh1 ga / ga genotype confer risk of death in patients with eac . 
 these data suggest that inter - individual differences in germ - line dna have an impact on dfs in patients with eac . 
 many therapeutic options are used to treat esophageal cancer , but traditionally , surgery is used most frequently to obtain loco - regional control and long - term survival.1,2 comprehensive preoperative staging has improved selection of patients for potentially curative surgery ; however , many patients present with recurrent disease within 2  years after operation . the majority of these patients develop not only loco - regional recurrences , but also distant metastases ( such as liver , lung , pleural , and/or peritoneal disease recurrences ) are common.35 despite attempts to improve the outcome of patients with esophageal cancer , prognosis remains poor with a 5-year overall survival of 2030%.5,6 well - known prognostic factors for esophageal cancer are summarized in tumor node metastasis ( tnm ) staging.7,8 although tnm parameters have the advantage of simplicity , they do not seem to completely reflect the biologic diversity of esophageal cancer.9,10 the true drivers of this clinical biologic diversity include the molecular aberrations of the cancer and the genetic make - up of the patient . in this respect , 
 the study of host genetic variability offers worthwhile potential to identify individuals that may have the best chance of survival . 
 single nucleotide polymorphisms ( snps ) in the germ - line are the most common type of host genetic variations . 
 gene - related functional snps can potentially lead to differences in protein expression and/or function . in this way , snps in proto - oncogenes and tumor suppressor genes can potentially alter the risk for metastatic or aggressive tumor , resulting in differences in clinical outcome . 
 altered expression of the aurka , erbb2 , mdm2 , cdh1 , cdkn2a , and tp73 proteins has been correlated to disease progression and clinical outcome in patients with esophageal cancer.1117 in addition , polymorphisms with effects on protein function have been identified in these proto - oncogenes and tumor suppressor genes.1823 based on these results , we postulated that functional snps in the aurka ( aurka_nm_003600.2 ; rs2273535 c.449 t > a ) , erbb2 ( erbb2_nm004448.2 ; rs1136201 c.655 a > g ) , mdm2 ( mdm2_nm002392.2 ; rs2279744 309 t > g ) , cdh1 ( cdh1_nm004360.3 ; rs5030625 -347 g > ga ) , cdkn2a ( cdkn2a_nm000077.3 ; rs11515 c.712 c > g ) , and tp73 ( tp73_nm005427.1 ; rs2273953 81 g  >  a ) genes may serve as molecular markers for clinical outcome in patients with esophageal adenocarcinoma ( eac ) or esophageal squamous cell carcinoma ( escc ) who underwent surgical resection . 
 patients between 1996 and 2001 , a total of 632 consecutive patients with esophageal cancer were evaluated for surgery with curative intent at the erasmus university medical center ( fig .  1 ) . 
 outcome for all patients with esophageal cancer referred to our hospital are collected prospectively and stored in a database by a data manager . 
 all patients were staged using esophago - gastroscopy with biopsies , ultrasonography of the cervical and upper abdominal region , and computed tomography of the thorax and abdomen . 
 endoscopic ultrasonography for evaluation of t - stage and nodal status was routinely performed . 
 1flowchart of patients with esophageal cancer referred to the erasmus mc for treatment between january 1996 and december 2001 . 
 patients excluded from the present study are shown flowchart of patients with esophageal cancer referred to the erasmus mc for treatment between january 1996 and december 2001 . 
 patients excluded from the present study are shown surgery for carcinomas of the upper half of the intra - thoracic esophagus , a right - sided thoracotomy was performed . for carcinomas of the lower half of the intra - thoracic esophagus , 
 the tumor and its adjacent lymph nodes were dissected en bloc ; however , no extended lymph node dissection was performed . 
 the continuity of the digestive tract was restored by means of a gastric tube reconstruction or colonic interposition with a cervical anastomosis . 
 resections were considered radical ( r0 ) if microscopic examination revealed no tumor tissue at or less than 1  mm from the circumferential , proximal , or distal margins . 
 pathological staging was done according to the union for international cancer control ( uicc ) sixth edition . 
 the tumor stage after resection was classified according to the tnm classification of the international union against cancer . 
 snp genotyping to determine the individual genotype for each snp , genomic dna was extracted from frozen or formalin - fixed and paraffin - embedded ( ffpe ) tissues . 
 normal tissue was obtained from the resection specimens ( i.e. , tumor - negative lymph nodes or tumor - negative resection margins ) . 
 all the archival tissue samples were used according to the code for adequate secondary use of tissue , code of conduct :  proper secondary use of human tissue  established by the dutch federation of medical scientific societies ( http://www.federa.org).polymerase chain reactions ( pcr ) were carried out in a volume of 15  l containing genomic dna , 8.3  l h2o , 5  l  mgfree buffer , 25  mm mgcl2 , 0.3  l of 10  mm deoxynucleotide triphosphates , 20  pmol of each primer , and 1  u taq polymerase ( promega , madison wi , usa ) . 
 pcr conditions were standardized at 35 cycles of 95c for 45  s , 61c for 45  s , 72c for 30  s , with a 10-min extension at 72c for 10  min following the last cycle . 
 for the polymorphism in cdh1 ( rs5030625 ) , amplified pcr products were visualized on a denaturing polyacrylamide gel . for detection of the restriction length polymorphisms in erbb2 ( rs1136201 ) and aurka ( rs2273535 ) , 
 pcr products were digested for 16  h at the appropriate temperature with10  u of restriction endonuclease bsmai , mspi , or apoi ( promega , madison , wi , usa ) , respectively . 
 the polymorphisms in cdkn2a ( rs11515 ) , mdm2 ( rs2279744 ) , and tp73 ( rs2273953 ) were genotyped by bi - directional sequencing . 
 table  1description of the polymorphisms located in different oncogenes and tumor suppressor genesgenerefsnp idchangeminor alleleminor allele frequencypotential effect on protein functionforward primerreverse primeraurkars2273535c.449 t > a ( phe > ile)a - allele0.25preferentially amplified ; faster growth of cultured cells175-tccattctaggctacagctc-35-aagaatttgaaggacacaagac-3erbb2rs1136201c.655 a > g ( ile > val)g - allele0.16increased dimerization , autophosphorylation of her-2 and tyrosine kinase activity185-agccctctgacgtccatc-35-ctgcagcagtctccgcatc-3mdm2rs2279744c.309 t > 
 gg - allelendassociated with higher levels of mdm2 expression195-gcggaggttttgttggactg-35-ctgagtcaacctgcccact-3cdh1rs5030625c.-347 g > gaga - allele0.14associated with decreased transcriptional activity205-ggccagaggaccgcttgag-35-gtttgttcgttttggaga-3cdkn2ars11515c.712 c > gg - allele0.13potential detrimental effect on rna stability215-ccccgattgaaagaaccagaga-35-aggaccttcggtgactgatgat-3tp73rs2273953c.-30 g 
 > aa - allele0.19influences the p73 translation225-gagcacgagttcccagggtg-35-ccaagcgcactcacagagag-3nd not determinedrefsnp i d ( http://www.ncbi.nlm.nih.gov/snp)minor allele frequency according 102 controls of the snp500cancer cohort ( http://snp500cancer.nci.nih.gov/snp.cfm ) description of the polymorphisms located in different oncogenes and tumor suppressor genes refsnp i d ( http://www.ncbi.nlm.nih.gov/snp ) minor allele frequency according 102 controls of the snp500cancer cohort ( http://snp500cancer.nci.nih.gov/snp.cfm ) statistical analysis data on follow - up were collected from the prospective database and the medical charts . 
 all patients were followed at an interval of 3 to 4  months during the first year , every 6  months for the second year , and then at the end of each year until 5  years after treatment . 
 recurrence or disease progression was diagnosed on clinical grounds . whenever a relapse was suspected , radiologic , endoscopic , or histologic confirmation was sought . 
 recurrent disease was classified as local  regional ( occurring in the upper abdomen or mediastinum ) or distant ( including cervical recurrences).study end - point was disease - free survival ( dfs ) that was defined as the time from surgery until recurrent disease or death from any cause . 
 the kaplan  meier survival function and log - rank tests were used to assess clinical outcome in relation to patient s characteristics and individual polymorphisms . 
 cox proportional hazard ratios for patients with eac were adjusted for weight loss prior to operation , tumor length , presence of barrett s epithelium , radicality of resection , and pathological tumor stage . for patients with escc 
 , cox proportional hazard ratios were adjusted for location of tumor , resection type , post - operative tnm stage , and radicality of resection . 
 we did not adjust for multiple testing since each gene outcome was prespecified and of interest in itself . 
 patients a total of 346 esophageal cancer patients underwent surgical resection with curative intent . of these , 25 patients were excluded from the current follow - up study because no tissue samples were available ( n  =  9 ) , genotyping failure ( n  =  3 ) , or incomplete follow - up ( n  =  13 ) ( fig .  1 ) . 
 of the 214 eac and 97 escc patients remaining for analysis , the majority were male , 85% in eac and 60% in escc . 
 of all patients with eac , 84% underwent primary surgery and 16% received preoperative chemotherapy with or without radiotherapy . 
 in contrast , 71% of patients with escc received preoperative chemotherapy with or without radiotherapy and 29% underwent primary surgical resection ( table  2 ) . 
 table  2survival according to patients and tumor characteristicsvariablepatients with eac ( n  =  214)patients with escc ( n  =  97)no . 
 ( % ) median dfsp valuesage in years0.230.33<65  years111 ( 52)1964 ( 66)2165  years103 ( 48)1233 ( 34)12gender0.410.44male182 ( 85)1658 ( 60)15female32 ( 15)1139 ( 40)20weight loss before operation0.0130.47no loss or < 5%127 ( 59)1946 ( 47)15510%35 ( 16)1131 ( 32)27>10%32 ( 15)817 ( 18)10not recorded20 ( 10)103 ( 3)5smoking status0.980.70current smoker54 ( 25)1443 ( 44)16no current smoker146 ( 68)1547 ( 49)20not recorded14 ( 7)87 ( 7)4location of tumor0.1500.008upper one third thoracic esophagus3 ( 3)4middle one third thoracic esophagus3 ( 1)1139 ( 40)12lower one third thoracic esophagus68 ( 32)2445 ( 47)20gej86 ( 40)1310 ( 10)60gastric cardia57 ( 27)12tumor length ( cm)0.0280.270234 ( 16)418 ( 8)373452 ( 24)1524 ( 25)104567 ( 31)1431 ( 32)24646 ( 22)925 ( 26)9not recorded15 ( 7)169 ( 9)11barrett s epithelium0.086no127 ( 59)12yes87 ( 41)24treatment0.830.18surgery alone180 ( 84)1428 ( 29)8chemotherapy  +  surgery23 ( 11)1565 ( 67)15chemoradiotherapy  +  surgery11 ( 5)214 ( 4)not reachedresection type0.830.042transhiatal187 ( 87)1553 ( 55)37transthoracic27 ( 13)944 ( 45)11post - operative uicc stage<0.001<0.001complete response8 ( 4)4013 ( 13)not reachedi24 ( 11)9813 ( 13)86iia43 ( 20)3734 ( 35)12iib8 ( 4)154 ( 4)26iii74 ( 34)1117 ( 18)8iv57 ( 27)716 ( 17)4radicality of resection<0.001<0.001r0141 ( 66)3465 ( 67)41r170 ( 33)729 ( 30)6  r23 ( 1)93 ( 3)5 survival according to patients and tumor characteristics snp genotyping genotyping was complete in 95% to 100% of eac and escc patients . the genotype distributions did not deviate from hwe ( p  >  0.05 ) . the genotype distribution of each snp is listed in table  3 . 
 tumor stage distributions were similar across all snp genotypes , and there was no association between genotypes and age at diagnosis , sex , weight loss , smoking status , or preoperative treatment . 
 table  3polymorphisms and clinical outcome in patients with resected eacgenotypedisease - free survival in eac patientsdisease - free survival in escc patientsnmps ( months)log - rank pahr[95% ci]nmps ( months)log - rank pahr[95% ci]aurka_rs2273535t / t129150.83reference62120.72referencea / t75141.1[0.761.4]29200.60[0.172.1]a / a9210.92[0.422.0]5550.63[0.182.1]erbb2_rs1136201a / a113140.25reference66120.66referencea / g86150.92[0.671.3]23260.73[0.411.3]g / g14120.68[0.331.4]681.3[0.493.2]mdm2_rs2279744t / t100110.076reference40100.63referencet / g84190.63[0.450.88]45210.98[0.591.6]g / g24120.95[0.581.6]7160.81[0.282.4]cdh1_rs5030625g / g166170.1468110.13referenceg / ga41111.2[0.781.7]18270.63[0.321.3]ga / ga474.0[1.411]1not reachedcdkn2a_rs11515c / c162130.79reference74120.67referencec / g47190.94[0.651.3]20200.67[0.361.3]g / g4191.7[0.525.6]1not reachedtp73_rs2273953g / g138160.44reference62240.48referenceg / a69130.98[0.711.4]32101.1[0.661.8]a / a5111.1[0.413.1]241.7[0.407.3]adjusted hazard ratio for weight loss prior to operation , tumor length , presence of barrett s epithelium , post - operative tnm stage , and radicality of resectionadjusted hazard ratio for location of the tumor , type of resection , post - operative tnm stage , and radicality of resection polymorphisms and clinical outcome in patients with resected eac adjusted hazard ratio for weight loss prior to operation , tumor length , presence of barrett s epithelium , post - operative tnm stage , and radicality of resection adjusted hazard ratio for location of the tumor , type of resection , post - operative tnm stage , and radicality of resection dfs and pattern of disease recurrence the median dfs of eac patients was 14  months ( range , 0.07138  months ) and for escc patients 16  months ( range , 0.5148  months ) . at the time of analysis , 37 ( 17% ) eac and 27 ( 28% ) 
 escc patients were alive with a median dfs time of 93  months ( range , 62138  months ) and 104  months ( range 79148  months ) , respectively ( fig  2a ) . 
 2a kaplan  meier analysis of dfs in patients with esophageal cancer , by histological subgroup . 
 b kaplan  meier analysis of dfs in patients with eac , by mdm2 ( rs2279744 ) . 
 c kaplan  meier analysis of dfs in patients with eac , by cdh1 genotype ( rs5030625)the pattern of disease recurrence is depicted in table  3 . 
 loco - regional recurrences were mediastinal or abdominal lymph node metastases and recurrences in the gastric tube . 
 distant metastases were found in liver , lung , brain , bone , adrenal gland , pleura , peritoneum , and skin.recurrent disease after surgery was found in 138 ( 78% ) eac patients ; 40 patients had loco - regional recurrence , 51 had distant metastasis , and 46 had both loco - regional recurrence and distant metastasis . 
 diseases recurrences were found in 51 escc patients ; 28 patients had loco - regional recurrence , 10 had distant metastasis , and 10 had both loco - regional recurrence and distant metastasis . 
 three patients had disease recurrence , but site of failure was not recorded . a kaplan  meier analysis of dfs in patients with esophageal cancer , by histological subgroup . 
 b kaplan  meier analysis of dfs in patients with eac , by mdm2 ( rs2279744 ) . 
 c kaplan  meier analysis of dfs in patients with eac , by cdh1 genotype ( rs5030625 ) snp genotype and dfs univariate analysis showed no significant associations between dfs in patients with eac or escc and the genotype distributions of the aurka , erbb2 , mdm2 , cdh1 , cdkn2a , and the tp73 gene polymorphisms ( table  4 ; fig  2b and c ) . 
 however , in a multivariate analysis , patients with eac carrying the heterozygous mdm2 ( rs2279744 ) t / g genotype had significantly improved dfs compared with patients carrying the wild - type t / t genotype ( adjusted hazard ratio ( ahr ) 0.63 , 95% confidence interval ( ci ) [ 0.450.88 ] , p  =  0.007 ) . 
 the post - operative tnm stage of the tumor and the radicality of resection were also found important factors for dfs ( hr 1.4 , 95% ci [ 1.21.6 ] , p  <  0.0001 and hr 2.3 , 95%ci [ 1.73.1 ] , p  <  0.0001 respectively ) . 
 table  4pattern of failureeac ( n  =  214)escc ( n  =  97)alive37 ( 17)27 ( 28)nature of first failure  local recurrence40 ( 29)28 ( 55 )  distant metastases51 ( 37)10 ( 20 )  local recurrence and distant metastases46 ( 33)10 ( 20 )  disease recurrence but site of failure not reported1 
 ( 1)3 ( 5)total deaths177 ( 83)70 ( 72)cause of death  cancer - related138 ( 78)51 ( 71 )  surgery - related14 ( 8)7 ( 11 )  2nd primary5 ( 3)6 ( 8)  death from other cause ( not cancer - related)20 ( 11)6 ( 8)also , patients with eac harboring the homozygous cdh1 ( rs5030625 ) ga / ga genotype had a significantly reduced survival as compared with patients carrying the wild - type g / g genotype ahr 4.0 , 95% ci [ 1.411 ] , p  =  0.008 . in multivariate analysis , the post - operative tnm stage of the tumor and the radicality of resection were found as important factors for dfs ( hr 1.4 , 95% ci [ 1.21.5 ] , p  <  0.0001 and hr 2.4 , 95%ci [ 1.73.2 ] , p  <  0.0001 respectively ) . 
 in the present study , we determined the relationship between inter - individual dna variations in six bona fide proto - oncogenes and tumor suppressor genes and dfs in a large cohort of caucasian patients with esophageal cancer . 
 after adjustment for potential confounders , the variant genotypes of snps located in the promoter region of the mdm2 and cdh1 gene were significantly associated with dfs in patients with eac . 
 the results of the present study showed a significant survival benefit for patients harboring the mdm2 t / g as compared with patients carrying the wild - type t / t genotype . 
 the mdm2 protein is a nuclear phosphoprotein that binds and inhibits the tumor suppressor tp53 as part of an autoregulatory negative feedback loop . 
 the most intensively characterized mdm2 polymorphism is the t309 g promoter snp located in the first intron.20 the g variant of this snp is known to increase promoter - binding affinity , leading to up - regulation of mdm2 and consequent inhibition and down - regulation of the p53 pathway . 
 therefore , it could be expected that the variant mdm2 genotypes ( t / g and g / g ) are associated with adverse outcome in esophageal cancer patients ( as shown in other cancer types).24 however , the present study showed improved survival in patients with the mdm2 t / g genotype compared with the wild - type t / t genotype . a possible explanation for our findings is provided by a large study in breast cancer patients that reported strong interaction between the mdm2 snp status and tumor tp53 status , which appeared to modify the association between tp53 status and breast cancer survival.25 among breast cancer patients with the wild - type mdm2 genotype ( t / t ) , a mutant tp53 status and aberrant tp53 expression in breast tumors were associated with poor survival . 
 the tumor tp53 status was not associated with breast cancer survival among carriers of the variant mdm2 allele ( t / g or g / g ) . 
 since tp53 is the most frequently mutated gene in eac , it could be hypothesized that the tumors of most patients with the t / t genotype harbor a tp53 mutation , which could lead to a reduced survival as observed in the present study . in a previously well - conducted study , the known tp53 codon 72 arg / pro and mdm2 polymorphisms were genotyped in 300 patients with eac and 63 patients with escc.26 as in concordance with the results of the present study , patients with eac harboring the mdm2 t / g genotype had a borderline improved overall survival as compared with patients carrying the wild - type genotype ( ahr for death 0.70 , 95% ci [ 0.500.99 ] , p  =  0.04 ) . 
 but unlike the present study , the mdm2 variant genotype did correlate with marked reduced survival in patients with escc . 
 this could be due to differences in study samples size , population selection , tissue handling , and genotyping methods . in this study , patients carrying the cdh1 ga / ga genotype had a significantly reduced survival as compared with patients with the wild - type g / g genotype . 
 however , it should be noted that only four eac patients carried the ga / ga genotype , which may represents a chance finding . nevertheless , this -347 g / ga insertion polymorphism located in the promoter of the cell 
 cell adhesion gene cdh1 has been reported to suppress cdh1 gene expression and was found to be associated with familial gastric cancer and sporadic colorectal cancer.27 the ga - allele has been associated with significant suppression of cdh1 promoter activity in colorectal and gastric cancer cell lines.27 it can be hypothesized that the ga - allele might enhance the progression of esophageal cancer by reducing cdh1 transcription resulting in a decrease in cdh1 protein expression and impairment of cell  cell adhesion . 
 all four patients harboring the ga / ga genotype died of recurrent disease ; three had loco - regional and distant metastasis , and one had only loco - regional disease recurrence . 
 to our knowledge , this is one of the first studies that investigated the relationship between polymorphisms and esophageal cancer outcome . here , we studied ( only ) six polymorphisms , whereas snp arrays can determine more than a million of snps in one sample . 
 although this technique is widely used on blood or fresh frozen samples , it is not very suitable for ffpe tissue samples ( our series consisted primarily of ffpe samples ) . therefore , collection of blood samples or fresh frozen tissue samples of esophageal cancer patients is necessary and should become standard procedure in order to perform genome - wide association studies . in this study , the majority of polymorphisms were not associated with dfs after esophagectomy 
 . it could be well that our study , among a relatively large population ( n  =  346 ) of esophageal cancer patients , failed to observe a difference due to under powering . since esophageal cancer has a relatively low incidence , consortia ( of multiple hospitals ) 
 recurrent cancer is the leading cause of death in patients undergoing surgical resection.35 although treatment options for esophageal cancer recurrences are limited , it could be proposed that early detection of recurrent disease is desirable because aggressive treatment may result in prolonged tumor - free survival or occasional cure . in this light , our findings could contribute to the identification of patients who are at high or low risk for the development of disease recurrences . 
 it can also be proposed that patients with a certain genetic constitution that is associated with a high chance of ( distant ) disease recurrence should be given systemic adjuvant treatment after surgical resection . 
 furthermore , identification of polymorphisms associated with dfs could serve well as hypothesis generating for prospective studies that evaluate the prognostic significance of germ - line variants . in summary , 
 our results indicate that two of six investigated functional polymorphisms are associated with dfs in patients who underwent esophagectomy for eac . 
 patients with eac carrying the heterozygous mdm2 t / g genotype had twofold reduced risk of disease recurrence , and patients with the homozygous cdh1 ga / ga had a fourfold increased risk of disease recurrence . 
 additional prospective studies are necessary to validate both associations and to study the prognostic significance of both germ - line variants .","purposethe aim of this study was to determine whether clinical outcome after surgical resection of esophageal adenocarcinoma ( eac ) or esophageal squamous cell carcinoma ( escc ) could be predicted by functional polymorphisms in different proto - oncogenes and tumor suppressor genes.experimental designsix single nucleotide polymorphisms ( snps ) in the aurka ( rs2273535 ) , erbb2 ( rs1136201 ) , mdm2 ( rs2279744 ) , cdh1 ( rs5030625 ) , cdkn2a ( rs11515 ) , and tp73 ( rs2273953 ) genes were genotyped in a consecutive cohort of 346 esophageal cancer patients , who had underwent surgical resection with curative intent . 
 associations with disease - free survival ( dfs ) were analyzed with kaplan  meier curves and cox regression , adjusting for potential confounders.resultsunivariate analysis showed no significant associations between the tested polymorphisms and dfs in patients with eac or escc . 
 however , in a multivariate analysis , patients with eac carrying the heterozygous mdm2 ( rs2279744 ) t / g genotype had significantly improved dfs compared with patients carrying the wild - type genotype ( adjusted hazard ratio ( ahr ) , 0.63 ; 95% confidence interval ( ci ) [ 0.450.88 ] ) . 
 patients with eac harboring the homozygous cdh1 ( rs5030625 ) ga / ga genotype had a significantly reduced survival as compared with patients carrying the wild - type genotype ahr 4.0 , 95% ci [ 1.411].conclusionsin a large cohort of esophageal cancer patients , the mdm2 t / g and cdh1 ga / ga genotype confer risk of death in patients with eac . 
 these data suggest that inter - individual differences in germ - line dna have an impact on dfs in patients with eac .",pubmed
"oral cavity is colonized by candida albicans or other candida species in 40 - 60% of healthy persons . in immune - compromised patients , candida species can trigger a variety of disease manifestations ranging from localized mild oral lesion to a disseminated candidiasis.1 many factors contribute to the development of oropharyngeal candidiasis ( opc ) including malnutrition , poor oral hygiene , dental malocclusion , and immunosuppression.2 diagnosis and treatment of oral lesions caused by candida species are of utmost importance in human immunodeficiency virus ( hiv)-positive patients who , despite the initiation of triple antiretroviral therapy ( art ) , continue to suffer from significant candida associated morbidity.3 - 5 according to the joint united nations program on hiv / acquired immune deficiency syndrome ( aids ) as of 2013 , approximately 35.3 millions people have hiv worldwide with the number of new infections that year being about 2.1 millions.6 opc has been described as the most frequent opportunistic fungal infection among hiv - positive patients , and it has been estimated that more than 90% of hiv - positive patients develop this infection at some time during the progression of their disease.7 - 9 opc is an opportunistic infection of soft buccal mucosa . 
 opc can appear as erythematous patches or white , scrapable lesions and is often one of the first clinical signs of hiv infection.9,10 opc is observed with a higher prevalence in patients with cd4 + counts below 200/mm or a high viral load ( > 10,000 copies / ml).4,5,7 - 9 opc caused by c. albicans is generally managed by judicious use of fluconazole.2,4,7 - 9 a rise in resistant organisms may be due to prolonged or frequent treatment with azoles.10 an epidemiologic shift of candida species could significantly impact the utility of fluconazole as empiric treatment for candidiasis in patients with hiv / aids.11 the present study was directed to evaluate the forms of opc and their correlation with cd4 + cell counts in hiv patients . 
 this was a descriptive and analytical cross - sectional study carried out for a 2-year period , in which quantitative data collection methods were used . 
 patient records , available at the odontology unit of saint - antoine hospital , were initially studied and then the patients were asked to visit the clinic for a further evaluation . a complete medical history was taken and a physical examination of the oral cavity , head , and neck area was performed on each patient . 
 the variables studied , including medical history , physical examination , socio - demographic characteristics , socio - behavioral factors , experience with oral lesions , and laboratory tests , were reviewed . 
 based on the findings of a physical examination and laboratory tests , patients were prescribed essential medication and repeated examination , and follow - up visits were considered . 
 one dental surgeon , who was blinded to the clinical staging , carried out all oral examinations . 
 patients were examined while seated in the dental chair and a well - illuminated room . 
 extra oral and perioral areas were examined first , followed by intraoral tissues for any abnormalities . 
 diagnosis of oral lesions was implemented using european community clearinghouse guidelines for presumptive diagnosis of opc.12 blood samples were obtained on the same day as the oral examinations , and their results were recorded onto each participant s questionnaire . 
 a single oral swab was collected from each study participant by passing a sterile swab firmly across buccal mucosa , floor of mouth , dorsal tongue in cases of asymptomatic patients , and from the base of the oral lesion in cases of symptomatic patients . 
 candida was identified by conventional tests and species identification was performed using the germ tube test , growth on chrom agar candida medium ( drg international inc . 
 dehydrated media , springfield , u.s.a ) , and sugar assimilation tests.12,13 all patients who had oral lesions and from whom candida species were isolated were considered to have opc . 
 recent cd4 + levels were analyzed by the flow cytometer method , and a history of intake of art was noted . 
 based on the who classification14 the cd4 + cell counts 500 cells / mm was classified as  type 1 , 
 cd4 + cell count of > 201 to < 499 cells / mm as  type 2  and cd4 + cell count of 200 cells / mm as  type 3 . 
 patient data , microbiological results , and cd4 + counts were collected and protected with a password . 
 data included age , gender , socio - behavioral factors , opc , antifungal use , art , cd4 + count , were entered in excel data sheet ( microsoft corporation seattle , usa ) . 
 all statistical calculations were performed using spss software version 20 ( ibm spss , chicago , usa ) . 
 one dental surgeon , who was blinded to the clinical staging , carried out all oral examinations . 
 patients were examined while seated in the dental chair and a well - illuminated room . 
 extra oral and perioral areas were examined first , followed by intraoral tissues for any abnormalities . 
 diagnosis of oral lesions was implemented using european community clearinghouse guidelines for presumptive diagnosis of opc.12 blood samples were obtained on the same day as the oral examinations , and their results were recorded onto each participant s questionnaire . 
 a single oral swab was collected from each study participant by passing a sterile swab firmly across buccal mucosa , floor of mouth , dorsal tongue in cases of asymptomatic patients , and from the base of the oral lesion in cases of symptomatic patients . 
 candida was identified by conventional tests and species identification was performed using the germ tube test , growth on chrom agar candida medium ( drg international inc . 
 dehydrated media , springfield , u.s.a ) , and sugar assimilation tests.12,13 all patients who had oral lesions and from whom candida species were isolated were considered to have opc . 
 recent cd4 + levels were analyzed by the flow cytometer method , and a history of intake of art was noted . 
 based on the who classification14 the cd4 + cell counts 500 cells / mm was classified as 
 cd4 + cell count of > 201 to < 499 cells / mm as  type 2  and cd4 + cell count of 200 cells / mm as  type 3 . 
 patient data , microbiological results , and cd4 + counts were collected and protected with a password . 
 data included age , gender , socio - behavioral factors , opc , antifungal use , art , cd4 + count , were entered in excel data sheet ( microsoft corporation seattle , usa ) . 
 all statistical calculations were performed using spss software version 20 ( ibm spss , chicago , usa ) . 
 of the 50 consenting participants , 5 ( 10% ) were females and 45 ( 90% ) males . 
 the median age in this study group was 39 years ( 28 - 57 ) . of the participants , 
 38 ( 76% ) had a history of sexual contamination , 11 ( 22% ) had a history of sharing intravenous needles , and one ( 2% ) had a history of homosexuality and sharing intravenous needles . 
 most participants had cd4 + count ( cells / mm ) was < 200 , 201 - 499 , and > 500 in 32 cases ( 64% ) , 16 cases ( 32% ) and 2 cases ( 4% ) , respectively , and the mean cd4 + count ( cells / mm ) was 167.12 . 
 median duration of art was 2 years ( range , 1.5 months to 6 years ) . 
 overall , candida species was isolated from the oral cavity in 38/50 ( 76% ) . 
 hiv - associated oral lesions were observed in all patients ( periodontal disease , herpetic lesions , hairy leukoplakia , gingivitis , oral ulceration , kaposi s sarcoma , and non - hodgkin lymphoma ) . 
 pseudomembranous candidiasis 20/38 ( p ) was the most common clinical presentation of opc ( 52.6% ) followed by erythematous 5/38 ( 13.15% ) , angular cheilitis 5/38 ( 13.15% ) ( ac ) and a combination of ac and e 4/38 ( 10.52% ) or ac , e and p 4/38 ( 10.52% ) ( figure 1 ) . 
 clinical forms of oropharyngeal candidiasis : ( a ) pseudomembranous , ( b ) erythematous ( c ) angular cheilitis . c. albicans was the most frequent species isolated in 35 cases of opc ( 92% ) . 
 candida tropicalis was isolated in 2 cases ( 5.26% ) and candida glabrata in 1 case ( 2.64% ) . 
 the distribution of oral lesions based on the cd4 + count of hiv - infected patients showed that 62% of the oral lesions occurred at cd4 + count < 200 cells / mm ( mean cd4 + ) , about 26% oral lesions were seen at cd4 + count of 201 - 499 cells / mm ( mean cd4 + ) whereas 12% cases of oral lesions were seen at cd4 + count > 500 cells / mm ( mean cd4 ) . 
 the majority of patients with opc had cell counts 28/38 ( 73% ) < 200 cells / mm , followed by 9/38 ( 23% ) at cd4 + cell counts of 201 - 499 cells / mm ( table 1 ) . profile of hiv patients with opc form distribution , cd4 + count and candida species 
 opc is the most common fungal infection in hiv patients and fluconazole efficiency in treating this infection has been proven . 
 however , certain non - albican species ( c. glabrata , candida krusei ) proved to be less susceptible to flunconazole than c. albicans . since they were isolated in hiv patients.7,13 impact of art on opportunistic infections in hiv patients is continuously evolving : patel and co - workers ( 2012 ) studied the changing aspect of opc epidemiology in 215 hiv / aids patients by assessing yeast colonization from oral rinse samples . c. albicans was the most prevalent and c. glabrata and candida dubliniensis were identified in 29% of cultures . 
 decrease susceptibility to flunonazole was studied and detected in 10% of isolates.9 in a prospective cross - sectional study of predisposing factors for oropharyngeal colonization of yeasts in hiv - infected patients , lin et al . 
 ( 2013 ) reviewed 105 patients , among whom , 54 ( 51.4% ) were colonized with yeasts and among the 68 isolates , c. albicans accounted for 73.5 % , c. tropicalis for 5.95% , c. glabrata for 5.9% , and c. dubliniensi for 4.4% , and there 7.5% candida isolates resistant to flunonazole and a higher prevalence of yeast colonization was observed in patients with cd4 + count < 200 cells / mm.15 thompson et al . 
 ( 2013 ) studied opc in 122 hiv - infected patients in whom infection was observed in one - third : c. albicans was the most implicated pathogen and oral yeast colonization in 81.1% , despite the availability of art , and resistant yeasts occurred in 25.3% of patients.4 in an observational cohort , arribas et al . 
 ( 2000 ) determined the relationship between art and changes in prevalence and amount of opc . 
 they found that the majority of 99 observed patient presented opc with a cd4 + count < 200 cells / mm.16 in our study , the mean age was 39 years , and male gender was 90% , and female was 10% . 
 about 76% of our patients were contaminated with hiv by sexual transmission and 22% by sharing intravenous needles and 2% by a combination of sexual and intravenous sharing needles . 
 similar figures were recorded by thompson et al.,4 castro et al.13 and lin et al.15 earlier studies2,5,15,16 showed that pseudomembranous candidiasis was the most common in clinical appearance of opc . 
 these findings shared our clinical observation . c. albicans was isolated in 92% of our cases followed by c. tropicalis and c. glabrata . 
 some of the recent report1,4,8 have also reported the same species while others12,13 described other species as c. dubliniensis beside the mentioned species . 
 these variations could be related to the differences in the socio - demographic characteristics and socio - behavioral factors or may be due to increased identification of the species , which can be mistaken phenotypically as c. albicans.17 distribution of opc lesions and the cd4 + cell counts , 73% associated with < 200 cells / mm is a common observation with other studies.4,5,18 the only given explication may be the decrease of the immunodeficiency of the immune system . 
 current guidelines note that for aids patients , 200 mg / day of fluconazole is acceptable and does not lead to resistance.19 
 oral candida colonization and invasive infection occur more frequently in hiv - positive patient and are significantly more common in patients with cd4 + cell counts < 200 cell / mm . 
 art reduces opc . c. albicans continues to be the most frequently isolated species in opc . 
 our results emphasize the need of continued awareness of opc as a major clinical sign in hiv population .","background : the present study was directed to evaluate the forms of oropharyngeal candidiasis ( opc ) and their correlation with cd4 + cell counts in human immunodeficiency virus ( hiv ) patients.materials and methods : this was a descriptive and analytical cross - sectional study carried out for a 2-year period , in which quantitative data collection methods were used . 
 50 patients with hiv infection were evaluated . 
 relationship between opc and cd4 + was investigated.results:five different clinical forms were noticed on examination : pseudomembranous candidiasis 20/38 ( p ) was the most common one ( 52.6% ) followed by erythematous 5/38 ( 13.15% ) , angular cheilitis 5/38 ( 13.15% ) ( ac ) , a combination of ac and e 4/38 ( 10.52% ) or ac , e and p 4/38 ( 10.52% ) . 
 candida albicans was the most frequent specie isolated in 35 cases of opc ( 92% ) . 
 candida tropicalis was isolated in 2 cases ( 5.26% ) and candida glabrata in 1 case ( 2.64% ) . 
 the majority of patients with opc had cell counts 28/38 ( 73% ) < 200 cells / mm3 , followed by 9/38 ( 23% ) at cd4 + cell counts of 201 - 499 cells / mm3.conclusion : oral candida colonization and invasive infection occur more frequently in hiv - positive patient and is significantly more common in patients with cd4 + cell counts < 200 cell / mm3 .",pubmed
"breast cancer is the most common cancer among women , with approximately 1.67 million new patients diagnosed annually worldwide and accounting for 25% of all cancer cases.1 breast cancer predominantly spreads via the lymphatic system to locoregional lymph nodes , meaning that the status of these lymph nodes is important for staging of the disease and determining prognosis.24 the sentinel lymph nodes ( slns ) are defined as the first draining nodes from a primary tumor and , therefore , are most likely to be the first site of lymphatic metastasis . 
 sentinel lymph node biopsy ( slnb ) with the  combined technique  of radioisotope and blue dye is the standard method for determining axillary staging in early - stage breast cancer patients with a clinically and radiologically negative axilla.3,59 the combined technique is performed by injecting a radioactive tracer ( tc nanocolloid ) and blue dye interstitially , either peritumorally or periareolarly . 
 the tracers are then detected within the slns by the surgeon using a handheld scintillation counter ( gamma probe ) and/or visually . 
 the gamma probe is first used to transcutaneously localize the slns and determine the optimal incision site . 
 postincision both the blue dye and gamma probe guide the surgeon to the slns . after identification 
 , the slns are removed and sent for histopathological examination . with an identification rate of 96% and a false - negative rate of 5%10% , 
 it is an effective procedure with low morbidity.3 although the currently used technique performs well , it has drawbacks . 
 the use of radioisotopes is subject to stringent regulations on training of staff and handling and disposal of radioactive material . 
 most importantly , many hospitals in the world do not have access to radioisotopes . consequently 
 , only approximately 60% of the patients in the western world and negligible numbers in the rest of the world have access to slnb.10 this has led to the search for other techniques , not reliant on the use of radioisotopes.11 recently the use of a magnetic tracer and a handheld magnetometer was evaluated against the  combined technique  as a radiation - free alternative in two separate clinical trials.12,13 a superparamagnetic iron oxide ( spio ) tracer was administered interstitially in the breast , followed by identification of the slns with a handheld magnetometer . 
 the magnetic technique was found to be noninferior to the  combined technique  with identification rates of 94.4%98.0%.12,13 however , there was a discordance in slns identified between the magnetic and combined techniques that ranged between 2.0%13 and 6.9%,12  potentially leading to false - negative staging with the magnetic technique . 
 a smaller study by shiozawa et al14 used a similar spio tracer and different magnetometer , comparing spio and blue dye only  no radioisotope was used . 
 they found an sln identification rate of 90% for the spio in combination with blue dye , with a discordance rate of 16.7% and demonstrated the viability of the technique for performing slnb in the absence of radioisotopes . 
 physical and chemical properties including shape , coating material , and particle size influence the distribution of nanoparticles in vivo.15,16 
 tc nanocolloid has a mean particle size of approximately 8 nm,17,18 while the particle size of the magnetic tracer used for the magnetic technique ( sienna+ , endomagnetics ltd , uk ) is much larger with a mean size of approximately 60 nm . 
 because particle size is the most important property influencing lymphatic uptake and lymph node retention,19 the identification of different slns by the two techniques could possibly arise due to differences in particle size of the radioisotope and magnetic tracer . in order to optimize the clinical application of the magnetic technique for slnb 
 , we evaluated the performance of three different - sized spio - based formulations  licensed for human use  within an in vivo porcine model . 
 three spio - based licensed formulations with a broad range in particle size were used as magnetic tracers in this study . 
 feraheme / rienso ( takeda , japan ) consists of ferumoxytol , 30 mg fe / ml and is an food and drug administration  approved drug used for the intravenous treatment of iron deficiency anemia in adult patients with chronic kidney disease . 
 the particles have a magnetic core of maghemite ( -fe2o3 ) surrounded by a polyglucose sorbitol carboxymethylether coating . 
 sienna+ contains 27 mg fe / ml and is a ce - marked magnetic tracer intended to mark and locate slns in cancer patients . 
 the magnetic core consists of magnetite and maghemite ( fe3o4/-fe2o3 ) , with a carboxydextran coating . finally , endorem / feridex ( guerbet , france ) consisting of ferumoxide , 11.2 mg fe / ml is licensed as a magnetic resonance imaging ( mri ) contrast agent for liver imaging , with a magnetite core coated with dextran . 
 all magnetic tracers were diluted to the same iron concentration of 11.2 mg fe / ml to facilitate comparison of performance . 
 the hydrodynamic particle size of all tracers was determined by dynamic light scattering using a zetasizer nano zs ( malvern instruments , uk ) . ethical permission for animal experimentation 
 was granted by the research institute against digestive cancer ( ircad ) ethics review board ( strasbourg , france ) under reference number 38.2012.01.047 . 
 a previously developed and validated porcine model that closely resembles human lymphatic drainage was used to evaluate the performance of the different magnetic tracers for slnb.20 at the ircad institute ( strasbourg , france ) , after induction of anesthesia , mini - pigs were injected with one of the three different magnetic tracers subcutaneously bilaterally into the areolas of the third inguinal mammary glands . 
 a total of 0.5 ml ( 5.6 mg fe ) of each diluted tracer was injected on each side , in six mini - pigs , for each tracer ( 18 mini - pigs in total ; 36 injections ) . 
 transcutaneous magnetometer measurements using a handheld magnetometer ( sentimag , endomagnetics ltd , uk ) were undertaken in the inguinal region , where the draining lymph nodes are located . 
 measurements were performed at 5 minutes , 10 minutes , 15 minutes , 30 minutes , 45 minutes , 60 minutes , and 240 minutes after administration of the tracer . 
 the sites at which uptake of spio was successfully detected ( hotspots ) were marked using a permanent marker . 
 after identification , lymph nodes demonstrating spio uptake were removed , and the ex vivo magnetometer count was recorded . 
 lymph nodes with a magnetometer count higher than 10% of the  hottest node  were defined as slns , as was used in the sentimag multicentre trial,12 and in accordance with standard clinical practice.21 after completion of the slnb procedure , a groin node clearance was performed . 
 ex vivo magnetometer measurements were performed on the groin node clearances to determine if there were any further slns that were missed initially . 
 all resected lymph nodes were fixed in formalin and sent to the university of twente ( enschede , the netherlands ) for iron quantification and subsequently to king s college london ( london , uk ) for histopathological evaluation . 
 the quantification of magnetic tracer in the excised and formalin - fixed sentinel lymph nodes was performed using vibrating sample magnetometry ( vsm ) on a physical properties measuring system ( quantum design inc . , san diego , ca , usa ) 
 . an applied magnetic field of 4.0 t was used to bring the magnetic iron oxide nanoparticles to saturation . 
 the amount of magnetic tracer in the lymph nodes was determined by comparing the obtained amplitude of the saturation magnetization to known calibration samples of each of the tracers . 
 the iron content was reported as the mass of iron ( fe ) in the node , present in the form of maghemite or magnetite . 
 vsm allows quantification with 0.5 g fe accuracy and is nondestructive , which allows subsequent histopathological analysis of the same samples.22 the nodes were then transferred intact to king s college london ( london , uk ) where they were embedded in wax , thinly sliced , and stained with hematoxylin and eosin and with perl s prussian blue for iron . 
 iron staining within each node was graded using a previously validated 5-point grading scale ( 0 = none , 1 = minimal , 2 = mild , 3 = moderate , and 4 = marked),20 by an experienced pathologist and a second observer ( sp , ba , or ma ) . 
 we conducted a two - sided test ( alpha = 0.05 ) expecting a difference of 50 g ( standard deviation [ sd ] = 30 ) in iron content readings between each magnetic tracer . 
 when performing a total of 12 procedures ( six mini - pigs ) for each magnetic tracer , these 36 procedures provided us with a power of 82% to detect this difference . 
 the relationship between continuous variables was calculated using pearson s correlation coefficient , associations between categorical data using the  test , and associations between categorical and continuous variables using analyses of variance . 
 all statistical analyses were performed using statistical analysis systems ( sas ) release 9.3 ( sas institute , cary , nc ) . 
 three spio - based licensed formulations with a broad range in particle size were used as magnetic tracers in this study . 
 feraheme / rienso ( takeda , japan ) consists of ferumoxytol , 30 mg fe / ml and is an food and drug administration  approved drug used for the intravenous treatment of iron deficiency anemia in adult patients with chronic kidney disease . 
 the particles have a magnetic core of maghemite ( -fe2o3 ) surrounded by a polyglucose sorbitol carboxymethylether coating . 
 sienna+ contains 27 mg fe / ml and is a ce - marked magnetic tracer intended to mark and locate slns in cancer patients . 
 the magnetic core consists of magnetite and maghemite ( fe3o4/-fe2o3 ) , with a carboxydextran coating . finally , endorem / feridex ( guerbet , france ) consisting of ferumoxide , 11.2 mg fe / ml is licensed as a magnetic resonance imaging ( mri ) contrast agent for liver imaging , with a magnetite core coated with dextran . 
 all magnetic tracers were diluted to the same iron concentration of 11.2 mg fe / ml to facilitate comparison of performance . 
 the hydrodynamic particle size of all tracers was determined by dynamic light scattering using a zetasizer nano zs ( malvern instruments , uk ) . 
 ethical permission for animal experimentation was granted by the research institute against digestive cancer ( ircad ) ethics review board ( strasbourg , france ) under reference number 38.2012.01.047 . 
 a previously developed and validated porcine model that closely resembles human lymphatic drainage was used to evaluate the performance of the different magnetic tracers for slnb.20 at the ircad institute ( strasbourg , france ) , after induction of anesthesia , mini - pigs were injected with one of the three different magnetic tracers subcutaneously bilaterally into the areolas of the third inguinal mammary glands . 
 a total of 0.5 ml ( 5.6 mg fe ) of each diluted tracer was injected on each side , in six mini - pigs , for each tracer ( 18 mini - pigs in total ; 36 injections ) . 
 transcutaneous magnetometer measurements using a handheld magnetometer ( sentimag , endomagnetics ltd , uk ) were undertaken in the inguinal region , where the draining lymph nodes are located . 
 measurements were performed at 5 minutes , 10 minutes , 15 minutes , 30 minutes , 45 minutes , 60 minutes , and 240 minutes after administration of the tracer . 
 the sites at which uptake of spio was successfully detected ( hotspots ) were marked using a permanent marker . 
 after identification , lymph nodes demonstrating spio uptake were removed , and the ex vivo magnetometer count was recorded . 
 lymph nodes with a magnetometer count higher than 10% of the  hottest node  were defined as slns , as was used in the sentimag multicentre trial,12 and in accordance with standard clinical practice.21 after completion of the slnb procedure , a groin node clearance was performed . 
 ex vivo magnetometer measurements were performed on the groin node clearances to determine if there were any further slns that were missed initially . 
 all resected lymph nodes were fixed in formalin and sent to the university of twente ( enschede , the netherlands ) for iron quantification and subsequently to king s college london ( london , uk ) for histopathological evaluation . 
 the quantification of magnetic tracer in the excised and formalin - fixed sentinel lymph nodes was performed using vibrating sample magnetometry ( vsm ) on a physical properties measuring system ( quantum design inc . , san diego , ca , usa ) 
 . an applied magnetic field of 4.0 t was used to bring the magnetic iron oxide nanoparticles to saturation . 
 the amount of magnetic tracer in the lymph nodes was determined by comparing the obtained amplitude of the saturation magnetization to known calibration samples of each of the tracers . 
 the iron content was reported as the mass of iron ( fe ) in the node , present in the form of maghemite or magnetite . 
 vsm allows quantification with 0.5 g fe accuracy and is nondestructive , which allows subsequent histopathological analysis of the same samples.22 
 the nodes were then transferred intact to king s college london ( london , uk ) where they were embedded in wax , thinly sliced , and stained with hematoxylin and eosin and with perl s prussian blue for iron . 
 iron staining within each node was graded using a previously validated 5-point grading scale ( 0 = none , 1 = minimal , 2 = mild , 3 = moderate , and 4 = marked),20 by an experienced pathologist and a second observer ( sp , ba , or ma ) . 
 we conducted a two - sided test ( alpha = 0.05 ) expecting a difference of 50 g ( standard deviation [ sd ] = 30 ) in iron content readings between each magnetic tracer . 
 when performing a total of 12 procedures ( six mini - pigs ) for each magnetic tracer , these 36 procedures provided us with a power of 82% to detect this difference . 
 the relationship between continuous variables was calculated using pearson s correlation coefficient , associations between categorical data using the  test , and associations between categorical and continuous variables using analyses of variance . 
 all statistical analyses were performed using statistical analysis systems ( sas ) release 9.3 ( sas institute , cary , nc ) . 
 the z - averaged hydrodynamic diameter of the magnetic tracers ferumoxytol , sienna+ , and ferumoxide was determined to be 32 nm , 59 nm , and 111 nm , respectively . 
 when the size distributions were evaluated graphically ( figure 1 ) , significant intratracer heterogeneity in particle size was observed . 
 this is reflected by a polydispersity index of 0.179 , 0.181 , and 0.266 for the 32 nm tracer , 59 nm tracer , and 111 nm tracer , respectively . 
 a total of 36 slnb procedures were performed in 18 mini - pigs , and sln identification was successful in all cases . in vivo magnetic hotspots from the draining inguinal lymph nodes 
 were successfully identified transcutaneously prior to surgical excision using the handheld magnetometer in all cases ( 12/12 ) for the 32 nm tracer and the 59 nm tracer and in all but one case ( 11/12 ) for the 111 nm tracer ( 240 minutes after injection ) . 
 the number of procedures in which a transcutaneous hotspot was successfully identified increased with time after injection for all three tracers ( figure 2 ) . 
 there were 7/12 and 8/12 successfully identified magnetic hotspots after 30 minutes using the 32 nm tracer and 111 nm tracer , respectively , compared to the 59 nm tracer with magnetic hotspots in all cases ( 12/12 ) . 
 a total of 77 slns were identified with the handheld magnetometer during surgery and subsequently excised . 
 thirty - five slns were identified using the 32 nm tracer ( mean 2.9 , sd 1.2 ) , 20 slns with the 111 nm tracer ( mean 1.7 , sd 0.9 ) , and 22 slns ( mean 1.8 , sd 0.8 ) with the 59 nm tracer ( figure 3a ) . a statistically significant difference between the tracer used , and the number of slns identified 
 more nodes per basin were excised with the 32 nm tracer compared to the 111 nm tracer ( p<0.001 ) and the 59 nm tracer ( p=0.03 ) . 
 the distribution of magnetometer counts demonstrated a statistically significant difference among the three tracers overall ( p<0.0001 ) ( figure 3b ) . 
 multicomparisons demonstrated that ex vivo magnetometer counts were significantly higher with the 59 nm tracer compared to the other two tracers ( p<0.05 ) . 
 the iron content of the ex vivo slns was recorded using vsm ( figure 3c ) . 
 multiple comparisons between the tracers demonstrated a significant higher iron content for the 59 nm tracer compared to the 32 nm tracer ( p<0.05 ) only . 
 the mean iron content of the slns was determined to be 106 ( sd 70 ) g , 179 ( sd 159 ) g , and 265 ( sd 206 ) g for the 32 nm tracer , 111 nm tracer , and 59 nm tracer , respectively . a linear relationship between the iron content of the ex vivo slns and the handheld magnetometer counts 
 was observed for all tracers ( figure 4 )  32 nm tracer ( r=0.93 ; p<0.001 ) , 111 nm tracer ( r=0.95 ; p<0.001 ) , and 59 nm tracer ( r=0.93 ; p<0.001 ) . 
 figure 4 demonstrates a difference in the sensitivity of the magnetometer for the different tracers . 
 the magnetometer count is 2.8 times higher for the 59 nm tracer compared to the same amount of iron for the 111 nm tracer and 1.3 times higher for the 32 nm tracer compared to the 111 nm tracer . 
 the iron was distributed predominantly in the subcapsular space peripherally within the cortex , subcapsular space , and sinuses ( figure 5 ) . the iron deposition from the 111 nm tracer 
 was mostly confined to macrophages within the nodes ( figure 5a ) , whereas the 59 nm tracer demonstrated more free iron granules ( particularly peripherally ) ( figure 5b ) . 
 only very sparse , small islands of iron within macrophages were visible for the 32 nm tracer ( figure 5c ) . 
 there was a significant difference in the grade of the iron content of excised slns between the three different tracers overall ( p<0.0001 ) ( figure 6 ) . 
 the amount of iron deposition was significantly greater with the 111 nm tracer and 59 nm tracer ( p<0.05 ) compared to the 32 nm tracer . 
 the z - averaged hydrodynamic diameter of the magnetic tracers ferumoxytol , sienna+ , and ferumoxide was determined to be 32 nm , 59 nm , and 111 nm , respectively . 
 when the size distributions were evaluated graphically ( figure 1 ) , significant intratracer heterogeneity in particle size was observed . 
 this is reflected by a polydispersity index of 0.179 , 0.181 , and 0.266 for the 32 nm tracer , 59 nm tracer , and 111 nm tracer , respectively . 
 a total of 36 slnb procedures were performed in 18 mini - pigs , and sln identification was successful in all cases . in vivo magnetic hotspots from the draining inguinal lymph nodes were successfully identified transcutaneously prior to surgical excision using the handheld magnetometer in all cases ( 12/12 ) for the 32 nm tracer and the 59 nm tracer and in all but one case ( 11/12 ) for the 111 nm tracer ( 240 minutes after injection ) . 
 the number of procedures in which a transcutaneous hotspot was successfully identified increased with time after injection for all three tracers ( figure 2 ) . 
 there were 7/12 and 8/12 successfully identified magnetic hotspots after 30 minutes using the 32 nm tracer and 111 nm tracer , respectively , compared to the 59 nm tracer with magnetic hotspots in all cases ( 12/12 ) . 
 a total of 77 slns were identified with the handheld magnetometer during surgery and subsequently excised . 
 thirty - five slns were identified using the 32 nm tracer ( mean 2.9 , sd 1.2 ) , 20 slns with the 111 nm tracer ( mean 1.7 , sd 0.9 ) , and 22 slns ( mean 1.8 , sd 0.8 ) with the 59 nm tracer ( figure 3a ) . a statistically significant difference between the tracer used , and the number of slns identified 
 more nodes per basin were excised with the 32 nm tracer compared to the 111 nm tracer ( p<0.001 ) and the 59 nm tracer ( p=0.03 ) . 
 the distribution of magnetometer counts demonstrated a statistically significant difference among the three tracers overall ( p<0.0001 ) ( figure 3b ) . 
 multicomparisons demonstrated that ex vivo magnetometer counts were significantly higher with the 59 nm tracer compared to the other two tracers ( p<0.05 ) . 
 the iron content of the ex vivo slns was recorded using vsm ( figure 3c ) . 
 multiple comparisons between the tracers demonstrated a significant higher iron content for the 59 nm tracer compared to the 32 nm tracer ( p<0.05 ) only . 
 the mean iron content of the slns was determined to be 106 ( sd 70 ) g , 179 ( sd 159 ) g , and 265 ( sd 206 ) g for the 32 nm tracer , 111 nm tracer , and 59 nm tracer , respectively . 
 a linear relationship between the iron content of the ex vivo slns and the handheld magnetometer counts was observed for all tracers ( figure 4 )  32 nm tracer ( r=0.93 ; p<0.001 ) , 111 nm tracer ( r=0.95 ; p<0.001 ) , and 59 nm tracer ( r=0.93 ; p<0.001 ) . 
 figure 4 demonstrates a difference in the sensitivity of the magnetometer for the different tracers . 
 the magnetometer count is 2.8 times higher for the 59 nm tracer compared to the same amount of iron for the 111 nm tracer and 1.3 times higher for the 32 nm tracer compared to the 111 nm tracer . 
 the iron was distributed predominantly in the subcapsular space peripherally within the cortex , subcapsular space , and sinuses ( figure 5 ) . the iron deposition from the 111 nm tracer 
 was mostly confined to macrophages within the nodes ( figure 5a ) , whereas the 59 nm tracer demonstrated more free iron granules ( particularly peripherally ) ( figure 5b ) . 
 only very sparse , small islands of iron within macrophages were visible for the 32 nm tracer ( figure 5c ) . 
 there was a significant difference in the grade of the iron content of excised slns between the three different tracers overall ( p<0.0001 ) ( figure 6 ) . 
 the amount of iron deposition was significantly greater with the 111 nm tracer and 59 nm tracer ( p<0.05 ) compared to the 32 nm tracer . 
 rapid uptake of a magnetic tracer after interstitial injection is important to maximize transcutaneous hotspot detection and enable injection in the theater suite , facilitating theater scheduling . 
 transcutaneous identification of hotspots was possible in most procedures with the 59 nm tracer within 10 minutes of the injection and in all except one procedure ( with the 111 nm tracer ) within 240 minutes . within 30 minutes of injection , all hotspots were already identifiable using the 59 nm tracer . 
 the greater sensitivity of the magnetometer for the 59 nm tracer for a given quantity of iron compared to the other two tracers ( figure 4 ) facilitates hotspot detection . 
 discoloration of the skin was present at the injection site using all tracers , but this was not quantified during this study . 
 our animal model did not allow to evaluate whether the skin discoloration would fade away over time or persist as a tattoo . 
 although superficial administration of an spio tracer can result in skin tattooing , the current clinical studies using spio have also not reported adequate follow - up data to fully quantify this.1214 however , any tattooing is anticipated to be temporary rather than permanent but may persist for several months  similar to that expected for blue dye.23 any tracer used for slnb must be retained by the slns and not pass on to higher echelon nodes in order to avoid excessive excision of normal nodes . within this study 
 , a statistically significantly greater number of nodes were excised using the 32 nm tracer compared to the other two tracers . 
 it suggests that rather than being retained within the sln after entering through the afferent duct , it passes through the efferent system and onto higher echelon nodes . 
 this is reflected by the small islands of sparsely distributed iron within the nodes after the administration of the 32 nm tracer on histopathology ( figure 5c ) . by applying the 
 10% rule  and excising those nodes with only 10% of the ex vivo count of the hottest excised node using radioisotope , martin et al24 demonstrated that a mean sln harvest of 1.96 nodes was associated with a false - negative rate of 5.8% . in the sentimag multicentre trial using the 59 nm tracer , 
 excising more than two slns during sentinel node biopsy is not proven to be beneficial except after primary systemic therapy.25 our data suggest that the small - sized 32 nm tracer would result in removal of an excessive number of normal nodes . 
 in addition to retention , the accumulation of magnetic tracer within the slns is essential for intraoperative identification . 
 the 59 nm tracer demonstrated statistically significant higher ex vivo magnetometer counts compared to the other two tracers , but only a statistically significant greater iron content over the 32 nm tracer . 
 this is explained by the higher sensitivity of the used magnetometer for the 59 nm tracer compared to the other two tracers . 
 the higher sensitivity for the 59 nm tracer is due to differences in magnetic properties of the tracers , arising from differences in size and material of the magnetic cores.26 by injecting the magnetic tracer 24 hours before surgery , one may be able to improve the iron uptake by the slns and thereby improve the performance of a tracer . 
 however , extending the period between injection and surgery could allow adverse spio migration to higher echelon nodes beyond the sln . 
 therefore , this is likely only suitable for the larger 111 nm tracer  within this context . 
 however , to optimize the time between injection and surgery for each tracer , formal assessment of individual tracers for slnb when injected at different times before surgery is needed . 
 the variation in the accumulation of iron within nodes , between different tracers was demonstrated on histopathology . 
 there was significantly less iron deposition in nodes from the 32 nm tracer compared to the other tracers , with only sparse , small islands of iron derived from the 32 nm tracer being observed in the nodes . 
 there was no difference in the grading of nodes between the 59 nm tracer and the 111 nm tracer on histopathology . in both cases , 
 the iron was distributed in the subcapsular space and sinuses of nodes , consistent with previous histopathological studies.20,27 however , the 111 nm tracer was found to be deposited mainly within macrophages  demonstrating its ability to activate the mononuclear phagocytic system  compared to the 59 nm tracer , which although demonstrating phagocytosis within the node also displayed extensive free iron granules peripherally . 
 these two processes of activating the mononuclear phagocytic system and free iron granule deposition facilitate iron accumulation and hence sln identification using a handheld magnetometer . 
 different studies have recently evaluated the influence of magnetic nanoparticle size on lymphatic uptake in rodent models.2830 tracers with particle sizes ranging from 4 to 1,000 nm were used in these studies . 
 the purpose of these studies was to optimize tracer uptake in the slns to facilitate preoperative localization with mri , rather than intraoperative detection . 
 mori et al30 used 50 nm , 100 nm , 200 nm , and 1,000 nm 
 sized particles and concluded that particles of 200 nm and larger are not suitable in view of lack of uptake within 24 hours . 
 iida et al28 compared the performance of 4 nm , 8 nm , and 20 nm citrate - coated nanoparticles and concluded that the 20 nm particles were best retained in the slns and accumulated in the highest concentration . 
 however , the 20 nm tracer was the largest used , and therefore , it is possible that uptake could be further optimized by increasing the particle size . 
 kjellman et al29 used monodisperse magnetic nanoparticles with a polyethylene glycol coating with sizes of 15 nm , 27 nm , and 57 nm . 
 the 15 nm particles were observed to accumulate in the slns the fastest and in the highest concentrations . a 15 nm polyethylene glycol 
 coated tracer approved for use in humans is currently not available and was not used in our study . 
 therefore , the efficiency of the 15 nm tracer can not be compared to our results . during the course of the animal experimentation 
 , there were no signs of adverse events developing within the animals as a consequence of the magnetic tracer administration . 
 when magnetic tracers are used as mri contrast agents , doses of 25100 mg are typically administered . 
 animal studies have demonstrated no acute or subacute toxicity when 150 times this dose has been administered.31 consequently , iron overload would not be possible within this model due to the small amounts of iron injected ( 5.6 mg ) . in this study 
 however , only the 59 nm tracer is currently ce - marked for the purpose of slnb in europe . 
 the 111 nm and 32 nm tracers are licensed as an mri contrast agent and an intravenous treatment of iron deficiency anemia , respectively . apart from the dilution of the tracers to standardize iron concentrations , no changes were made to the constituents to facilitate the translation of the results to clinical practice . 
 the formulation of the tracers is very similar ; all are aqueous suspensions of magnetic nanoparticles in water , with a ph level ranging from 5 to 9 . 
 the most remarkable difference between the tracers is the particle size ; however , differences in coating and other constituents are also evident . 
 the coating of the 111 nm tracer and 59 nm tracer are both dextran based ( dextran and carboxydextran , respectively ) ; however , a polyglucose sorbitol carboxymethylether coating is used in the 32 nm tracer . 
 although the coating is known to be a factor influencing in vivo nanoparticle distribution , particle size was demonstrated to be the most significant factor for interstitially administered tracers.19,29,3234 the poor performance of the polyethylene glycol  coated 32 nm tracer compared to the other tracers is therefore likely explained by a combination of the particle size distribution and the coating . 
 the observed differences between the dextran - coated tracers are most likely explained by the different particle size distributions of these tracers . 
 currently , the 59 nm tracer is the best performing magnetic nanoparticle tracer , approved for human use , in slnb with a handheld magnetometer . 
 the particles of the 59 nm tracer distribute rapidly from the injection site to the slns , allowing transcutaneous localization within 30 minutes in contrast to the 240 minutes for the 32 nm tracer and 111 nm tracer . 
 the particles of the 59 nm tracer are retained in the first nodes reached in contrast to the particles of the 32 nm tracer . finally , the particles accumulate in high concentrations , facilitating intraoperative localization . 
 the rapid distribution , retention in the first nodes reached , and accumulation in high concentration make the 59 nm tracer the most suitable magnetic tracer for slnb .","introductionbreast cancer staging with sentinel lymph node biopsy relies on the use of radioisotopes , which limits the availability of the procedure worldwide . 
 the use of a magnetic nanoparticle tracer and a handheld magnetometer provides a radiation - free alternative , which was recently evaluated in two clinical trials . 
 the hydrodynamic particle size of the used magnetic tracer differs substantially from the radioisotope tracer and could therefore benefit from optimization . 
 the aim of this study was to assess the performance of three different - sized magnetic nanoparticle tracers for sentinel lymph node biopsy within an in vivo porcine model.materials and methodssentinel lymph node biopsy was performed within a validated porcine model using three magnetic nanoparticle tracers , approved for use in humans ( ferumoxytol , with hydrodynamic diameter dh = 32 nm ; sienna+ , dh = 59 nm ; and ferumoxide , dh = 111 nm ) , and a handheld magnetometer . 
 magnetometer counts ( transcutaneous and ex vivo ) , iron quantification ( vibrating sample magnetometry ) , and histopathological assessments were performed on all ex vivo nodes.resultstranscutaneous  hotspots  were present in 12/12 cases within 30 minutes of injection for the 59 nm tracer , compared to 7/12 for the 32 nm tracer and 8/12 for the 111 nm tracer , at the same time point . 
 ex vivo magnetometer counts were significantly greater for the 59 nm tracer than for the other tracers . 
 significantly more nodes per basin were excised for the 32 nm tracer compared to other tracers , indicating poor retention of the 32 nm tracer . using 
 the 59 nm tracer resulted in a significantly higher iron accumulation compared to the 32 nm tracer.conclusionthe 59 nm tracer demonstrated rapid lymphatic uptake , retention in the first nodes reached , and accumulation in high concentration , making it the most suitable tracer for intraoperative sentinel lymph node localization .",pubmed
"visceral adipose tissue - derived serine proteinase inhibitor ( vaspin ) was identified in the visceral adipose tissue of oletf ( otsuka long - evans tokushim a fatty ) rats , an animal model of obesity and type 2 diabetes mellitus ( t2 dm ) . 
 vaspin is also expressed in the skin , hypothalamus , pancreatic islets , and stomach . 
 circulating levels of adiponectin , a hormone produced predominantly by adipocytes , are highly heritable and are inversely associated with t2 dm and other metabolic traits . 
 some studies have shown that plasma vaspin concentrations are significantly higher in men with the metabolic syndrome compared with those without the metabolic syndrome . 
 the serum vaspin levels in the patients with type 2 diabetes have been demonstrated to be higher than [ 5 , 6 ] or similar to   those observed in subjects with normal glucose tolerance . in recent , 
 inoue et al . reported that the serum vaspin levels are negatively correlated with the creatinine levels and were significantly reduced in the japanese chronic hemodialysis ( hd ) patients . 
 thus , it was interesting to ask whether serum vaspin levels are elevated or reduced in diabetic patients with renal insufficiency . 
 we measured serum vaspin and adiponectin levels in subjects with normal renal function and renal insufficiency with or without t2 dm and compared various clinical parameters . 
 all subjects were local residents of han ethnicity in shanghai and consecutively recruited in department of endocrinology and cardiology , xinhua hospital and shanghai tenth people 's hospital from june 2011 to december 2011 . 
 the study was approved by the ethical committee of shanghai tenth people 's hospital and xinhua hospital . 
 clinical characteristics were recorded with respect to age , sex , body mass index ( bmi ) , risk factors for renal function , and diabetic mellitus ( dm ) . 
 dm was defined as fasting serum glucose level 7.0  mmol / l or 2-hour postprandial glucose 11.1  mmol / l , or taking hypoglycemic drugs . those with type 1 diabetes , acute myocardial infarction , systemic inflammatory disorders , severe heart failure ( left ventricular ejection fraction < 30% ) , advanced renal insufficiency , and malignant tumor were excluded . 
 human total adiponectin ( r&d systems , minneapolis , american ) and vaspin ( adipogen , seoul , south korea ) plasma levels were measured with commercially available elisas as indicated before . while high - sensitive c - reactive protein ( hscrp ) , fibrinogen ( fib ) , creatinine ( cr ) , blood urea nitrogen ( bun ) , uric acid ( ua ) , fasting blood glucose ( fbg ) , 2-hour postprandial glucose ( pg2h ) , hemoglobin a1c ( hba1c ) , and lipid profiles , including total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , apolipoprotein a ( apoa ) , and apolipoprotein b ( apob ) were measured by colorimetric enzymatic assay systems ( roche modular p-800 , swiss confederation ) . 
 416 subjects were divided into 4 groups according to the presence or absence of ri and t2 dm : 247 individuals without t2 dm who had ri ( n = 27 ; group i ) or normal renal function ( n = 220 ; group ii ) and 169 patients with t2 dm who had ri ( n = 23 ; group iii ) or normal renal function ( n = 146 ; group iv ) . 
 renal function was assessed by the estimated creatinine clearance ( crcl ) derived from cockroft - gault formula , where crcl ( ml / min ) = ( [ 140  age ( years ) ]  weight ( kg))/(0.818  serum creatinine ( mol / l ) ) , corrected in women by a factor of 0.85 . 
 a calculated crcl less than 60  ml / min was defined as ri , according to guidelines . 
 statistical analyses were performed using the pasw statistics 20 software ( ibm corporation , armonk , ny , usa ) . 
 comparisons of variables with a normal distribution were made using student 's t - test , and values were provided as mean  s.d . for parameters with an abnormal distribution , the mann - whitney u test was used for comparisons , and values were given as median ( interquartile range ) . 
 categorical variables were presented as frequencies . to evaluate the correlation between serum vaspin and adiponectin levels with other variables such as age , bmi , hscrp , crcl , cr , fbg , and tc single linear univariate correlations and multivariate regression analyses were performed . 
 in the population either with t2 dm or without t2 dm , patients with ri ( group i or group iii ) were older and had lower bmi , higher systolic blood pressure ( sbp ) than those without ri ( group ii or group iv ) ( all p < 0.05 ) ( table 1 ) . 
 the serum vaspin showed no significant differences either between non - t2 dm patients with ri ( group i ) and without ri ( group ii ) ( 0.548 ( 0.4281.136 ) ng / ml versus 0.517 ( 0.2321.324 ) ng / ml , p = 0.402 ) or between diabetic patients with ri ( group iii ) and without ( group iv ) ( 0.497 ( 0.2880.907 )  ng / ml versus 0.381 ( 0.2320.740 )  ng / ml , p = 0.245 ) . 
 interestingly , the serum vaspin was lower in 169 patients with t2 dm than in 247 individuals without t2 dm ( 0.399 ( 0.2240.737  ng / ml versus 0.523 ( 0.1821.172 )  ng / ml , p < 0.05 ) ( table 2 ) 
 . however , no significant difference for the serum vaspin level was observed between 50 patients with ri ( combined by group i and group iii ) and 366 individuals without ri ( combined by group ii and group iv ) ( 0.540 ( 0.3991.020 )  ng / ml versus 0.432 ( 0.2340.925 )  ng / ml , p = 0.141 ) ( table 2 ) . 
 compared with the nondiabetic individuals without ri ( group ii ) , the serum adiponectin was higher in the non - diabetic individuals with ri ( group i ) ( 16082.437  9926.203 
 ng / ml , p < 0.01 ) but did not differ between diabetic patients with ri ( group iii ) and without ri ( group iv ) ( 12987.336  14623.762 
 ng / ml versus 8744.920  5628.952  ng / ml , p = 0.349 ) . 
 regardless of the individuals with or without t2 dm , the serum adiponectin level was significantly higher in 50 individuals with ri ( combined by group i and iii ) than 366 individuals without ri ( combined by group ii and iv ) ( 10549.706 ( 5589.71822429.025 )  ng / ml versus 6955.130 ( 2590.05213533.955 )  ng / ml , p < 0.01 ) ( tables 1 and 2 ) . 
 when compared with the individuals without t2 dm ( combined by group i and ii ) in our tall 416 samples , the serum adiponectin was significantly higher in 169 patients with t2 dm ( combined by group iii and iv ) ( 7599.068 ( 4100.63313827.261 )  ng / ml versus 5553.476 ( 12.09614225.162 )  ng / ml , p < 0.05 ) . 
 spearmen 's analysis showed that serum vaspin level was only related to cr in patients without t2 dm . in patients with t2 dm , 
 serum vaspin levels were related to age , sbp , and ua . in all subjects , serum vaspin levels were related to crcl , tc , and hba1c ( tables 3 and 4 ) . 
 the serum adiponectin levels were related to age , sbp , hscrp , crcl , and cr in subjects without t2 dm . in patients with t2 dm , 
 serum adiponectin levels were related to fbg , ldl - c . in all subjects , 
 serum adiponectin levels were related to age , sbp , crcl , cr , bun , and hba1c ( tables 3 and 4 ) . 
 multiple regression analyses were performed to test whether any of the following factors influence the serum vaspin and adiponectin concentrations : age , sbp , fbg , ua , crcl , cr , tg , tc , hba1c levels , and so on . when correcting for these variables using stepwise regression analyses after adjustment for age and bmi , the level of hba1c was found to be independently and significantly associated with the vaspin concentration in 427 total subjects . 
 however , no significant correlations between the serum vaspin level and any parameter were detected in both groups of the subjects with t2 dm and without t2 dm . 
 the level of crcl was found to be independently and significantly associated with the adiponectin concentration in all subjects . 
 however , in the groups of the subjects with or without t2 dm , no significant correlations were detected between the serum adiponectin level and any parameter ( tables 5 and 6 ) . 
 mann - whitney u test showed that the serum vaspin and adiponectin levels were both higher in women than in men whether in total subjects or in patients with t2 dm ( tables 7 and 8) . 
 adipose tissue is a highly active endocrine organ secreting a number of bioactive molecules called adipokines [ 5 , 6 , 12 ] , such as vaspin and adiponectin . 
 vaspin is a member serpin a12 of the serine protease inhibitor family [ 1315 ] , and a large number of clinical data have showed that vaspin played an important role in occurrence and development in the metabolic syndrome [ 1 , 16 , 17 ] . 
 upregulation of vaspin can improve the insulin resistance , which also inhibit inflammation and progression of arthrosclerosis . 
 some studies show that vaspin increases nitric oxide bioavailability through the reduction of asymmetric dimethylarginine in vascular endothelial cells   or inhibits platelet - derived growth factor - bb - induced migration of vascular smooth muscle cells . 
 the decrease of vaspin in the ri patients will increase the risk of coronary heart disease ( chd ) . 
 therefore , measuring the levels of adipokines in patients with ri may be beneficial for predicting cardiovascular events and patient survival . in patients with ckd , the levels of adipokines appear to increase in association with declines in the gfr . 
 this is most likely due to reduced renal metabolism of adipokines , which may not increase vascular risks in patients with a reduced renal function . 
 since vaspin is a small protein size with the molecular mass 50  kda , which would be freely filtered by the kidneys ; thus it is reasonable to presume that vaspin levels should be increased in patients with renal insufficiency . 
 but our data suggested that the serum vaspin levels have no significant difference between patients with ri and subjects without ri due to the fact that many factors might affect the serum levels of vaspin concentration . 
 previous studies have shown that , in the diabetic patients , serum vaspin level is higher than that in normal glucose tolerance subjects in women , and rosiglitazone therapy decreased plasma vaspin levels through glucose and insulin sensitivity regulation . 
 our study also shows that the serum vaspin level was higher in women than in men . 
 however , we have found that the serum vaspin level was lower in 169 diabetic patients than 247 individuals without t2 dm . 
 the paradox results may be caused by two reasons : one is the sex difference in the subjects between their study and our study ; another reason is that there are differences in the diabetic patients with vascular complications between the two population , since some reports have demonstrated that low vaspin serum concentrations have been found to be correlated with recently experienced ischemic events in patients with carotid stenosis   and coronary artery disease ( cad ) . 
 adiponectin is a fat - derived hormone whose reduction plays central roles in obesity - linked diseases including insulin resistance , type 2 diabetes , and atherosclerosis . 
 adiponectin multimers are secreted by white adipose tissue [ 2527 ] , which is associated with body weight and markers of cardiovascular risk in adolescence . 
 adiponectin has recently attracted much attention because of its antidiabetic and antiatherogenic effects as well as its antiproliferative effects in cancer cells ; thus , adiponectin is expected to be a therapeutic tool for diabetes , metabolic syndrome , cardiovascular diseases , and cancers [ 29 , 30 ] . 
 plasma adiponectin levels have also been reported to be reduced in obese humans , particularly those with visceral obesity [ 31 , 32 ] . 
 so , in the ri patients , lower bmi may lead to the high level of adiponectin 
 but in the diabetic patients , there are no differences between ri and non - ri . 
 some studies have shown that rosiglitazone can increase serum adiponectin levels in patients with type 2 diabetes mellitus [ 33 , 34 ] . in all patients with diabetes in our current study , only 19 patients have been treated with thiazolidinediones . after removing the patients , 
 both of t2 dm and renal insufficiency were correlated with the serum level of adiponectin . 
 although the serum vaspin levels showed no significant difference between the patients with renal insufficiency which did not required treatment by chronic hemodialysis and subjects without renal insufficiency , we have found that the serum vaspin level was lower in 169 diabetic patients than in 247 individuals without t2 dm .","vaspin and adiponectin are two adipocytokines with antidiabetic effects . 
 some studies reported that levels of adiponectin and vaspin were correlated with decreased glomerular filtration rate ( fgr ) and increased albuminuria . 
 we therefore evaluated the vaspin and adiponectin levels in renal insufficiency ( ri ) patients with or without t2 dm . 
 serum vaspin , adiponectin levels were measured in 416 subjects with or without t2 dm . 
 analysis was made between groups divided by these subjects presence or absence of ri . 
 we found that serum adiponectin level was significantly higher in nondiabetic patients with ri than in nondiabetic subjects without ri ; however , there were no statistical differences between the diabetic patients with ri and without ri . 
 in all the subjects , the serum adiponectin level was also higher in 50 individuals with ri than that in 366 subjects without ri . 
 the serum vaspin levels showed no significant differences between the diabetic patients or nondiabetics subjects with ri and without ri . 
 contrary to adiponectin , the serum vaspin level was lower in 169 patients with t2 dm than in 247 individuals without t2 dm . 
 our data suggested that both of t2 dm and renal insufficiency were correlated with the serum level of adiponectin . 
 however , the serum vaspin levels showed no significant difference between the individuals with renal insufficiency and without renal insufficiency .",pubmed
"data from two multicenter , double - blind , randomized clinical trials were used to test the surrogate variables proposed for neuropathic dfus and to explore the correlation between granulation tissue development ( the main efficacy variable of the studies ) and complete healing during follow - up for 1 year after the eot ( median time to complete closure , 15 weeks ; 25th percentile , 9.6 weeks ; 75th percentile , 21.4 weeks ) . in the first study , a dose - exploratory trial in five hospitals ( 13 ) , patients were randomized to receive intralesional injections of rhegf at 75 ( 23 subjects ) or 25 g ( 18 subjects ) , three times per week . in the second study , a confirmatory trial in 20 hospitals ( 16 ) , 
 53 subjects were randomized to rhegf 75 g , 48 to rhegf 25 g , and 48 to a placebo group . 
 each patient had only one lesion , so the total number of ulcers included in the analyses was 190 . 
 for confirmation in a larger sample from common clinical practice , the results of an active postmarketing surveillance of rhegf treatment in 1,440 dfus in 60 healthcare units ( 41 hospitals and 19 primary care polyclinics ) were taken into account ( unpublished data ) . in this series , 
 the 25- and 75-g dose levels were used , according to the product label ( 17 ) . in all series , rhegf treatment was administered up to complete granulation or a maximum of 8 weeks . 
 diabetic patients ( type 1 or 2 ) , both sexes , > 18 years of age were included in the trials , with wagner grade 3 or 4 dfus > 1 cm . 
 intralesional rhegf was administered adjuvant to standard care , which included thorough debridement ( sometimes minor amputation ) , moist dressing , pressure offloading , and antibiotics , if necessary . 
 patients with chronic , uncontrolled illness , psychiatric or neurologic diseases that could impair proper reasoning for consent , or presence or suspicion of neoplasia or who were pregnant or breastfeeding were excluded . 
 ( 2 ) as surrogate markers of complete healing were generated : percent change in area , log healing rate , and ratio of log areas at the 2nd week and eot ( median , 5 weeks).additionally the variablewas taken into account . 
 considering that the median treatment duration was 5 weeks , and that there was no area evaluation at 4 weeks for most of the patients ( > 50% missing values ) , the eot data were taken as the comparison points with the 4-week area change proposed by other authors ( 24 ) . 
 ulcer areas and percent granulation were measured by planimetry from manual tracing on a transparent grid sheet , using a validated portable wound measurement device ( visitrack ; smith & nephew , london , u.k . ) ( 18 ) . for comparability with the results in margolis et al . 
 ( 2 ) , the variables defined in eqs . 2 and 3 were calculated using the area in mm and t in days . 
 continuous variables are expressed as mean  sd or median ( 25th and 75th percentiles ) . 
 comparison between healed and nonhealed was assessed by the  test for categorical variables and mann - whitney u test for continuous variables . since missing data can constitute deviations from the intention - to - treat principle , clinical trial analyses require the use of imputation ( 19 ) . 
 for the purpose of this work , 18 missing data at the 2nd week ( 9.5% ) were imputed using the last observation carried forward ( locf ) or the average between the two contiguous evaluations that included the missing one . 
 the area under the receiver operating characteristic ( roc ) curve was calculated ( 20 ) for each candidate surrogate variable to evaluate its capacity and quality of discrimination among individuals . 
 cutoff points were generated in order to maximize both sensitivity ( probability to detect a positive result if the outcome is positive ) and specificity ( probability to detect a negative result if the outcome is negative ) . 
 the candidate surrogates were then codified , and their association with the final outcome ( complete ulcer closure ) was assessed using the sensitivity , specificity , positive predictive values ( pv+ , conditional probability to have a positive result if the test was positive ) , negative pvs ( pv , conditional probability to have a negative result if the test was negative ) , and the correct classification index ( percent of individuals correctly classified ) , also named validity index . 
 test validation and sensitivity were performed only for the placebo - controlled trial ( 16 ) for the sake of homogeneity and balance of the samples compared . to quantify the contribution of the candidate surrogates to the final outcome , the product coefficient ( pc ) 
 this is one of the methods to assess mediation in the case of binary - dependent outcome that treats it as the product of two regression coefficients : the regression of the independent treatment variable on the mediator ( t in eq . 
 6 , below ) and the partial regression coefficient of the independent treatment variable on the final outcome adjusted for the mediator ( s in eq . 7 , below ) . in order to estimate the pc , 
 closure  ( 1 , yes ; 0 , no ) , x the baseline covariates x1 ( etiology : neuropathic and ischemic ) and x2 ( initial area ) , t the intervention status ( 1 for rhegf treatment and 0 for placebo ) , and s the granulation tissue covering the wound area ( at 2nd week or at the eot ) . 
 the following models were adjusted : which measures the effect of treatment on healing without the influence of the surrogate , which measures the effect of treatment on the surrogate , taking into account the same covariates , andwhich measures the effect of treatment on wound closure , mediated by the surrogate , taking into account the covariates . 
 the pc estimator   and the proportion mediated   and its 95% ci ( 21 ) were used to quantify the contribution of the surrogate ( s : granulation and area change variables ) on ulcer closure . 
 the association with some baseline influencing factors between healing and candidate surrogate variables was estimated using the odds ratio and the 95% cis in binary logistic regression . 
 confirmation was performed only for the granulation variable percent of granulation tissue covering the ulcer area ( > 75% or not ) , since during medical practice it was not possible to standardize a precise measurement of the wound area and partial percent of granulation . 
 variables proposed by margolis et al . ( 2 ) as surrogate markers of complete healing were generated : percent change in area , log healing rate , and ratio of log areas at the 2nd week and eot ( median , 5 weeks).additionally the variablewas taken into account . 
 considering that the median treatment duration was 5 weeks , and that there was no area evaluation at 4 weeks for most of the patients ( > 50% missing values ) , the eot data were taken as the comparison points with the 4-week area change proposed by other authors ( 24 ) . 
 ulcer areas and percent granulation were measured by planimetry from manual tracing on a transparent grid sheet , using a validated portable wound measurement device ( visitrack ; smith & nephew , london , u.k . ) ( 18 ) . for comparability with the results in margolis 
 et al . ( 2 ) , the variables defined in eqs . 2 and 3 were calculated using the area in mm and t in days . 
 continuous variables are expressed as mean  sd or median ( 25th and 75th percentiles ) . 
 comparison between healed and nonhealed was assessed by the  test for categorical variables and mann - whitney u test for continuous variables . since missing data can constitute deviations from the intention - to - treat principle , clinical trial analyses require the use of imputation ( 19 ) . for the purpose of this work , 
 18 missing data at the 2nd week ( 9.5% ) were imputed using the last observation carried forward ( locf ) or the average between the two contiguous evaluations that included the missing one . 
 the area under the receiver operating characteristic ( roc ) curve was calculated ( 20 ) for each candidate surrogate variable to evaluate its capacity and quality of discrimination among individuals . 
 cutoff points were generated in order to maximize both sensitivity ( probability to detect a positive result if the outcome is positive ) and specificity ( probability to detect a negative result if the outcome is negative ) . 
 the candidate surrogates were then codified , and their association with the final outcome ( complete ulcer closure ) was assessed using the sensitivity , specificity , positive predictive values ( pv+ , conditional probability to have a positive result if the test was positive ) , negative pvs ( pv , conditional probability to have a negative result if the test was negative ) , and the correct classification index ( percent of individuals correctly classified ) , also named validity index . 
 test validation and sensitivity were performed only for the placebo - controlled trial ( 16 ) for the sake of homogeneity and balance of the samples compared . to quantify the contribution of the candidate surrogates to the final outcome , the product coefficient ( pc ) 
 this is one of the methods to assess mediation in the case of binary - dependent outcome that treats it as the product of two regression coefficients : the regression of the independent treatment variable on the mediator ( t in eq . 
 6 , below ) and the partial regression coefficient of the independent treatment variable on the final outcome adjusted for the mediator ( s in eq . 
 ( 1 , yes ; 0 , no ) , x the baseline covariates x1 ( etiology : neuropathic and ischemic ) and x2 ( initial area ) , t the intervention status ( 1 for rhegf treatment and 0 for placebo ) , and s the granulation tissue covering the wound area ( at 2nd week or at the eot ) . 
 the following models were adjusted : which measures the effect of treatment on healing without the influence of the surrogate , which measures the effect of treatment on the surrogate , taking into account the same covariates , andwhich measures the effect of treatment on wound closure , mediated by the surrogate , taking into account the covariates . 
 the pc estimator   and the proportion mediated   and its 95% ci ( 21 ) were used to quantify the contribution of the surrogate ( s : granulation and area change variables ) on ulcer closure . 
 the association with some baseline influencing factors between healing and candidate surrogate variables was estimated using the odds ratio and the 95% cis in binary logistic regression . 
 confirmation was performed only for the granulation variable percent of granulation tissue covering the ulcer area ( > 75% or not ) , since during medical practice it was not possible to standardize a precise measurement of the wound area and partial percent of granulation . 
 continuous variables are expressed as mean  sd or median ( 25th and 75th percentiles ) . 
 comparison between healed and nonhealed was assessed by the  test for categorical variables and mann - whitney u test for continuous variables . since missing data can constitute deviations from the intention - to - treat principle , clinical trial analyses require the use of imputation ( 19 ) . for the purpose of this work , 
 18 missing data at the 2nd week ( 9.5% ) were imputed using the last observation carried forward ( locf ) or the average between the two contiguous evaluations that included the missing one . 
 the area under the receiver operating characteristic ( roc ) curve was calculated ( 20 ) for each candidate surrogate variable to evaluate its capacity and quality of discrimination among individuals . 
 cutoff points were generated in order to maximize both sensitivity ( probability to detect a positive result if the outcome is positive ) and specificity ( probability to detect a negative result if the outcome is negative ) . 
 the candidate surrogates were then codified , and their association with the final outcome ( complete ulcer closure ) was assessed using the sensitivity , specificity , positive predictive values ( pv+ , conditional probability to have a positive result if the test was positive ) , negative pvs ( pv , conditional probability to have a negative result if the test was negative ) , and the correct classification index ( percent of individuals correctly classified ) , also named validity index . 
 test validation and sensitivity were performed only for the placebo - controlled trial ( 16 ) for the sake of homogeneity and balance of the samples compared . to quantify the contribution of the candidate surrogates to the final outcome , the product coefficient ( pc ) 
 this is one of the methods to assess mediation in the case of binary - dependent outcome that treats it as the product of two regression coefficients : the regression of the independent treatment variable on the mediator ( t in eq . 
 6 , below ) and the partial regression coefficient of the independent treatment variable on the final outcome adjusted for the mediator ( s in eq . 7 , below ) . in order to estimate the pc , 
 ( 1 , yes ; 0 , no ) , x the baseline covariates x1 ( etiology : neuropathic and ischemic ) and x2 ( initial area ) , t the intervention status ( 1 for rhegf treatment and 0 for placebo ) , and s the granulation tissue covering the wound area ( at 2nd week or at the eot ) . 
 the following models were adjusted : which measures the effect of treatment on healing without the influence of the surrogate , which measures the effect of treatment on the surrogate , taking into account the same covariates , andwhich measures the effect of treatment on wound closure , mediated by the surrogate , taking into account the covariates . 
 the pc estimator   and the proportion mediated   and its 95% ci ( 21 ) were used to quantify the contribution of the surrogate ( s : granulation and area change variables ) on ulcer closure . 
 the association with some baseline influencing factors between healing and candidate surrogate variables was estimated using the odds ratio and the 95% cis in binary logistic regression . 
 confirmation was performed only for the granulation variable percent of granulation tissue covering the ulcer area ( > 75% or not ) , since during medical practice it was not possible to standardize a precise measurement of the wound area and partial percent of granulation . 
 the median age was > 60 years , median ulcer size was 22 cm ( 75% > 10 cm ) , lesions were 57% ischemic , and wagner classification grades 3 and 4 were 73.7 and 26.3% , respectively . 
 the inferential summary of baseline characteristics and candidate surrogates by healing is shown in table 1 . globally , 58.4% of the wounds achieved closure . 
 complete healing showed dependence on all variables except for sex , ethnicity , treatment duration , and wagner classification . 
 patient characteristics in terms of healing ( all treatment groups combined ) all variables tested had a good discrimination capacity , with roc curve areas > 0.7 at the 2nd week and > 0.8 at the eot ( see supplementary table 2 for details ) . those with the lower limit of the 95% ci > 0.7 were selected as the best ones : the three variables based on the area change at the eot and granulation tissue formation at the 2nd week and at the eot . 
 table 2 shows these , as well as the sensitivity , specificity , positive and negative pvs , and percent correct classification calculated from them . 
 the 95% cis were not shown for simplicity of the table , but they were all < 2% around the estimators , showing high precision . diagnostic test for candidate surrogate variables and complete healing the most efficient predictor was the granulation tissue covering > 75% of the ulcer area at the end of the treatment , with 79.5% correct classification and a high negative pv ( 93.5% ) . 
 an earlier evaluation of this surrogate marker ( granulation tissue covering > 50% of the ulcer area at 2 weeks ) was also adequate , with 73.7% correct classification , pvs ( positive and negative ) > 70% , and acceptable sensitivity and specificity values . 
 the best cutoff points for variables based on the area change at the eot were selected too . 
 the correct classification was also high ( > 70% ) , with suitable diagnostic indicators . 
 at this point , all candidates can be used to discriminate healing . the proportion - mediated ( using pc estimators )   for 2 weeks > 50% granulation and eot 
 > 75% granulation were 1.008 ( 95% ci 0.9331.083 ) and 1.043 ( 0.9461.142 ) , respectively , both very close to 1 , suggesting that almost 100% of the effect of the treatment on complete healing may have been mediated by the formation of granulation tissue . with the area change 
 the mediation explained by this variable was very low : 0.003 ( 95% ci 0.002 to 0.008 ) . 
 the effect of the treatment on healing of this type of complicated ulcers is not necessarily mediated by an area reduction in the earlier weeks . 
 the models given by eqs . 6 and 7 for percent area change and ratio of log areas 
 table 3 shows the association between healing and candidate surrogate variables with some baseline influencing factors . 
 all the models with the granulation - based candidates were adequate ( hosmer - lemeshow goodness - of - fit statistics ) . 
 treatment had a significant influence on healing and the two granulation - dependent variables considered . for simplicity , only the 75-g rhegf is shown , since the 25-g dose had no significant effect on healing in the clinical trial ( 16 ) . 
 the effect of the intervention on healing was lost when either 2 weeks > 50% granulation or eot > 75% granulation is incorporated in the model . 
 the odds ratios for the influence of the covariates ( initial area and etiology ) were similar for the three dependent outcomes ( 2-week and eot granulation and healing ) . 
 association between covariates and outcomes ( granulation and healing ) the results of the model with the eot percent area change are also shown in table 3 as an example . 
 models with the log healing rate variable are not shown , but their interpretation was the same . 
 no effect of treatment on the surrogate was found ( 95% ci 0.714.03 ) , and the effect of treatment on complete healing was not lost when the percent area change was incorporated in the model . 
 the results of the granulation - healing correlation analysis in the postmarketing study in current medical practice were obtained from the data of 1,440 ulcers . 
 the result confirms that granulation tissue development at the eot was an early predictor of final healing , with a 72.9% certainty and positive / negative pvs of 71.7/75.8% , respectively ( details in supplementary table 4 ) . 
 figure 1 illustrates the discrimination capacity of the granulation variables in clinical trials and in the postmarketing series . 
 discrimination capacity of the granulation variables in clinical trials ( cts ) and in the postmarketing ( pm ) series . 
 the results of this study show that several surrogate variables can be taken into consideration for the final outcome in advanced dfus , including ischemic , wagner grade 3 or 4 , and median area > 20 cm . 
 granulation tissue development has been taken previously as a secondary outcome in dfu trials ( 14,15 ) . 
 it was also proposed as a potential intermediate variable ( 9 ) , but its use as a surrogate of complete wound closure has not been proposed before , despite being part of the healing process and necessarily preceding final re - epithelization . the granulation process showed good capacity to discriminate healers from nonhealers with roc curve areas > 70% at the 2nd week and eot . 
 the validation tests using multivariate analyses were developed with the data of the randomized placebo - controlled clinical trial of intralesional rhegf in advanced dfus ( 16 ) . 
 the proportion mediated for granulation variables ( at 2 weeks and at the eot ) was very high , which agrees with the idea that granulation constitutes an essential part of the healing process , particularly in larger and advanced ulcers . 
 the egf - induced mitogenic , motogenic , and cytoprotective actions are instrumental for healing events that may be summarized as follows : 1 ) stimulation of productive cell migration toward the injured area ; 2 ) stimulation of granulation tissue outgrowth , including extracellular matrix accumulation , maturation , and de novo angiogenesis ; 3 ) stimulation of wound contraction by myofibroblast activation and proliferation ; and 4 ) stimulation of the damaged area resurfacing by epithelial cell migration and proliferation ( 23 ) . 
 since treatment with rhegf was given only up to complete granulation , mechanisms 1 and 2 took place completely but not 3 and 4 , which require a previous granulating wound bed . for area change  dependent candidates , the proportion mediated was small , which could be explained by a more exigent condition for these lesions . had the treatment continued after complete granulation , maybe a better proportion mediated would be obtained , given that mechanisms 3 and 4 could have worked longer . the models for final cicatrization and the granulation surrogates were adequate and fulfilled the original prentice ( 24 ) criteria for the definition of a surrogate end point : 1 ) treatment had a significant effect upon the surrogate end point ; 
 2 ) treatment had a significant effect upon the true end point ; 3 ) the surrogate end point had a significant effect upon the true end point ; and 4 ) the full effect of treatment upon the true end point was mediated by the surrogate . 
 additionally , the effect of the covariates ( wound area and ulcer etiology ) was similar in all models . 
 criterion 4 is highly dependent on the power of the model and the sample size used . 
 patients with such advanced dfus are usually not included in clinical trials , which limits the possibility of meta - analysis at present . the larger sample of the postmarketing series could not be included since it was not a controlled study . nevertheless , it was useful to confirm the predictive character of the eot granulation on final wound closure . 
 on the other hand , as a measure of statistical power , 10 events per predictor is considered suitable , taking all the candidates and the other covariates ( 25,26 ) . in this work , only in the placebo - controlled confirmatory trial ( 16 ) , there were 93 healing events and 9 variables , so the criterion is fulfilled . 
 eot > 75% granulation had a higher pv with respect to final re - epithelization and thus should be a better surrogate for clinical trial design . 
 this can be useful since it would not be necessary to wait for complete re - epithelization due to treatment , and alternative wound closure procedures could be used after complete granulation , such as grafts or engineered skin . 
 the 2 weeks > 50% granulation , being an earlier surrogate , may also have practical value for treatment algorithm decisions and/or pharmacoeconomic considerations . 
 several variables based on wound area change have been proposed as surrogates for complete healing in neuropathic noncomplicated lesions . 
 ( 2 ) described the predictive capacity of 61% area reduction at 4 weeks as well as several transformations of the area change . 
 sheehan ( 27 ) , based on previous work ( 3 ) , proposes 50% wound closure at 4 weeks as a good surrogate for final healing and thus a decision point in a foot ulcer management algorithm . 
 the results of the present work suggest that the predictive property of percent area change , log healing rate , and ratio of log area can be extrapolated to more complicated , ischemic , larger , wagner grade 3 or 4 ulcers . however , the cutoff points for classification were not the same . with the cutoff proposed by the above - mentioned authors , 
 the correct classification percent in the present series was < 70% , with negative pvs < 60 . 
 this is reasonable since larger and more complex ulcers take longer to heal , and thus the value to expect as predictive around 4 weeks should be smaller , 16% area change , and much lower for the other transformed variables . 
 it was not possible to validate the surrogate property for these variables in this series since the percent area change did not fulfill the prentice criteria ( 24 ) 1 and 4 . additionally , the proportion mediated by these variables was very low ( discussed above ) . 
 the extrapolation of the cutoff points proposed in the present work has the limitation of the small sample size analyzed , so it requires further research . 
 it could not be performed in the confirmatory , postmarketing series since the partial areas were not measured in routine clinical practice . 
 this work provides the first evidence for the use of granulation tissue development as a predictor of wound healing in advanced dfus . 
 these results can be useful for clinical trial design , particularly during the exploratory phase of development of new products . 
 the surrogate markers that we report can easily be applied by clinicians because the measurement of the proportion of wound area covered by granulation tissue is part of routine clinical care . correctly classifying patients with advanced diabetic foot ulcers into those who will heal or not heal by the 2nd week of care or at the end of granulation 
 may have relevant implications in new wound healing products and for healthcare programs managing diabetic foot ulcers . 
 ","objectiveto determine if partial wound closure surrogate markers proposed for neuropathic , small diabetic foot ulcers ( dfus ) can be extended to advanced lesions and if the development of granulation tissue can be used to predict complete healing.research design and methodsdata from two multicenter , double - blind , randomized clinical trials ( one of them placebo controlled ) that used intralesional recombinant human epidermal growth factor ( rhegf ) to promote granulation and healing were used . 
 for confirmation in a larger sample from common clinical practice , the results of an active postmarketing surveillance of rhegf treatment of dfus in 60 healthcare units was included . 
 the surrogates evaluated were percent area change , log healing rate , ratio of log areas , and percent of granulation tissue covering the wound area . 
 the tests used were surrogate final end point correlation , receiver operating characteristic curves to discriminate healers from nonhealers , validation tests using logistic regression models , and the proportion - mediated estimation.resultstwo weeks > 50% granulation , end of treatment > 75% granulation , and 16.1% area change showed significant predictive value ( > 70% correct classification ) for final wound closure . 
 the granulation - based variables fulfilled the criterion that the effect of rhegf treatment on wound closure was mediated by the surrogate.conclusionsthis work provides the first evidence for the use of granulation tissue development as a predictor of wound healing in advanced dfus . 
 these results can be useful for clinical trial design , particularly during the exploratory phase of new products .",pubmed
"to date , laparoscopic transabdominal preperitoneal ( tapp ) mesh repair has been regarded as one of the standard treatments for inguinal hernia in adults . 
 however , patients who undergo the procedure experience a significantly higher incidence of early postoperative pain , which is associated with subsequent chronic pain . surgical trauma to the abdominal wall , mesh implantation , and mesh fixation in the groin region have been indicated as possible causes of the pain . 
 the ideal mesh material is still being debated , but mesh fixation with fibrin glue or the alternate choice of self - fixating mesh with microhooks has obvious advantages in reducing chronic groin pain . in the context of surgical tissue trauma at trocar sites , 
 guidelines suggest that the use of a 10-mm trocar in the umbilical region or in the oblique abdominal wall predisposes patients to local nerve or vessel injury and port - site hernia . 
 thus , an improvement in current laparoscopic hernia surgery has been the further reduction of the size of the abdominal wall incision for trocar insertion . 
 modified tapp repairs by needlescopic surgery ( ntapp ) , with the use of smaller trocars than the standard 10-mm optical trocars and 5-mm working trocars , have been shown to be superior in patient outcomes over conventional tapp repairs . 
 also tapp repairs in single - incision laparoscopic surgery ( stapp ) , with a reduction in the number of ports to only 1 umbilical port , have been put forward with good initial results . however , a direct comparison between these 2 advanced minimally invasive approaches is still missing . 
 the purpose of this study was to compare the perioperative extent of surgical trauma and its postoperative impact on the abdominal wall after ntapp and stapp repair . 
 for this retrospective cohort study with institutional review board approval , 85 patients with a single - sided primary inguinal hernia were recruited at a community hospital between november 1 , 2009 , and july 31 , 2012 . in this period of 32 months 
 demographic and surgical data , including gender , age , body mass index , location and size of the inguinal hernia , length of the umbilical skin incision for the trocar port , operative time , and length of hospital stay were retrospectively retrieved from surgical records . 
 on postoperative day 3 , all patients were scheduled for a wound check and removal of skin staples . in october 2013 , 
 the patients were asked to complete a questionnaire and undergo a physical examination in the outpatient clinic . 
 the following information was then recorded : hernia recurrence ; postoperative pain , according to the visual analog scale ( vas ) ( 0 = no pain ; 10 = worst pain ) ; postoperative mobility of the abdominal wall , determined by janda 's test , a sit - up exercise ( 0 = no recovery of normal muscle power ; 5 = full recovery of normal muscle power ) ; postoperative cosmetic satisfaction , according to the cosmetic vas ( 0 = completely dissatisfied ; 10 = completely satisfied ) ; and period of sick leave after hospital discharge for employed workers . 
 the operative procedure in laparoscopic tapp mesh repair was described in depth in our previous study . in brief 
 , it involved establishing pneumoperitoneum , dissecting the peritoneal flap and the preperitoneal space with an electric hook , placing a 15  9-cm parietene progrip self - fixating monofilament polypropylene mesh ( covidien , dublin , ireland ) , and closing the peritoneal flap with a v - loc barbed suture ( covidien ) . in the ntapp group , 
 the length of the transverse skin incision at the umbilicus was first measured , and an 8-mm trocar was inserted for the use of a 5-mm optical instrument . under optical view , a 5-mm trocar for the electric hook on the right side of the abdomen and a 2-mm trocar for the grasping forceps on the left side of the abdomen were placed at the level of the umbilicus on the midclavicular line . in the stapp group , 
 the umbilical skin incision length was also first measured , and a gelpoint advanced - access platform ( applied medical , rancho santa margarita , california ) was placed through the umbilicus . 
 a 5-mm optical trocar and two 5-mm working trocars were then positioned in this single - port - access device . 
 the self - fixating parietene progrip mesh , with microhooks on its underside , was folded in a special manner , as demonstrated in our previous publication . 
 it was then inserted into the abdominal cavity through the 8-mm umbilical trocar in the ntapp group or through the 510-mm compatible cannula of the gelpoint advanced - access platform in the stapp group . 
 once hernia repair was completed , the umbilical wound was closed with an absorbable fascia suture and skin staples in all ntapp and stapp procedures . 
 the lateral 5- and 2-mm trocar wounds in the ntapp group were closed with steri - strips adhesive skin closures ( 3 m health center , st . 
 all operations were performed on an elective basis in patients under general anesthesia by the same experienced surgeon . 
 spss 22 for windows ( ibm corp . , somers , new york ) was used for statistical analysis . 
 data are expressed as percentages for categorical variables and as the mean  sd for continuous variables . 
 comparisons between the 2 operative approaches were performed with the  test and student 's t test , as applicable . 
 for this retrospective cohort study with institutional review board approval , 85 patients with a single - sided primary inguinal hernia were recruited at a community hospital between november 1 , 2009 , and july 31 , 2012 . in this period of 32 months 
 demographic and surgical data , including gender , age , body mass index , location and size of the inguinal hernia , length of the umbilical skin incision for the trocar port , operative time , and length of hospital stay were retrospectively retrieved from surgical records . 
 on postoperative day 3 , all patients were scheduled for a wound check and removal of skin staples . in october 2013 , 
 the patients were asked to complete a questionnaire and undergo a physical examination in the outpatient clinic . 
 the following information was then recorded : hernia recurrence ; postoperative pain , according to the visual analog scale ( vas ) ( 0 = no pain ; 10 = worst pain ) ; postoperative mobility of the abdominal wall , determined by janda 's test , a sit - up exercise ( 0 = no recovery of normal muscle power ; 5 = full recovery of normal muscle power ) ; postoperative cosmetic satisfaction , according to the cosmetic vas ( 0 = completely dissatisfied ; 10 = completely satisfied ) ; and period of sick leave after hospital discharge for employed workers . 
 the operative procedure in laparoscopic tapp mesh repair was described in depth in our previous study . in brief 
 , it involved establishing pneumoperitoneum , dissecting the peritoneal flap and the preperitoneal space with an electric hook , placing a 15  9-cm parietene progrip self - fixating monofilament polypropylene mesh ( covidien , dublin , ireland ) , and closing the peritoneal flap with a v - loc barbed suture ( covidien ) . in the ntapp group , 
 the length of the transverse skin incision at the umbilicus was first measured , and an 8-mm trocar was inserted for the use of a 5-mm optical instrument . under optical view , a 5-mm trocar for the electric hook on the right side of the abdomen and a 2-mm trocar for the grasping forceps on the left side of the abdomen were placed at the level of the umbilicus on the midclavicular line . in the stapp group , 
 the umbilical skin incision length was also first measured , and a gelpoint advanced - access platform ( applied medical , rancho santa margarita , california ) was placed through the umbilicus . 
 a 5-mm optical trocar and two 5-mm working trocars were then positioned in this single - port - access device . 
 the self - fixating parietene progrip mesh , with microhooks on its underside , was folded in a special manner , as demonstrated in our previous publication . 
 it was then inserted into the abdominal cavity through the 8-mm umbilical trocar in the ntapp group or through the 510-mm compatible cannula of the gelpoint advanced - access platform in the stapp group . once hernia repair was completed , the umbilical wound was closed with an absorbable fascia suture and skin staples in all ntapp and stapp procedures . 
 the lateral 5- and 2-mm trocar wounds in the ntapp group were closed with steri - strips adhesive skin closures ( 3 m health center , st . 
 all operations were performed on an elective basis in patients under general anesthesia by the same experienced surgeon . 
 spss 22 for windows ( ibm corp . , somers , new york ) was used for statistical analysis . 
 data are expressed as percentages for categorical variables and as the mean  sd for continuous variables . 
 comparisons between the 2 operative approaches were performed with the  test and student 's t test , as applicable . 
 patient characteristics in the ntapp and stapp groups were comparable at baseline ( male : female 5.4:1 vs 5.3:1 ; age 52.5 y vs 48.3 y ; bmi 25.3 kg / m vs 24.4 kg / m ; direct : indirect : pantaloon hernia , 32:58:10% vs 31.4:54.3:14.3% ; and hernia diameter , 2.4 cm vs 2.3 cm ) . 
 only 2 patients in the stapp group required an additional 5-mm trocar port in the lateral abdomen , to create a better view of the operative field . 
 the mean measured umbilical skin incision length was 13  4 mm in the ntapp group , compared with 27  3 mm in the stapp group ( p < .001 ) . 
 the ntapp procedure required significantly less operating time than the stapp procedure ( ntapp 54.8  16.9 minutes vs stapp 85.9  19.7 minutes ; p < .001 ) . 
 the reduction in operative time was particularly seen in cases of direct hernia ( figure 1 ) . 
 the mean length of postoperative hospital stay was 1.8  0.7 days for the ntapp group and 2.0  0.9 d for the stapp group ( p = .21 ) . by the time of the wound check on postoperative day 3 
 , one patient in the stapp group had a superficial hematoma in the umbilical region that required only conservative treatment . 
 operative time of ntapp and stapp hernia repair . the mean operative time for a direct or an indirect hernia was significantly shorter in ntapp repair ( direct : ntapp 
 .001 ; indirect : ntapp 59.4 minutes vs stapp 88.7 minutes ; p < .001 ) . after a mean follow - up time of 33.5 months for the ntapp group and 30.1 months for the stapp group , 
 40 ntapp patients ( 80% ) and 26 stapp patients ( 74% ) were re - examined in the outpatient clinic . 
 neither inguinal nor port - site hernia occurred in either group . regarding immediate postoperative pain , there was a tendency for longer pain duration in the wound region after the stapp procedure ( ntapp , 5.6  5.5 days vs stapp 9.8  8.4 days ; p = .072 ) . 
 also stapp patients experienced greater wound pain intensity when they began to resume their regular daily activities after hospital discharge ( ntapp 2.7  2.1 vas vs stapp 4.4  1.9 vas ; p = .016 ) . 
 one patient in the ntapp group had chronic discomfort , with paresthesia in the groin region . 
 one patient in the stapp group developed postherniorrhaphy pain syndrome and remained on paid sick leave at home for approximately 2 months . at the time of the follow - up examination , both groups achieved similar results in janda 's clinical test for abdominal wall mobility ( ntapp 4.7  0.5 vs stapp 4.1  1.1 ; p = .101 ) . 
 however , 7 ( 20% ) patients who underwent stapp repair noted a subjective deterioration in abdominal wall mobility in the questionnaire , whereas no patients who underwent ntapp repair had such an experience . 
 cosmetic satisfaction was equally high , with a vas score of 9.7  0.6 in the ntapp group and 9.6  0.7 in the stapp group ( p = .836 ) . 
 sick leave records showed that the ntapp group had a significantly shorter mean period of sick leave , compared with the stapp group ( ntapp 11.2  8.4 days vs stapp 24.1  20.1 days ; p = .02 ) . 
 notably , even with cessation of postoperative wound pain , 27 ntapp patients ( 54% ) and 32 ( stapp patients 91.4% ) remained on sick leave at the follow - up assessment ( p = .045 ) . 
 patient characteristics in the ntapp and stapp groups were comparable at baseline ( male : female 5.4:1 vs 5.3:1 ; age 52.5 y vs 48.3 y ; bmi 25.3 kg / m vs 24.4 kg / m ; direct : indirect : pantaloon hernia , 32:58:10% vs 31.4:54.3:14.3% ; and hernia diameter , 2.4 cm vs 2.3 cm ) . 
 only 2 patients in the stapp group required an additional 5-mm trocar port in the lateral abdomen , to create a better view of the operative field . 
 the mean measured umbilical skin incision length was 13  4 mm in the ntapp group , compared with 27  3 mm in the stapp group ( p < .001 ) . 
 the ntapp procedure required significantly less operating time than the stapp procedure ( ntapp 54.8  16.9 minutes vs stapp 85.9  19.7 minutes ; p < .001 ) . 
 the reduction in operative time was particularly seen in cases of direct hernia ( figure 1 ) . 
 the mean length of postoperative hospital stay was 1.8  0.7 days for the ntapp group and 2.0  0.9 d for the stapp group ( p = .21 ) . by the time of the wound check on postoperative day 3 
 , one patient in the stapp group had a superficial hematoma in the umbilical region that required only conservative treatment . 
 operative time of ntapp and stapp hernia repair . the mean operative time for a direct or an indirect hernia was significantly shorter in ntapp repair ( direct : ntapp 
 47.1 minutes vs stapp 82.5 minutes ; p < .001 ; indirect : ntapp 59.4 minutes vs stapp 88.7 minutes ; p < .001 ) . 
 after a mean follow - up time of 33.5 months for the ntapp group and 30.1 months for the stapp group , 40 ntapp patients ( 80% ) and 26 stapp patients ( 74% ) were re - examined in the outpatient clinic . 
 neither inguinal nor port - site hernia occurred in either group . regarding immediate postoperative pain , there was a tendency for longer pain duration in the wound region after the stapp procedure ( ntapp , 5.6  5.5 days vs stapp 9.8  8.4 days ; p = .072 ) . 
 also stapp patients experienced greater wound pain intensity when they began to resume their regular daily activities after hospital discharge ( ntapp 2.7  2.1 vas vs stapp 4.4  1.9 vas ; p = .016 ) . 
 one patient in the ntapp group had chronic discomfort , with paresthesia in the groin region . 
 one patient in the stapp group developed postherniorrhaphy pain syndrome and remained on paid sick leave at home for approximately 2 months . at the time of the follow - up examination 
 , both groups achieved similar results in janda 's clinical test for abdominal wall mobility ( ntapp 4.7  0.5 vs stapp 4.1  1.1 ; p = .101 ) . 
 however , 7 ( 20% ) patients who underwent stapp repair noted a subjective deterioration in abdominal wall mobility in the questionnaire , whereas no patients who underwent ntapp repair had such an experience . 
 cosmetic satisfaction was equally high , with a vas score of 9.7  0.6 in the ntapp group and 9.6  0.7 in the stapp group ( p = .836 ) . 
 sick leave records showed that the ntapp group had a significantly shorter mean period of sick leave , compared with the stapp group ( ntapp 11.2  8.4 days vs stapp 24.1  20.1 days ; p = .02 ) . 
 notably , even with cessation of postoperative wound pain , 27 ntapp patients ( 54% ) and 32 ( stapp patients 91.4% ) remained on sick leave at the follow - up assessment ( p = .045 ) . 
 in the present study , only patients with uncomplicated inguinal hernias without strangulation or acute pain were included in the investigation , to avoid the possibility that memory of preoperative pain would influence the postoperative pain assessment . also needlescopic trocars and instruments , per se , 
 are not suitable for bowel resections and removal of any extensive tissue from the abdominal cavity . to avoid technical errors and the effects of the learning curve , 
 the operating surgeon in this study was experienced , having performed approximately 20 ntapp repairs with an 8-mm optical trocar and two 5-mm working trocars . before the study 
 , the same surgeon had also performed at least 20 single - incision laparoscopic surgeries for inguinal hernia or cholecystectomy . 
 the results presented in this study should therefore apply to cases involving common hernia and treatment by an experienced surgeon . for the assessment of perioperative surgical trauma to the abdominal wall 
 , we focused on the umbilical skin incision length and total operative time for each procedure . in the ntapp group , a larger ( 8-mm ) umbilical trocar 
 the purpose was to allow for gentle mesh insertion without inadvertent adherence of the folded parietene progrip mesh to itself within the trocar cannula . 
 still , the incisional trauma at the umbilicus in stapp patients was twice that in ntapp patients . 
 although we encountered only 1 stapp patient with postoperative hematoma , there is evidence that a longer surgical skin cut in single - incision laparoscopic surgery is associated with wound complications . 
 furthermore , the operative time for stapp repair of a unilateral hernia ( 85 minutes ) was also significantly longer on average than that required for ntapp ( 54 minutes ) or conventional tapp ( 60 minutes in our previous publication ) repair . 
 the increased operative time for stapp repair indicates an increase in technical difficulty without obvious benefits in surgical capability , as 2 stapp cases ( 5.7% ) in our study still needed an additional working trocar for the creation of a sufficient preperitoneal space . as mentioned in the literature , operative difficulties for single - incision laparoscopic surgery lie in the lack of triangulation and the restricted movement of surgical instruments . in this study , we did not use curved or articulating instruments to improve triangulation in the stapp procedure , as such instruments would add another factor of variation of surgical equipment between the 2 procedures . 
 the smaller jaws and lesser contact area would theoretically exert more force and tear tissue , particularly under the control of the nondominant left hand in our study . 
 some surgeons in our department prefer to perform ntapp repairs with one 5-mm optical trocar and two 5-mm working trocars , with mesh fixation via absorbable tacks and closure of the peritoneum via extracorporeally knotted running sutures . 
 particularly in such procedures , no fascial closure ( a potential source of postoperative wound pain ) was needed for the 5-mm trocar wounds . 
 such ntapp repair variation also seems to be safe and feasible , according to unpublished data . 
 concerning technique variations in single - incision laparoscopic surgery , one study group reported using a single skin incision with 3 separate adjacent fascia punctures in laparoscopic ventral hernia operations . 
 this single - incision multiport technique reduces incisional trauma on the abdominal wall , but preformed proximate fascia defects still have a greater potential to cause a  swiss cheese  hernia . apart from investigating the surgical trauma in ntapp and stapp repair , we investigated postoperative pain , mobility , and cosmesis of the abdominal wall , as well as length of sick leave . 
 we found that the ntapp procedure was able to further reduce immediate postoperative pain in wound regions . 
 these results of wound pain reduction in ntapp repair are in concurrence with available studies comparing pain experienced after needlescopic and conventional laparoscopic inguinal hernia repair . 
 in contrast , a current review referenced 4 available stapp trials and could not verify superiority in pain reduction over conventional tapp repair . 
 however , reduction of postoperative wound pain in patients who underwent ntapp did not significantly improve incidence of chronic groin pain . 
 such pain still occurred in 2% of our ntapp group , compared with 2.8% in the stapp group . 
 moreover , functional mobility of the abdominal wall is the result of intact motor activity and simultaneous load transfer . 
 scar tissues with less loading capacity at the umbilicus in the midline therefore suggests a mechanical mismatch in the latter component and predisposes the patient to a functional impairment of the abdominal wall . despite the greater extent of such scar tissue in the stapp group 
 , the groups regained equal abdominal wall mobility and satisfactory cosmetic results after an approximate observation time of 30 months . 
 taking into account the length of hospital stay added to the subsequent sick leave days , the total time until return to work in the ntapp group was half that in the stapp group . 
 it is also worth noting that more stapp than ntapp patients remained on sick leave , even with an absence of postoperative wound pain . 
 there are nonetheless multiple factors that influence the period of sick leave , including psychological factors related to the larger scar caused by stapp repair . 
 finally , such patient - related outcomes in pain , cosmetic satisfaction , and length of disability are motivation dependent and therefore are subjective measurements . 
 this effect is especially seen in our data on pain duration and sick leave time , with high standard deviations indicating more polarized responses . 
 in a direct comparison of 2 advanced techniques for laparoscopic tapp hernia repair , the ntapp procedure with smaller caliber trocars can further minimize surgical trauma to the abdominal wall tissue . 
 it also requires significantly less operating time than the stapp procedure , as it still adheres to the basic principles of triangulation in laparoscopic technique . in uncomplicated hernia repair , 
 the ntapp procedure has distinct advantages in the immediate postoperative phase in reduced postoperative wound pain and shorter sick leave . 
 however , it offers comparable long - term results in hernia recurrence , abdominal wall mobility , and cosmetic satisfaction . randomized controlled trials comparing ntapp , stapp , and conventional tapp repairs are still needed to validate the benefits of needlescopic surgery .","background and objectives : in recent years , 2 modifications of laparoscopic transabdominal preperitoneal ( tapp ) inguinal hernia repair  needlescopic ( ntapp ) surgery and single - port ( stapp ) surgery  have greatly improved patient outcomes over traditional approaches . for a comparison of these 2 modifications 
 , we sought to investigate and compare the extent of surgical trauma and postoperative consequences for the abdominal wall in these two procedures.methods:in a retrospective study , 50 ntapp and 35 stapp procedures occurring at a community hospital from november 1 , 2009 , through july 31 , 2012 were reviewed . 
 intraoperative data , including length of the umbilical skin incision and operative time , were recorded . 
 a follow - up evaluation included investigation of hernia recurrence , postoperative pain , abdominal wall mobility , cosmetic satisfaction , and period of sick leave.results:the mean umbilical skin incision was 13  4 mm in ntapp vs 27  3 mm in stapp ( p < .001 ) . 
 the ntapp procedure required less operating time than the stapp procedure ( 54.8  16.9 minutes vs 85.9  19.7 minutes ; p < .001 ) . 
 the mean immediate postoperative pain score on the visual analog scale was 2.7  2.1 in the ntapp group and 4.4  1.9 in the stapp group ( p = .016 ) . 
 in addition , patients who underwent ntapp had a shorter period of sick leave ( 11.2  8.4 days vs 24.1  20.1 days ; p = .02 ) . at the follow - up evaluation after approximately 30 months , abdominal wall mobility and cosmetic satisfaction were equally positive , with no hernia recurrence.conclusion:in patients with uncomplicated inguinal hernia , the ntapp procedure , with less surgical trauma and operating time , has distinct advantages in reduction of immediate postoperative pain and sick leave time .",pubmed
"the breathing process , which begins at the upper airway and culminates in gas exchange inside the alveoli , is essential for the human organism to survive . 
 when breathing begins through the nose , the air is prepared in order to reach the lungs with the ideal conditions , that is , warmed , moistened , and filtered , thus activating immunological processes such as mucociliary transport and microbicidal activity that protect the lower airway . 
 when breathing begins through the mouth , despite air warming and moistening , no filtering or immunological activity occurs1 . 
 when mouth breathing occurs in childhood , a period during which intense facial muscular and skeletal growth occurs , it promotes a pathological adaptation of the structures of the stomatognathic system , to the detriment of the usually harmonious morphological and functional growth of these structures2 . 
 the main causes of mouth breathing in childhood are the hypertrophy of the pharyngeal and/or palatine tonsil , nasal mucosa and turbinate edema , nasal septal deviation , extended suction habits , and others3 . 
 morphological adaptations in mouth - breathing children occur with the aim of facilitating the necessary arrival of the air to the alveoli . 
 thus , it is possible to observe maxillary hypoplasia and posterior mandibular demotion / rotation , which lead to dental occlusion alterations , higher mandibular inclination , and a vertical facial growth pattern , with alterations in normal facial proportions and hard palate elevation , head anteriorization , and muscular deharmonization , occurring mainly in the nasofacial region . 
 these adaptations generate functional changes in the stomatognathic system , which are observed through alterations in speech , chewing , and deglutition4 
 6 . 
 the complexity of the consequences of mouth breathing that are associated with various etiologies justifies the participation of several professionals , including otorhinolaryngologists , odontologists , phonoaudiologists , physiotherapists , pediatricians , among others , along the different phases of the care of mouth - breathers , including the diagnosis , treatment , rehabilitation , and the prevention of mouth breathing in childhood . 
 multidisciplinary staff integration is essential , as it is desirable to use a uniform categorization of mouth breathers , consisting of the same terminology and the same complementary exams . 
 accurate diagnosis of the cause of mouth breathing is essential to the effectiveness of treatment . in phonoaudiology , the type of care that is used for mouth - breathing patients is typically determined by the etiology of the altered respiratory mode . 
 therefore , the accurate use of the available diagnostic options contributes to early diagnosis , and will aid in the establishment of a multidisciplinary therapeutics that are more appropriate to each case and minimize relapse during mouth breathing rehabilitation7 
 8 . 
 most causes of mouth breathing are diagnosed through a disarmed otorhinolaryngologic exam , except for the diagnosis of hyperplasia of the pharyngeal tonsils , which demands that complementary exams be used3 . in the diagnosis of pharyngeal hyperplasia , 
 nasopharyngoscopy is a procedure that is used to visualize the pharyngeal tonsil and its relation to other nasopharyngeal structures , and is considered by many authors as the most reliable exam in the diagnosis of nasopharyngeal obstruction9 
 10 
 11 
 12 
 13 . 
 cephalometry is an exam that is similar to cavum radiography ; however , it is carried out through the use of a cephalostat , which enables the most appropriate patient positioning , and provides data related to craniofacial growth and the myofunctional status of these structures14 . since it is a noninvasive exam 
 , it is more comfortable for the patient , and is often more accepted by children . 
 moreover , is associated with a lower cost and greater availability relative to nasopharyngoscopy . with the aim of contributing to the accurate diagnosis of mouth breathing , 
 this study was carried out with the purpose of verifying the correlation between nasopharyngoscopy and cephalometry in the diagnosis of hyperplasia of the pharyngeal tonsils . 
 the preset study was approved by the research ethics committee of the institution of origin under the protocol number 220.0.243.000 - 8 . 
 the children agreed to take part in the study and the informed consent forms were signed by their representatives . the study sample consisted of children from 3 public schools who were evaluated from september 2008 through december 2009 . 
 the inclusion criteria were included complaints related to mouth breathing , such as night drooling , snoring , and restless sleep ; the group age ranged from 7.0 to 11.11 years . 
 children that presented evident neurological disorders , craniofacial malformation , syndromes , or history of pharyngeal surgery were excluded from the study . 
 after employing the inclusion and exclusion criteria , 55 children , including 25 males and 30 females , with an average age 9.8 years were selected . 
 these children underwent otorhinolaryngologic assessment , as well as nasopharyngoscopy and cephalometry to check for the presence or absence of nasopharyngeal obstruction . 
 any other causes of obstruction of the upper airway , such as nasal septal deviation , primary hypertrophy of the nasal turbinates , polyps , and malformations were not diagnosed in the study sample . for the patients with bacterial rhinosinusitis and/or symptomatic allergic rhinitis , 
 appropriate treatments were prescribed and subsequent revaluation was carried out during a period ranging from 30 to 60 days . after this period was completed , the nasopharyn - goscopy and cephalometry exams were performed . 
 otorhinolaryngologic evaluation was carried out in the presence of and with the help of the parents or sponsors . 
 after a general and specific otorhinolaryngologic anamnesis was conducted , giving emphasis to the aspects related to mouth / nasal breathing , a physical exam consisting of otoscopy , anterior rhinoscopy , cervical palpation , and nasoscopy was carried out . 
 this evaluation allowed for the determination of whether the patient had either an oral or mouth - breathing mode . immediately after the completion of the anamnesis and the otorhinolaryngologic clinical exam , 
 nasopharyngoscopy was carried out , always by the same assessor and in the same environment , employing topical anesthesia ( lidocaine 5% ) and a nasal vasoconstrictor ( oxymetazoline 0.05% ) , with a flexible nasofibroscope ( 3.2 mm mashida brand , microcamera asap ) , and recorded on dvd . through this exam , 
 apart from studying the size and the relation of the pharyngeal tonsil to the nasopharynx , the nasal septum positioning , turbinate size , upper pole of the palatine tonsils , presence of secretions , and other kinds of lesions in the nasal cavity and hypopharynx were assessed . 
 when using nasopharyngoscopy to determine the level of hyperplasia of the pharyngeal tonsils , we used a graded scale in which the relation of the pharyngeal tonsils to the other structures that are present in the nasopharynx was assessed15 , as follows : grade 1tonsil without contact with the nasopharyngeal structures ( figure 1 ) ; grade 2tonsil in contact with the torus tubarius ( figure 2 ) ; grade 3tonsil in contact with the torus tubarius and vomer ( figure 3 ) ; grade 4tonsil in contact with the torus tubarius , vomer , and the soft palate at rest ( figure 4 ) . 
 grade 1 hyperplasia of the pharyngeal tonsils by nasofibropharyngoscopy and cephalometry ( cephalometric analysis ) . 
 grade 2 hyperplasia of the pharyngeal tonsils by nasofibropharyngoscopy and cephalometry ( cephalometric analysis ) . 
 grade 3 hyperplasia of the pharyngeal tonsils by nasofibropharyngoscopy and cephalometry ( cephalometric analysis ) . 
 grade 4 hyperplasia of the pharyngeal tonsils by nasofibropharyngoscopy and cephalometry ( cephalometric analysis ) subsequently , children were referred to the odontological radiology service center to undergo a cephalometric assessment , through which it was possible to obtain a left profile cranium teleradiograph by using the cephalostat for positioning the patient at a distance of 1.5 meters . for the standard lateral radiography assessment , 
 computerized cephalometric tracing was performed with cef x software ( a computerized cephalometry system version 2.4.0.0 from cdt software for informatics consulting , development , and training ) . 
 the exam was carried out with the same equipment in each patient , and was analyzed by the same professional expert in odontological radiology . when using cephalometric analysis , in order to determine the level of hyperplasia of the pharyngeal tonsils , we used a graded scale in which the airway between the palate pharynx border and the point that was most proximal to the pharynx was measured in millimeters16 , as follows : grade 1nasopharyngeal space larger than 6  mm ( figure 1 ) ; grade 2nasopharyngeal space between 4.1 and 6  mm ( figure 2 ) ; grade 3nasopharyngeal space between 2.1 and 4  mm ( figure 3 ) ; grade 4nasopharyngeal space between 0 and 2  mm ( figure 4 ) . in order to verify the correlation between the nasopharyngoscopy and cephalometry examinations , 
 the test results in a correlation coefficient ranging from -1 to + 1 , with zero indicating that there is no correlation , -1 indicating a perfect negative correlation , and + 1 denoting a perfect positive correlation ; the closer to 1 or + 1 , the greater the correlation between the variables that are being tested . the possible results for a positive correlation are17 : 0 when there is no correlation ; between 0 and 0.3 - poor correlation ; between 0.3 and 0.6 - regular correlation ; between 0.6 and 0.9 - a very strong correlation ; equal to 1a perfect correlation between the data . 
 for calculating the sensitivity , specificity , the positive predictive value , and the negative predictive value of radiologic exams , nasopharyngoscopy was considered to be the reference exam ( the gold standard ) . 
 it was also necessary to establish limit values in order to determine whether the exams that were studied were either positive or negative . 
 therefore , the grade 1 and 2 nasopharyngoscopic and cephalometric exams were considered to be negative , and the grade 3 and 4 exams were considered to be positive . 
 table 1 shows the results of the correlation analysis between the nasopharyngoscopy and cephalometry examinations in diagnosing the grade of hyperplasia in the pharyngeal tonsils . 
 n  =  number of children ; p  =  significance level at 5% ; r  =  spearman 's rank correlation coefficient . from the analysis of the results presented in table 1 , it was possible to verify that the spearman 's rank correlation coefficient , rho ( r ) , was equal to 0.52 . 
 this result indicates a significant regular and positive association between the nasopharyngoscopy and cephalometry assessments ( p  =  0.000 ) at a significance level of 5% . 
 table 2 describes the nasopharyngoscopy and cephalometry distribution results that were used for assessing the diagnostic tests . in table 2 , using nasopharyngoscopy as the gold standard for the diagnosis of hyperplasia of the pharyngeal tonsils , it was possible to verify that the sensitivity of cephalometry ( determined by the proportion between the number of correct positive radiographic cephalometry diagnoses and the total number of positive cases ) was 35% . 
 the negative predictive value of cephalometry ( determined by the proportion between the number of correct negative diagnoses and the total number of negative cases ) was 97% . 
 the positive predictive value of the cephalometric exam ( determined by the ratio of the number of correct positive cephalometric diagnoses and the total number of positive cephalometric diagnoses ) was found to be 87% . 
 the negative predictive value ( determined by the ratio of the number of correct negative cephalometric diagnoses and the total number of negative cephalometric diagnoses ) was 72% . 
 hyperplasia of the pharyngeal tonsils is one of the main causes of upper airway obstruction . for this reason , 
 studies have been carried out with the objective of assessing the reliability of the available diagnostic procedures that are used for the detection of nasopharyngeal obstruction resulting from the increased size of the pharyngeal tonsils . 
 the most commonly used exams are cavum radiographs , flexible and rigid nasopharyngoscopy , and cephalometry11 
 13 
 18 . 
 reports in the literature indicate that there is concern regarding the best method for diagnosing and treating children in which hyperplasia of pharyngeal tonsils is suspected , which a frequently occurring situation in the otorhinolaryngological practice13 
 19 . 
 therefore , this study aimed to establish a correlation between the nasopharyngoscopy and cephalometry examinations in the diagnosis of hyperplasia of the pharyngeal tonsils , while highlighting their main contributions . 
 the findings of this study demonstrated that it was possible to verify a regular and positive correlation , r  =  0.52 ; however , a perfect correlation between the exams was not found . 
 the results revealed that most of the children showed grade 2 and 3 hyperplasia of the pharyngeal tonsils , followed by grade 1 in the nasopharyngoscopic exam , and most of the children showed grade 1 hyperplasia of the pharyngeal tonsils , followed by grade 2 in the cephalometric assessment ( table 1 ) . after analyzing these results , it was possible to infer that cephalometry , as compared to nasopharyngoscopy , presented a tendency to underestimate the pharyngeal tonsil size in the nasopharynx . 
 the sensitivity of cephalometric exam , which indicates the probability of a positive result when the patient presents with the condition in question , was 35% . the negative predictive value , which expresses the probability that the individual does not present with the condition in question when the exam result is negative , was 72% . 
 these results suggested that a negative cephalometric exam might not provide a proper assessment of the nasopharynx when compared to the nasopharyngoscopic exam . 
 similar results were obtained when cavum radiography and flexible nasopharyngoscopy for the assessment of the grade of nasopharyngeal obstruction were compared in other similar study13 . 
 the authors concluded that children with classical symptoms of respiratory obstruction , in the absence of hypertrophy of the pharyngeal tonsils by a radiological exam , should undergo flexible nasopharyngoscopy in order to achieve a more accurate diagnosis . 
 several published studies consider nasopharyngoscopy to be the gold standard exam for assessing the nasopharynx12 
 13 
 20 
 21 
 22 . 
 this is justified , among other reasons , by the dynamic and tridimensional nasopharyngeal view provided by this assessment , which makes it possible to analyze not only the pharyngeal tonsil size , but also its relation with other structures in the region . 
 this may explain the reason why , in the comparison between nasopharyngoscopy and cephalometry , the latter tended to underestimate the size and position of the pharyngeal tonsils in relation to the nasopharynx . 
 frequently in clinical practice , it is possible to verify that patients with mouth breathing symptoms who show normal or next to normal pharyngeal tonsil size with radiological exams , may present hyperplasia of the pharyngeal tonsils and other alterations , such as hypertrophy of the lower turbinate and posterior nasal septal deviation , when assessed by nasopharyngoscopy . 
 this statement is reinforced by another study11 in which 45 children between 4 and 12 years of age were assessed with nasopharyngoscopy ; all of them presented chronic nasal obstruction due to hyperplasia of the pharyngeal tonsils , and cavum radiography revealed no alterations . 
 the results demonstrated that 27% ( 17 cases ) of the pharyngeal tonsils were estimated to be large , 42% ( 24 cases ) of the pharyngeal tonsils were of moderate size , and 31% ( 19 cases ) of the pharyngeal tonsils were small . 
 furthermore , the exams detected 8 cases of hypertrophy of the lower turbinate ( 13.3% ) and 4 cases of posterior nasal septal deviation ( 6.6% ) . 
 these findings reinforced the importance of the indication of nasopharyngoscopy for children with nasal obstruction and normal radiological exams , as this exam allows direct , tridimensional , and dynamic assessment of the cavum area . 
 on the other hand , another study23 that verified the efficacy of lateral cephalometric radiography in the diagnosis of hyperplasia of the pharyngeal tonsils compared with nasal endoscopy , differs from the results of the present study , as the authors substantiated that lateral cephalometric radiography proved to be an effective exam because of its sensitivity , specificity , and high positive and negative predictive values for diagnosis . 
 they concluded that in spite of the superiority of nasopharyngoscopy in assessing the nasopharynx , a cephalometric exam that was negative for hypertrophy of the pharyngeal tonsils would be sufficient to exclude this pathology . 
 based on information that was derived from 3 units that comprise the sus ( sistema nico de sade ) , another study reported that mouth breathing is the most frequently cited complaint and that cavum radiography was the most commonly requested exam by otorhinolaryngologists19 . from the results of the present study , it can be inferred that if only the radiographic exam is used for the detection of hyperplasia of the pharyngeal tonsils , a proper mouth - breather diagnosis and treatment might be compromised , as in some cases the grade of pharyngeal tonsil obstruction was underestimated relative to the nasopharyngoscopic exam . 
 when not treated in childhood , mouth breathing causes many alterations , including craniofacial , dental , and phonoarticulatory changes , as well as alterations in body posture , oral functions , sleep , nutrition , behavior , and others24 . 
 all of these alterations that are presented by mouth breathers require multidisciplinary intervention , including physicians , phonoaudiologists , odontologists , physiotherapists , among others1 . 
 although cephalometry was only regularly correlated with nasopharyngoscopy in diagnosing mouth breathing , that exam was able to complement the endoscopic exam , thus providing information regarding facial growth that could be documented and monitored . 
 this information will be useful for other professionals involved in the care of mouth - breathing patients . 
 cephalometry provides early identification of many alterations that are present in mouth - breathing patients and provides the otorhinolaryngologist with information about the nasopharynx , informs the phonoaudiologist about morphological alterations of the stomatognathic system , and informs the odontologists about facial growth and dental occlusion3 
 14 
 23 . 
 therefore , cephalometry is useful not only for the initial diagnosis , but also in cost reduction and promoting collaboration between the multidisciplinary staff members that are involved in the care of mouth - breathing patients . a research study comparing cavum radiography with cephalometry found that in the first exam 61% of the patients were incorrectly positioned25 . 
 importantly , the cephalostat is used in cephalometry to control the positioning of the patient , which explains the superiority of this exam compared to cavum radiography . 
 although nasopharyngoscopy is the gold standard exam for nasopharyngeal assessments , cephalometry is a complementary exam that provides information about craniofacial growth , thus making it possible to document and monitor the complex morphofunctional modifications related to mouth breathing . in this study , 
 cephalometry showed a tendency to underestimate the size of pharyngeal tonsil compared with nasopharyngoscopy ; therefore , we suggest that patients presenting with mouth - breathing symptoms and normal radiography be referred for a complementary assessment with nasopharyngoscopy , which is associated with greater diagnostic accuracy due to its tridimensional and dynamic nature . 
 nasopharyngeal assessment of children can be performed with nasopharyngoscopy and cephalometry , as these exams are associated with a regular and positive correlation . 
 however , we found that cephalometry tends to underestimate the size of the pharyngeal tonsil compared with nasopharyngoscopy . 
 therefore , nasopharyngoscopy is suggested when the cephalometry results for nasopharyngeal obstruction are negative in patients with mouth - breathing symptoms .","summary 
 introduction :  hyperplasia of the pharyngeal tonsil is one of the main causes of mouth breathing , and accurate diagnosis of this alteration is important for proper therapeutic planning . 
 therefore , studies have been conducted in order to provide information regarding the procedures that can be used for the diagnosis of pharyngeal obstruction . 
 objective :  to verify the correlation between nasopharyngoscopy and cephalometric examinations in the diagnosis of pharyngeal tonsil hyperplasia . 
 method :  this was a cross - sectional , clinical , experimental , and quantitative study . 
 fifty - five children took part in this study , 30 girls and 25 boys , aged between 7 and 11 years . 
 the children underwent nasofibropharyngoscopic and cephalometric evaluation to determine the grade of nasopharyngeal obstruction . 
 the spearman 's rank correlation coefficient at the 5% significance level was used to verify the correlation between these exams . 
 results :  in the nasopharyngoscopy evaluation , most children showed grade 2 and 3 hyperplasia of the pharyngeal tonsil , which was followed by grade 1 . in the cephalometry assessment , most children showed grade 1 hyperplasia of the pharyngeal tonsil , which was followed by grade 2 . a statistically significant regular positive correlation was observed between the exams . 
 conclusion :  it was concluded that the evaluation of the pharyngeal tonsil hyperplasia could be carried out by fiber optic nasopharyngoscopy and cephalometry , as these examinations were regularly correlated . 
 however , it was found that cephalometry tended to underestimate the size of the pharyngeal tonsil relative to nasopharyngoscopy .",pubmed
"the incidence of ipsilateral breast tumor relapse ( ibtr ) in patients previously treated for breast carcinoma through breast - conserving surgery , systemic adjuvant therapy and radiation therapy , is estimated to be less than 6% . however , as each case has different clinical relevance ( evolution , prognosis and therapeutic management ) , it is necessary to distinguish in these patients between a true local recurrence ( tr ) of a previous tumor , and the appearance of a new primary tumor ( npt ) [ 1 , 2 , 3 , 4 ] . to do this 
 , we must study the location of the new tumor ( distance from the previous tumor ) , staging ( by imaging and biopsy ) and determine their histological subtype ( through immunohistochemical studies , and the determination of her-2 and hormone receptor expression ) . 
 a 35-year - old premenopausal woman with no relevant medical history of interest , except for a family history of a maternal cousin with breast cancer at 28 years , was referred to the oncologic gynecology department by the palpation of a 1-cm nodule in the right axilla . 
 fine needle aspiration cytology was performed , with the pathological result of suspicion of malignancy . in february 2004 
 the pathological diagnosis was moderately differentiated invasive ductal carcinoma ( idc ) of the right breast of 0.8 cm in size , with no metastasis detected in 20 lymph nodes removed ( pt1 pn0 m0 ) . 
 immunohistochemical study of the tumor cells showed positive staining for both estrogen receptors ( er ) and progesterone receptors ( pr ) in 50 and 15% , respectively , and showed negative membrane staining of her-2 marker . 
 fluorescence in situ hybridization ( fish ) analysis found no her-2 amplification in the primary tumor . 
 the computer tomography performed after surgical intervention revealed no other organs compromised or distant metastatic extension . 
 preoperative serum levels of the marker ca 15 - 3 were normal . with this diagnosis , the patient was referred to our medical oncology department where she received postoperative adjuvant chemotherapy consisting of 4 cycles of adriamycin 60 mg / m and cyclophosphamide 600 mg / m every 3 weeks . 
 then , adjuvant radiotherapy ( 50 gy ) was performed on right - breast residual tissue , followed by adjuvant hormone therapy with tamoxifen 20 mg daily during the following 4 years . 
 meanwhile , the patient continued routine follow - up visits in our department . in january 2008 ( 
 4 years after surgery ) , the patient noticed the presence of periareolar thickening in the right breast with redness and heat . in the physical examination 
 a mammography showed the presence of breast architectural distortion with high suspicion of malignancy ( birads-4 ) . 
 the result of the biopsy of the lesion confirmed the presence of an idc consistent with the diagnosis of inflammatory breast cancer ( pt4 nx m0 ) in the same breast where the previous tumor was treated . 
 immunohistochemical study of biopsy sample showed negative staining for both er and pr , and showed positive membrane staining of her-2 marker . 
 then , the patient received treatment with a weekly schedule of paclitaxel ( 80 mg / m ) plus carboplatin ( auc = 2 ) 3 weeks on and 1 week off , in combination with weekly trastuzumab ( initially 4 mg / kg followed by 2 mg / kg every week ) . 
 she received a total of 11 cycles of chemotherapy schedule and 15 cycles of trastuzumab . during that time period , 
 , there was a progressive decrease of inflammatory signs , with disappearance of the redness and heat from the affected area . 
 after surgery , the patient received adjuvant chemotherapy consisting of 8 cycles of weekly paclitaxel ( 80 mg / m ) in combination with weekly trastuzumab ( 2 mg / kg ) , followed by trastuzumab monotherapy ( 6 mg / kg every 3 weeks ) up to 1 year of treatment . 
 later , the patient underwent breast reconstruction surgery , and currently , after 4 years and 8 months of follow - up after the last surgery , the patient remains in complete clinical remission , developing her life normally . 
 however , given the age of the patient at the time of first diagnosis and due to the presence of a second primary tumor in the same breast , a genetic study was performed with negative results for currently known breast cancer - associated genetic mutations . 
 breast cancer is a major public health problem for women throughout the world , and idc is the most frequent form of invasive breast cancer . 
 it accounts for 7080% of all cases of invasive carcinomas , and , in global , is the histologic type of breast cancer with the worst prognosis of all [ 6 , 7 ] . 
 its treatment is based on a multidisciplinary approach consisting in primary tumor surgery , adjuvant chemotherapy and radiotherapy , and hormone therapy if indicated . currently , the treatment of choice for idc is a combination therapy based on neoadjuvant chemotherapy , targeted therapy , surgery , adjuvant chemotherapy , hormone therapy and radiation therapy . 
 numerous randomized prospective studies have shown that breast - conserving treatment in idc is as effective as mastectomy in terms of overall survival , disease - free survival and long - term disease - free survival [ 8 , 9 ] . 
 inflammatory breast carcinoma is a rare and very aggressive form of locally advanced breast carcinomas . 
 it represents 0.55% of primary invasive breast tumors , being more common in caucasian women under 60 years . in these cases , it is important to make a differential diagnosis with locally advanced 
 noninflammatory breast carcinomas that subsequently evolve with inflammation , as well as other non - neoplastic diseases ( mastitis and breast abscess ) , by using biopsies and imaging tests to confirm the differential clinical diagnosis . 
 after breast - conserving treatment , ibtr may represent two distinct types of lesion that it is important to define , a tr or an npt . 
 tr and npt have different natural histories , prognosis , and in turn different implications for therapeutic management [ 1 , 2 , 3 , 4 ] . in a retrospective study , designed by bouchardy et al . 
 , about second primary contralateral breast cancer , it is concluded that women with er - positive first tumors have a decreased risk of second breast cancer occurrence ( standardized incidence ratio ( sir ) : 0.67 ; 95% confidence interval ( ci ) , 0.480.90 ) , whereas patients with er - negative primary tumors have an increased risk limited to er - negative second tumors ( sir : 7.94 ; 95% ci , 3.8114.60 ) . 
 patients with positive family history had a tenfold higher risk of er - negative second tumor which increased to nearly 50-fold when the first tumor was er negative . 
 so the risk of second er - negative breast cancer is really very high after a first er - negative tumor , particularly in women with strong family history . 
 certain studies provide additional evidence on differences between er - positive and er - negative breast cancer , not only in presentation , prognosis , and treatment , but also in etiology and natural history . 
 overall , the risk of developing a second breast cancer among women diagnosed with a first breast cancer of any er status was similar to the risk of developing a first breast cancer in the general population . 
 but it is important to analyze the first breast cancer by means of immunohistochemical markers , because risk of a second tumor depends on er status , period of diagnosis , and family history of breast and/or ovarian cancer ; and consequently , surveillance and prevention of second cancer occurrence should consider these risk factors . the risk of a second breast cancer is the same whether the patient had previously underwent a mastectomy or bct . and 
 with bct , the risk for developing a second primary breast cancer in the preserved breast is similar to the contralateral one . unlike disease recurrence 
 , second primary breast cancer often occurs after the first 36 months , with an average range from 34 to 60 months , requiring a long - term follow - up . 
 secondary malignancies of the breast are rare with a reported frequency of 0.4 to 2.16% . according to the national surgical adjuvant breast and bowel project ( nsabp ) , there is a 14.3% cumulative incidence of ibtr over 20 years since primary operation . 
 ibtr can be defined as the re - emergence of tumor in the previously treated breast . 
 ibtr rate is under 1% per year , but it is about 5 to 10% at 5 years and 10 to 15% at 10 years . when evaluating ibtr , it is important to consider whether it is a tr or an npt . on the one hand , 
 tr are cases consistent with the regrowth of malignant cells not removed by surgery or not killed by radiotherapy , as veronesi et al .   defined them . on the other hand , 
 npts are de novo cases of malignancies arising from mammary epithelial cells of the residual breast tissue [ 4 , 8 ] . 
 the complex behavior of ibtr may be related to the fact that the ibtr patient population is composed by these two different entities . 
 each ibtr can be classified as either tr or as npt based on the following criteria : according to pathologic features , npt is designated when it is a distinct histology type ( e.g. from an infiltrating ductal carcinoma to an infiltrating lobular , tubular , or medullar carcinoma ) , or when it has a change from a more invasive to a less invasive carcinoma . 
 by contrast , a change in histology from ductal carcinoma in situ to an infiltrating ductal carcinoma is considered as tr because it is consistent with the natural progression of the disease . depending on whether the relapse occurs at or near the original site of the primary tumor . 
 a tumor is classified as npt if it changes from an aneuploid primary to a diploid relapse ; otherwise , it would be considered as a probable progression representing the natural history . 
 the best way to differentiate npt and tr would be genetic sequencing ( to establish true clonality ) . 
 recently , some molecular techniques such as dna finger printing , loss of heterozygosity pattern or allelic imbalances profile have been used to distinguish npt from tr , but the classification criteria are not standardized yet . 
 patients with npt were significantly younger at initial diagnosis than those who experienced tr . however , there are no age differences at relapse . 
 the rate of np in the ipsilateral breast does not differ significantly from the rate of primary contralateral tumors . 
 currently , salvage mastectomy is the standard treatment for all types of ibtr , which provides locoregional control in 90% of patients . 
 this recommendation is based on an elevated risk of further in - breast relapse with conservative surgeries . 
 but , there is no conclusive evidence of its superiority compared to conservative surgery , the number of repeat lumpectomies is small and the follow - up is not long enough to draw definitive conclusions . 
 further studies are needed to determine the indications . after the surgical management of ibtr , the optimal systemic therapy is also unknown . 
 tr has a poor prognosis in terms of survival rates and development of other metastases , and may benefit from more aggressive adjuvant treatment , with additional radiotherapy , hormone therapy and chemotherapy . 
 by contrast , patients with npt generally have a favorable prognosis , and therapeutic decisions concerning systemic therapy should be similar to those in patients with de novo breast carcinoma , according to the equivalent stage . however , because of the higher risk of developing contralateral breast carcinoma ( genetic predisposition ) there is a need for better chemoprevention strategies . 
 two trials have shown that patients with npt benefit from adjuvant tamoxifen , if indicated , because it reduces contralateral and ipsilateral disease recurrence with minimal side effects . 
 due to declining mortality rates that are , in part , attributable to the use of screening mammography and effective adjuvant therapy , more women are surviving their breast cancer . 
 the care of breast cancer survivors is an important issue that requires an understanding of relapse patterns , establishing appropriate follow - up visits and screening tests . 
 nowadays , there exists a good follow - up among cancer survivors , with higher screening rates . 
 according to pathologic features , npt is designated when it is a distinct histology type ( e.g. from an infiltrating ductal carcinoma to an infiltrating lobular , tubular , or medullar carcinoma ) , or when it has a change from a more invasive to a less invasive carcinoma . by contrast , a change in histology from ductal carcinoma in situ to an infiltrating ductal carcinoma is considered as tr because it is consistent with the natural progression of the disease . 
 depending on whether the relapse occurs at or near the original site of the primary tumor . 
 a tumor is classified as npt if it changes from an aneuploid primary to a diploid relapse ; otherwise , it would be considered as a probable progression representing the natural history . 
 the best way to differentiate npt and tr would be genetic sequencing ( to establish true clonality ) . 
 recently , some molecular techniques such as dna finger printing , loss of heterozygosity pattern or allelic imbalances profile have been used to distinguish npt from tr , but the classification criteria are not standardized yet . 
 patients with npt were significantly younger at initial diagnosis than those who experienced tr . however , there are no age differences at relapse 
 . the rate of np in the ipsilateral breast does not differ significantly from the rate of primary contralateral tumors . 
 currently , salvage mastectomy is the standard treatment for all types of ibtr , which provides locoregional control in 90% of patients . 
 this recommendation is based on an elevated risk of further in - breast relapse with conservative surgeries . 
 but , there is no conclusive evidence of its superiority compared to conservative surgery , the number of repeat lumpectomies is small and the follow - up is not long enough to draw definitive conclusions . 
 further studies are needed to determine the indications . after the surgical management of ibtr , the optimal systemic therapy is also unknown . 
 tr has a poor prognosis in terms of survival rates and development of other metastases , and may benefit from more aggressive adjuvant treatment , with additional radiotherapy , hormone therapy and chemotherapy . 
 by contrast , patients with npt generally have a favorable prognosis , and therapeutic decisions concerning systemic therapy should be similar to those in patients with de novo breast carcinoma , according to the equivalent stage . 
 however , because of the higher risk of developing contralateral breast carcinoma ( genetic predisposition ) there is a need for better chemoprevention strategies . 
 two trials have shown that patients with npt benefit from adjuvant tamoxifen , if indicated , because it reduces contralateral and ipsilateral disease recurrence with minimal side effects . 
 due to declining mortality rates that are , in part , attributable to the use of screening mammography and effective adjuvant therapy , more women are surviving their breast cancer . 
 the care of breast cancer survivors is an important issue that requires an understanding of relapse patterns , establishing appropriate follow - up visits and screening tests . 
 nowadays , there exists a good follow - up among cancer survivors , with higher screening rates .","after breast - conserving treatment , the occurrence of ipsilateral breast tumor relapse raises the concern regarding whether it may represent two distinct types of lesion that it is important to define , a true recurrence ( tr ) or a new primary tumor ( npt ) . 
 tr and npt have different natural histories , prognosis , and in turn different implications for therapeutic management . 
 we report the case of a 35-year - old woman who developed a breast invasive ductal carcinoma , which after receiving breast - conserving treatment with adjuvant chemotherapy , radiotherapy and hormone therapy , developed four years after an inflammatory carcinoma in the same breast , with different expression of immunohistochemical markers than the first breast cancer . 
 the patient was treated with neoadjuvant chemotherapy that allowed the realization of a radical mastectomy with a complete pathological response . 
 we describe the diagnostic and therapeutic approach of ipsilateral breast tumor relapses , along with a review of existing literature .",pubmed
"age - related macular degeneration ( amd ) is a leading cause of severe vision loss in people aged 55 years and older in developed countries.1,2 the neovascular form of amd , involving choroidal neovascularization ( cnv ) associated with fluid exudation or bleeding , accounts for the majority of cases of amd - related severe central vision loss.3 upregulation of angiogenic factors , including vascular endothelial growth factor ( vegf ) , has been implicated in the development and progression of amd.4 the management of neovascular amd has been transformed over the past decade with the development and advent of intravitreal anti - vegf treatment , which is significantly more effective in preventing progressive vision loss from neovascular amd than previous alternative treatment modalities , including laser photocoagulation and photodynamic therapy . 
 moreover , anti - vegf therapy has been shown to significantly improve visual acuity ( va ) in many patients with neovascular amd or other retinal diseases.5 current management of neovascular amd involves the use of continuing anti - vegf treatment to control signs of exudation by blocking the growth of abnormal blood vessels to prevent vision loss and potentially improve va . 
 intravitreal anti - vegf agents ranibizumab ( lucentis ; novartis pharmaceuticals canada inc , dorval , qc , canada ) and aflibercept ( eylea ; bayer inc , mississauga , on , canada ) are health canada - approved options for the treatment of neovascular amd . 
 large multicenter clinical trials have demonstrated that monthly ranibizumab , a recombinant vegf - specific monoclonal antibody fragment , prevents vision loss in most patients with neovascular amd , with significant visual gain achieved in some.6,7 the phase 3 randomized view clinical studies demonstrated that intravitreal aflibercept injection every 8 weeks , following 3 initial monthly injections , was noninferior to monthly ranibizumab , which was measured by the primary end point of proportion of subjects who maintained vision ( loss of < 15 letters compared to baseline ) at week 52.8 intravitreal aflibercept injection is a recombinant fusion protein of key domains from human vegf receptors 1 and 2 with the constant region ( fc ) of human immunoglobulin g. aflibercept binds to both vegf - a and proangiogenic placental growth factor . 
 the binding affinity of aflibercept to vegf is substantially higher than that of ranibizumab,9 with a greater potency in vitro and a longer duration of action allowing potentially for less frequent dosing.8 research findings in eyes with neovascular amd demonstrate a mean duration of complete vegf suppression of 36.4 days for ranibizumab compared with ~70 days for aflibercept injection.10,11 switching treatment - resistant neovascular amd patients to aflibercept is an option that may improve visual and anatomic outcomes.12,13 assessment of the overall interaction with the external environment through the visual system is measured using various methods , including va ( 100% contrast , early treatment diabetic retinopathy study [ etdrs ] , and snellen charts ) , contrast sensitivity ( cs ) testing ( 100%0.6% contrast ) , humphrey visual field test , and color vision ( eg , isihara charts , farnsworth dichotomous test ) . 
 the ability to perform everyday visual tasks , such as reading , recognizing faces , driving , telephone use , and using household appliances , depends on visual components beyond high - contrast visual distance acuity , including cs , near vision , color vision , and sensitivity to glare . 
 the snellen eye chart is primarily used to measure va and is depicted as a series of high contrast black - on - white letters in different sizes . 
 relatively small changes in refractive status can be detected by this test , and it is a useful standard for defining vision changes caused by spherical blur . as various types of vision loss are not caused by spherical blur ( eg , cataracts , glaucoma , and irregular astigmatism ) , the snellen va test may not be an adequate or appropriate measure in all cases . 
 cs measures an individual s ability to detect low contrast images and to perceive differences between light and dark , whereas the snellen va chart test measures the level at which an individual can identify fine detail at high contrast using black - on - white letters . 
 using low contrast images , the cs method can detect subtle vision changes that may be concealed by acuity.14 cs could be considered as an adjunct to standard acuity testing , to better assess visual capability , as well as being a useful measure for evaluating a wide range of ocular conditions ( eg , diabetic eye disease , macular degeneration , cataracts , glaucoma , traumatic brain injury , amblyopia , and optic nerve disorders).14 cs is a sensitive and arguably essential outcome end point for a complete assessment of visual function and visual performance , including driving , mobility , reading speed , and general vision - related quality of life.15 additionally , cs has potential applications when measuring the vision of patients with high visual requirements , including athletes and public service personnel . cs charts that utilize letters are familiar to both the patient and the practitioner , in comparison to other nonletter - based cs charts.16 the pelli 
 robson cs test is considered a quick and reliable method in clinical settings.17,18 patients scoring below 1.65 log on the pelli 
 robson cs test are considered to have impaired visual function , with a 6-letter change in cs equating to a 15-letter ( or 3 lines ) change in va.19 both va and cs are associated with the ability to perform daily vision - related activities20 and are considered as significant independent parameters in the determination of visual impairment and quality of life . however , in clinical studies , change in va over time is commonly used as the main primary outcome measure as an effective and reliable indicator of functional deficit or improvement . 
 when measuring va , single optotypes ( usually letters , numbers , or geometric symbols ) with high contrast are presented to the patient ; however , the environment is not always seen in high contrast as presented on a standard acuity chart . 
 therefore , using cs determination to measure the ability to recognize low contrast patterns may detect functional impairment not evident when measuring va only.21 study results have indicated that va and cs do not reliably demonstrate the same parallel progression in visual function loss , although they show a moderate correlation in eyes with neovascular amd.21 both parameters provide important and supplementary information about visual disability,21 supporting the role of multiple visual end point evaluations in interventional studies.21,22 to better assess the overall visual functioning following anti - vegf treatment switch , this study was conducted to determine and evaluate change from baseline to week 12 in pelli 
 robson cs as the primary clinical end point in patients with recalcitrant exudative amd , despite previous ranibizumab treatment who were switched to aflibercept therapy . 
 other outcome measures recorded and assessed were mean change in best corrected visual acuity ( bcva ) , mean change in central retinal thickness ( crt ) , and vision - related quality of life ( or visual functioning ) assessed using the national eye institute 25-item visual function questionnaire ( nei vfq-25 ) single composite score . the latter captures key dimensions of self - reported vision - targeted health status and visual functioning in patients with chronic eye disease . 
 assessment of the overall interaction with the external environment through the visual system is measured using various methods , including va ( 100% contrast , early treatment diabetic retinopathy study [ etdrs ] , and snellen charts ) , contrast sensitivity ( cs ) testing ( 100%0.6% contrast ) , humphrey visual field test , and color vision ( eg , isihara charts , farnsworth dichotomous test ) . 
 the ability to perform everyday visual tasks , such as reading , recognizing faces , driving , telephone use , and using household appliances , depends on visual components beyond high - contrast visual distance acuity , including cs , near vision , color vision , and sensitivity to glare . 
 the snellen eye chart is primarily used to measure va and is depicted as a series of high contrast black - on - white letters in different sizes . relatively small changes in refractive status can be detected by this test , and it is a useful standard for defining vision changes caused by spherical blur . as various types of vision loss 
 are not caused by spherical blur ( eg , cataracts , glaucoma , and irregular astigmatism ) , the snellen va test may not be an adequate or appropriate measure in all cases . 
 cs measures an individual s ability to detect low contrast images and to perceive differences between light and dark , whereas the snellen va chart test measures the level at which an individual can identify fine detail at high contrast using black - on - white letters . 
 using low contrast images , the cs method can detect subtle vision changes that may be concealed by acuity.14 cs could be considered as an adjunct to standard acuity testing , to better assess visual capability , as well as being a useful measure for evaluating a wide range of ocular conditions ( eg , diabetic eye disease , macular degeneration , cataracts , glaucoma , traumatic brain injury , amblyopia , and optic nerve disorders).14 cs is a sensitive and arguably essential outcome end point for a complete assessment of visual function and visual performance , including driving , mobility , reading speed , and general vision - related quality of life.15 additionally , cs has potential applications when measuring the vision of patients with high visual requirements , including athletes and public service personnel . 
 cs charts that utilize letters are familiar to both the patient and the practitioner , in comparison to other nonletter - based cs charts.16 the pelli 
 robson cs test is considered a quick and reliable method in clinical settings.17,18 patients scoring below 1.65 log on the pelli 
 robson cs test are considered to have impaired visual function , with a 6-letter change in cs equating to a 15-letter ( or 3 lines ) change in va.19 both va and cs are associated with the ability to perform daily vision - related activities20 and are considered as significant independent parameters in the determination of visual impairment and quality of life . however , in clinical studies , change in va over time is commonly used as the main primary outcome measure as an effective and reliable indicator of functional deficit or improvement . when measuring va , single optotypes ( usually letters , numbers , or geometric symbols ) with high contrast 
 are presented to the patient ; however , the environment is not always seen in high contrast as presented on a standard acuity chart . 
 therefore , using cs determination to measure the ability to recognize low contrast patterns may detect functional impairment not evident when measuring va only.21 study results have indicated that va and cs do not reliably demonstrate the same parallel progression in visual function loss , although they show a moderate correlation in eyes with neovascular amd.21 both parameters provide important and supplementary information about visual disability,21 supporting the role of multiple visual end point evaluations in interventional studies.21,22 to better assess the overall visual functioning following anti - vegf treatment switch , this study was conducted to determine and evaluate change from baseline to week 12 in pelli 
 robson cs as the primary clinical end point in patients with recalcitrant exudative amd , despite previous ranibizumab treatment who were switched to aflibercept therapy . other outcome measures recorded and assessed 
 were mean change in best corrected visual acuity ( bcva ) , mean change in central retinal thickness ( crt ) , and vision - related quality of life ( or visual functioning ) assessed using the national eye institute 25-item visual function questionnaire ( nei vfq-25 ) single composite score . 
 the latter captures key dimensions of self - reported vision - targeted health status and visual functioning in patients with chronic eye disease . 
 this single - center study was an institutional review board ( irb)-approved , investigator - masked , prospective , interventional , noncontrolled , single - arm study that included 40 randomly selected neovascular amd patients with persistent fluid on spectral domain optical coherence tomography ( sd - oct ) following at least three intravitreal injections of ranibizumab in the previous 6 months . 
 if both eyes of individual subjects met the study inclusion criteria , they were included in the study population dataset ( table 1 ) . 
 subjects were switched to 2 mg aflibercept injection on a dosing protocol that matched the recommended treatment posology for neovascular amd , involving treatment initiation with three doses administered once a month for the first 3 months ( weeks 0 , 4 , and 8) , followed by an injection every 2 months for the first 12 months of treatment . 
 assessments included the pelli  robson log cs measurement at 1 m , bcva converted to logarithm of minimum angle of resolution ( logmar ) using standard methods,23 crt using sd - oct ( heidelberg spectralis ; heidelberg engineering inc . , vista , ca , usa ) , and the nei vfq-25 questionnaire with composite score . 
 statistical data analysis was performed once all patients had completed the week 12 visit , using microsoft excel ( microsoft corporation , redmond , wa , usa ) . 
 a two - sided paired t - test was used to compare the means of continuous variables . 
 if not otherwise stated , all values are presented as mean  standard deviation ( sd ) . 
 the study was conducted in accordance with the principles of the declaration of helsinki and in compliance with good clinical practice and applicable regulatory requirements . 
 the study procedure and informed consent document were approved by irb services ( aurora , on , canada ) prior to initiating the study . written informed consent was obtained from all participants before study enrollment . 
 a total of 49 eyes of 40 patients ( 27 male and 13 female ) with treatment - resistant neovascular amd were evaluated at baseline and at the week 12 visit following treatment switch to aflibercept from ranibizumab . 
 no patients were lost to follow - up , and there were no treatment discontinuations . 
 there were no ocular or nonocular adverse events in the patient population during the duration of the study . 
 the baseline age was 80.26.8 years ( mean  sd , range 6893 years ) . 
 the mean number of ranibizumab injections in the previous 6 months before switch to aflibercept was 5.1 ( range 36 ) . 
 ranibizumab injections ( mean  sd 28.222.1 [ range 386 ] ) were administered before initiation of aflibercept treatment . 
 the duration of prior ranibizumab therapy was 36.824.7 months ( mean  sd , range 694 ) , with a treatment start date from 2008 to 2015 . 
 the mean cs increased from 1.32 log units at baseline to 1.40 log units at week 12 ( p=0.00079 ) . 
 mean va was stable from baseline to week 12 : mean logmar va was 0.51878 at baseline and 0.53204 at week 12 ( median 0.4 and 0.42 , respectively ) . at week 12 , there was a statistically significant decrease of 22 m in mean crt from 354 m at baseline to 332 m at week 12 ( p=0.004 ) . 
 there was no demonstrated relationship between change in crt and va measurements ( p=0.88 ) . 
 there was however an associated relationship between improvement in cs and mean change in crt ( p=0.000046 ) . 
 of 40 patients , 26 ( 65% ) experienced an overall improvement in nei vfq-25 composite score from baseline to week 12 ; 8 of 40 ( 20% ) patients experienced no change ; and 6 of 40 ( 15% ) experienced an overall decrease in composite score . 
 a total of 49 eyes of 40 patients ( 27 male and 13 female ) with treatment - resistant neovascular amd were evaluated at baseline and at the week 12 visit following treatment switch to aflibercept from ranibizumab . 
 no patients were lost to follow - up , and there were no treatment discontinuations . 
 there were no ocular or nonocular adverse events in the patient population during the duration of the study . 
 the baseline age was 80.26.8 years ( mean  sd , range 6893 years ) . 
 the mean number of ranibizumab injections in the previous 6 months before switch to aflibercept was 5.1 ( range 36 ) . 
 ranibizumab injections ( mean  sd 28.222.1 [ range 386 ] ) were administered before initiation of aflibercept treatment . 
 the duration of prior ranibizumab therapy was 36.824.7 months ( mean  sd , range 694 ) , with a treatment start date from 2008 to 2015 . 
 the mean cs increased from 1.32 log units at baseline to 1.40 log units at week 12 ( p=0.00079 ) . 
 mean va was stable from baseline to week 12 : mean logmar va was 0.51878 at baseline and 0.53204 at week 12 ( median 0.4 and 0.42 , respectively ) . at week 12 , there was a statistically significant decrease of 22 m in mean crt from 354 m at baseline to 332 m at week 12 ( p=0.004 ) . 
 there was no demonstrated relationship between change in crt and va measurements ( p=0.88 ) . 
 there was however an associated relationship between improvement in cs and mean change in crt ( p=0.000046 ) . 
 of 40 patients , 26 ( 65% ) experienced an overall improvement in nei vfq-25 composite score from baseline to week 12 ; 8 of 40 ( 20% ) patients experienced no change ; and 6 of 40 ( 15% ) experienced an overall decrease in composite score . 
 the efficacy evaluation following treatment switch to aflibercept from ranibizumab incorporated both cs and va measurement as independent outcome measures at 12 weeks following an initial treatment - loading phase of 3 consecutive monthly aflibercept injections . 
 overall , the study population received close to the planned 4-weekly aflibercept dosing after switch initiation . 
 adherence to strict inclusion criteria ensured that the study population included only suboptimal responders with persistent fluid , despite previous near monthly ranibizumab treatment . 
 it was shown that in this switch population , there was a statistically significant improvement in the primary outcome end point of change in cs from baseline to week 12 , with an average cs change of two letters broadly comparable to a five - letter ( one line on etdrs chart ) difference in va in terms of vision function performance . 
 it has been reported , for example , that a 6-letter difference in cs had a similar impact on self - reported difficulty with everyday vision - related tasks as a 15-letter ( 3 lines on etdrs chart ) va difference in a population - based sample of individuals aged 65 years and older.19 the evaluation of change in cs may be a clinically meaningful measure of treatment benefit in terms of quality of vision on anti - vegf therapy . 
 published literature has shown that cs is an important measure of visual function in patients with subfoveal cnv due to amd , and the potential benefits of treatment may not be completely characterized by va measurement alone.24 obtaining information on the overall quality of vision that includes va and cs as efficacy outcomes may provide physicians , optometrists , and general ophthalmologists with a better and more complete understanding of their patient s visual status and help improve patient care.20,25 pauleikhoff 26 noted in a review of natural history and treatment outcomes in neovascular amd that reducing the risk of further va and cs loss might allow neovascular amd patients to maintain better vision - related functional abilities . 
 this study demonstrated that there is a general and immediate decreasing trend in mean crt after treatment switch to aflibercept , while no statistically significant change from baseline va was noted . in this neovascular amd population of suboptimal responders 
 to ranibizumab , persistence of fluid is associated with impairment in visual function as measured by cs . the fluid reduction seen following a switch to aflibercept 
 numerous studies have evaluated the effectiveness of switching to aflibercept from another anti - vegf agent in treatment - resistant neovascular amd patients with persistent or recurrent fluid , despite prior multiple anti - vegf injections . 
 wykoff et al27 reported 6-month results from the turf trial providing evidence that aflibercept treatment may be anatomically valuable in some recalcitrant exudative amd eyes , while maintaining prior visual gains attained with ranibizumab treatment . 
 a decrease in the proportion of cnv lesions that were graded as active at 12 months after switch from ranibizumab to aflibercept was noted by barthelmes et al.28 chang et al29 prospectively assessed the efficacy of aflibercept in a cohort of 40 treatment - resistant neovascular amd patients ; at 12 months after switch , mean bcva improved from baseline by 4.7 letters ( 95% ci : 2.17.3 , p<0.001 ) and crt decreased by 97.2 m ( 95% ci : 54.4140.1 , p<0.001 ) . 
 a meta - analysis noted a small but statistically significant improvement in bcva 6 months following treatment switch to aflibercept in patients with treatment - resistant neovascular amd on another anti - vegf agent.12 the nei vfq-25 questionnaire captures patient - reported visual function and was used to assess vision - related quality of life before and after treatment switch . 
 the fact that two - thirds of patients experienced an overall improvement in nei vfq-25 total score at week 12 compared with baseline score is encouraging . 
 however , the study sample is too small to allow evaluation of the statistical relationship between different subgroup responses , although this might usefully be addressed in a future study analysis . 
 in the view studies , involving treatment - naive neovascular amd patients , total nei vfq-25 score was a prespecified secondary efficacy variable and improved scores were observed for both ranibizumab and aflibercept treatment groups , with the greatest improvements observed for mental health and general vision.8,30 yuzawa et al30 reported results evaluating change in composite nei vfq-25 score based on categorical change in va ( worsened , unchanged , and improved ) over 52 weeks in the view studies , comparing the approved dosing for each agent . 
 meaningful improvement in nei vfq-25 composite score was attained only in patients who gained five etdrs letters or more from baseline va to week 52 . 
 when reviewing the results of our study , differences in participant selection , baseline features , and inclusion criteria limit generalizability and direct comparisons between reported anti - vegf switch studies in neovascular amd . 
 the sample size of the switch cohort in this study was relatively small , many patients had received chronic long - term ranibizumab treatment , and follow - up of participants was limited at 12 weeks only . 
 comparative analyses involving a control arm of patients continuing on ranibizumab despite recurrent or persistent fluid might have strengthened the overall analysis . 
 a crossover study design incorporating a reswitch arm from aflibercept back to ranibizumab may be a useful consideration in the development of future anti - vegf treatment switch studies . 
 it would also be of interest to conduct a larger study to evaluate long - term outcomes , including the potential for greater improvement in vision - related functional benefit with continuing treatment , after a treatment switch to aflibercept from ranibizumab . despite limitations , the study results reported herein demonstrate interesting findings with regard to disease progression that may be useful in the design of future switch studies in neovascular amd . 
 va and cs should be considered as significant independent parameters for determining degree of visual impairment and treatment response , as they provide predictive information on visual disability and vision - related quality of life . using the pelli  robson chart , 
 cs determination is a standardized technique that is straightforward and reproducible ; therefore , we suggest that cs be more widely considered as a valuable additional outcome end point in future clinical studies of neovascular amd , diabetic macular edema ( dme ) , and retinal vein occlusion . 
 switching of anti - vegf agents in neovascular amd patients is a viable treatment strategy to improve outcomes among initial nonresponders or for patients exhibiting recalcitrant exudative disease activity on continued anti - vegf treatment . 
 the 2016 preferences and trends membership survey by the american society of retina specialists found that more than 80% of respondents considered that persistent or recurrent fluid on oct best determines inadequate response to an anti - vegf treatment in neovascular amd , and more than 75% considered it reasonable to switch due to inadequate response after three to six initial injections . 
 other studies have shown that improvements in anatomic parameters and stable or moderately improved va outcomes may be attained in treatment - resistant neovascular amd patients switched to intravitreal aflibercept from another anti - vegf agent . 
 results of this prospective , interventional investigation reveal a statistically significant improvement in cs and a statistically significant reduction in crt over 12 weeks after treatment switch to aflibercept from ranibizumab in recalcitrant neovascular amd patients , with no meaningful change from baseline va observed . in our study 
 series , a demonstrated correlation was observed between crt decrease and improvement in pelli  robson log cs score at 12 weeks after treatment switch to aflibercept . 
 vision functioning , assessed using the nei vfq-25 score , improved from baseline to week 12 in two - thirds of the anti - vegf switch population . 
 the observed positive correlation between improvement in cs and reduction in crt supports the argument that measurement of cs may provide a more complete understanding of the benefits of anti - vegf treatment on vision - related functional performance in patients with neovascular amd . 
 as emphasized earlier , measurement of cs could provide useful early information on visual impairment not identifiable on va assessment alone , as well as provide another method for monitoring global treatment benefit and individual patient responses . 
 overall , cs measurement may provide a better understanding of visual performance challenges faced by individuals with vision impairment . 
 an extended study duration involving a larger patient population would allow for further investigation and potential validation of results with regard to improvement in cs and other visual functioning parameters . 
 further analyses exploring multiple measures of visual function as efficacy end points in treatment studies of dme and other retinal diseases are warranted .","purposethis study evaluated visual function and anatomic and vision - related quality - of - life outcomes in recalcitrant neovascular age - related macular degeneration ( amd ) subjects switched to aflibercept ( eylea ) from ranibizumab ( lucentis).methodsin a single - center study conducted in barrie , on , 40 patients with persistent fluid despite previous ranibizumab treatment were switched to aflibercept with 3 consecutive monthly doses . 
 main outcome measure was mean change from baseline to week 12 in pelli 
 robson contrast sensitivity ( cs ) . 
 secondary outcomes were mean change in best corrected visual acuity ( bcva ) , central retinal thickness ( crt ) , and national eye institute 25-item visual function questionnaire ( nei vfq-25 ) score . a two - sided paired t - test was used in the statistical data analysis to compare the means of continuous variables.resultsforty-nine eyes ( baseline visual acuity [ va ] > 6/120 ) were evaluated . 
 ranibizumab injections ( mean  standard deviation [ sd ] 28.222.1 [ range 386 ] ) were administered prior to treatment switch . 
 mean cs improved from 1.32 at baseline to 1.40 log units at week 12 . 
 va was stable throughout . 
 mean crt decreased from 354 m at baseline to 332 m at week 12 ( 22 m , p=0.004 ) . 
 twenty - six ( 65% ) patients experienced an overall improvement in nei vfq-25 score . 
 interestingly , a correlation was observed between improvement in log cs and crt change ( p=0.000046).conclusioncontrast sensitivity improved statistically and significantly , and crt decreased from baseline to week 12 after a switch to aflibercept from ranibizumab . 
 analysis of cs as an independent outcome end point in neovascular amd treatment switch studies may provide a more complete understanding of visual response .",pubmed
"multiple sclerosis ( ms ) is a common autoimmune system disease which affects the central nervous system with network vascular inflammation , demyelination and axonal damage symptoms ( 1 , 2 ) . 
 it is a chronic disease of the central nervous system and causes severe cognitive and physical impairments ( 3 , 4 ) . 
 accordingly , it has been documented that white matter lesion may affect the cognition of ms patients ( 5 ) . among the various clinical forms , 
 relapsing - remitting is the most common type , found in approximately 85% to 90% of cases ( 6 ) . 
 although , it is difficult to differentiate primary progressive multiple sclerosis ( ppms ) from relapsing remitting multiple sclerosis ( rrms ) in the early phases , yet previous studies have demonstrated that mrs can be used as a potential way to diagnose ppms and rrms ( 2 ) . 
 in fact , after traumatic and rheumatic diseases , ms is the third factor for asthenic diseases , and has been the subject of extensive studies worldwide ( 7 ) . 
 multiple sclerosis is a disease of young adults and most of patients are diagnosed between the ages of 20 and 40 ( 9 ) . although the exact reason for this disease is unknown , it has been reported that several factors such as genetic and environmental factors ( especially viruses ) are involved in the pathogenesis of ms ( 1 , 10 , 11 ) . 
 accordingly , several investigators are trying to improve symptoms of ms by alteration in the behavior of ms patients including exercise ( 4 ) . in spite of such efforts 
 interestingly , researchers do not limit this disease just to a single gene and candidate several immune and non - immune genes in the pathogenesis of ms . 
 recently , scientists have investigated interleukin-25 ( il-25 ) including its effects on the immune system . 
 this cytokine has a similar structure to il-17 and plays a key role in stimulating and development of t helper 2 ( th2 ) responses ( 12 ) . 
 furthermore , il-25 is involved in the control function of endothelial cell , th1 and th17 , which are the main cells involved in the pathogenic activities of the immune system ( 12 ) . 
 therefore , any factor , which can regulate the expression of the cytokine , can be considered as a candidate for investigation in the ms disease process . 
 it has been reported that there is a known polymorphism ( c424c / a polymorphism ) within exon 2 of the il-25 gene ( 13 ) , which may be associated with immune system - related disease . 
 therefore , according to this explanation , it seems that the polymorphism may be associated with ms . 
 thus , the main aim of this study was to evaluate the association between c424c / a polymorphism within the il-25 gene with ms in an iranian population . 
 in this case - control study , according to the study of arababadi et al . 
 ( 1 ) and using sample - size estimation we compared two binomial independent proportions with the following equation : where significance level was 0.05 , the expected power was considered 90% , p1 = 1% ( frequency of aa genotype in ms patients ) , p2 = 17% ( frequency of aa genotype in healthy controls ) , k = 1 ( sample size was considered equal in both groups ) , and  = 15% ( the minimum difference in frequency of aa genotype in the two groups , which was considered clinically important ) . based on the above parameters , sample size was determined as 75 subjects in either group . in this study , 80 patients with ms were recruited for blood sampling yet according to the inclusion and exclusion criteria , 74 patients with relapse and remitting ms ( rrms ) and 75 healthy controls were finally enrolled during august 2013 to september 2014 . "" 
 blood samples were collected from consecutively admitted patients to the sina hospital , the main referral hospital for ms patients from all provinces of iran . 
 age and gender matched control samples were also collected from the tehran blood transfusion services . 
 the occurrence of ms was confirmed based on mcdonald s criteria ( diplopia , blurry vision , finger pruritus and disability ) ( 14 ) and brain magnetic resonance imaging ( mri ) results by an expert neurologist , according to clinical and preclinical findings . 
 having at least one central nervous system ( cns ) demyelinating event and being at least 18 years old to give informed consent were considered as the main inclusion criteria . 
 the two groups were matched in terms of age and sex and subjects with genetic diseases , diabetes , autoimmune disease and smokers were excluded from the study . 
 informed consent was filled out by all participants and this study was approved by the ethics committee of rafsanjan university of medical sciences ( rums ) with number 9/1077 on the 15th of july 2013 . regarding ethical considerations , 
 patients data were collected by their nurses and the data and blood samples were trans - located to the laboratory without names . 
 dna was purified from peripheral blood immune cells of participants using a commercial kit ( cinnaclon , tehran , iran ) , according to the manufacturer s instructions . 
 the pcr amplification of exon 2 of the il-25 gene and rflp ( using bsrfi restriction enzyme ) was performed according to the study of buning and et al . 
 numerical variables are presented as mean  sd , while categorical variables are summarized by absolute frequencies and percentages . 
 continuous variables were compared using independent two - sample t - test , and categorical variables were compared using the chi - square test across the two study groups . 
 one - sample kolmogorov - smirnov test was applied to test for normal distribution in cases and controls . 
 the logistic regression model was established to estimate the odds ratio ( or ) and 95% confidence interval ( ci ) of ms development in patients with the ac and aa compared to patients with the cc genotype . for the statistical analysis , the spss version 18.0 software for windows ( spss inc . , chicago , il ) was used . 
 all p values were two - tailed with statistical significance defined by p  0.05 . 
 the results also revealed 1 to 2% missing values in the evaluated genotypes and alleles . 
 in this case - control study , according to the study of arababadi et al . 
 ( 1 ) and using sample - size estimation we compared two binomial independent proportions with the following equation : where significance level was 0.05 , the expected power was considered 90% , p1 = 1% ( frequency of aa genotype in ms patients ) , p2 = 17% ( frequency of aa genotype in healthy controls ) , k = 1 ( sample size was considered equal in both groups ) , and  = 15% ( the minimum difference in frequency of aa genotype in the two groups , which was considered clinically important ) . based on the above parameters , sample size was determined as 75 subjects in either group . in this study , 80 patients with ms were recruited for blood sampling yet according to the inclusion and exclusion criteria , 74 patients with relapse and remitting ms ( rrms ) and 75 healthy controls were finally enrolled during august 2013 to september 2014 . "" 
 blood samples were collected from consecutively admitted patients to the sina hospital , the main referral hospital for ms patients from all provinces of iran . 
 age and gender matched control samples were also collected from the tehran blood transfusion services . 
 the occurrence of ms was confirmed based on mcdonald s criteria ( diplopia , blurry vision , finger pruritus and disability ) ( 14 ) and brain magnetic resonance imaging ( mri ) results by an expert neurologist , according to clinical and preclinical findings . 
 having at least one central nervous system ( cns ) demyelinating event and being at least 18 years old to give informed consent were considered as the main inclusion criteria . 
 the two groups were matched in terms of age and sex and subjects with genetic diseases , diabetes , autoimmune disease and smokers were excluded from the study . 
 informed consent was filled out by all participants and this study was approved by the ethics committee of rafsanjan university of medical sciences ( rums ) with number 9/1077 on the 15th of july 2013 . regarding ethical considerations , 
 patients data were collected by their nurses and the data and blood samples were trans - located to the laboratory without names . 
 dna was purified from peripheral blood immune cells of participants using a commercial kit ( cinnaclon , tehran , iran ) , according to the manufacturer s instructions . 
 the pcr amplification of exon 2 of the il-25 gene and rflp ( using bsrfi restriction enzyme ) was performed according to the study of buning and et al . 
 numerical variables are presented as mean  sd , while categorical variables are summarized by absolute frequencies and percentages . 
 continuous variables were compared using independent two - sample t - test , and categorical variables were compared using the chi - square test across the two study groups . 
 one - sample kolmogorov - smirnov test was applied to test for normal distribution in cases and controls . 
 the logistic regression model was established to estimate the odds ratio ( or ) and 95% confidence interval ( ci ) of ms development in patients with the ac and aa compared to patients with the cc genotype . for the statistical analysis , the spss version 18.0 software for windows ( spss inc . , chicago , il ) was used . 
 all p values were two - tailed with statistical significance defined by p  0.05 . 
 the results also revealed 1 to 2% missing values in the evaluated genotypes and alleles . 
 the results revealed that eight ( 11.3% ) out of 74 patients with ms had the aa genotype , while 16 ( 22.5% ) had ac and 47 ( 66.2% ) had cc genotypes ( table 2 ) . 
 the results also showed that 8 ( 11.4% ) , 18 ( 25.7% ) and 44 ( 62.9% ) of the healthy controls had the aa , ac and cc genotypes , respectively . 
 the chi - square test revealed that the frequency distribution of the genotypes was not statistically different between patients with ms and the controls ( p = 0.901 ) ( table 2 ) . 
 according to the logistic regression model , the or of ms development in patients with ac versus patients with cc genotypes was 0.832 ( 95% ic : 0.378 - 1.832 ) ( p = 0.648 ) . 
 the corresponding results for aa versus cc genotypes was 0.936 ( 95% ic : 0.323 - 2.710 ) ( p = 0.903 ) . 
 the results also indicated that the frequencies of a and c alleles were 110 ( 77.5% ) and 32 ( 22.5% ) , respectively , in the patients with ms and were 106 ( 75.7% ) and 34 ( 24.3% ) , respectively , in the healthy controls . 
 the statistical analysis found that the differences in distribution of a and c alleles between groups were not statistically significant ( p = 0.728 ) ( table 2 ) . 
 the or of ms development in patients with the a allele versus patients with the c allele was 0.907 ( 95% ic : 0.522 - 1.574 ) , which did not reach statistical significance level ( p = 0.729 ) . 
 multiple sclerosis is an autoimmune disease , with several immune - related genes involved in its pathogenesis ( 15 ) . 
 it has been confirmed that th1 and th17 responses are the main causes of progression of ms ( 16 ) . therefore , th2 responses , which regulate th1 and th17 , can improve the clinical and laboratory outcome of the disease ( 16 ) . 
 it is believed that il-25 , as a th2 cytokine , can participate in the regulation of immune responses during inflammatory diseases including ms . 
 accordingly , previous studies suggested that serum levels of il-25 were decreased in ms patients when compared with the healthy controls ( 17 ) . 
 it has been proposed that polymorphisms within cytokine genes are associated with their expression ( 18 , 19 ) . 
 the authors of this study hypothesized that the polymorphisms within the il-25 gene may be associated with ms disease ; hence , the c424c / a polymorphism within the il-25 gene was evaluated in this study . 
 the results of our study identified that neither aa , ac and cc genotypes nor a and c alleles were associated with ms in the iranian population . to the best of our knowledge , this is a unique study , which evaluated the c424c / a polymorphism within the il-25 gene in patients with ms , yet there is a study , which evaluated this polymorphism in inflammatory bowel disease ( ibd ) , as an inflammatory disease ( 13 ) . 
 parallel to our results , researchers have reported that the c424c / a polymorphism within the il-25 gene was not associated with ibd ( 13 ) . 
 interestingly , this study documented that the expression level of il-25 decreased in ibd patients ( 13 ) . again , as mentioned previously , it has also been revealed that expression of il-25 decreased in ms patients ( 17 ) . 
 based on our results and the mentioned study it may be concluded that although il-25 may play significant roles in the pathogenesis of inflammatory diseases such as ms and ibd , c424c / a polymorphism is not important in the pathogenesis of these diseases . on the other hand , another study on iranian patients with ms revealed that other polymorphisms , except c424c / a , within il-25 are associated with ms ( 17 ) . 
 thus , it may be concluded that other polymorphisms like 4076a > a , 3672 t > ta , 3712 g > ga , and 3463c > ca , but not the c424c / a polymorphism , may regulate il-25 expression . according to our and previous studies it appears that evaluation of the c424c / a polymorphism , as the first report , and lack of evaluation of other polymorphisms within the il-25 gene as well as lack of evaluation of serum levels of il-25 are the strong and weak points , respectively , of our study . 
 additionally , it can be proposed that using a large sample size may be better for understanding the roles of c424c / a polymorphism in the pathogenesis of ms . according to the earned results of our investigation 
 it can be hypothesized that c424c / a polymorphism within the il-25 gene is not associated with ms and can not be considered as a risk factor for ms development .","backgroundmultiple sclerosis ( ms ) is a common autoimmune system disease which affects the central nervous system . 
 it has been documented that interleukin-25 ( il-25 ) plays key roles in suppressing th1 responses , which is increased during ms.objectivesthe aim of this study was to investigate the c424c / a polymorphism within the il-25 gene in ms patients in comparison to healthy controls.patients and methodsin this case - control study , 74 patients with ms and 75 healthy controls were selected . 
 polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) was used in order to determine c424c / a polymorphism within the il-25 gene.resultsthe results showed that there was no statistical significant difference in distribution of genotype ( aa , ac and cc ) and allele ( a and c ) frequencies between ms patients and healthy controls ( p = 0.901 and p = 0.728 , respectively).conclusionsin conclusion , it appears that the c424c / a polymorphism within the il-25 gene has no significant relationship with ms , and this polymorphism is probably not associated with ms complications , its onset and gender distribution .",pubmed
"aortic aneurysm ( aa ) can be life - threatening and aorta size is the best criterion for determining if an intervention is necessary to prevent ruptures , dissections or aneurysm - related deaths.1 ) however , even large aas seldom cause symptoms,2)3 ) although the incidence of aortic disease increases with age.4)5 ) therefore , it is important for clinicians to be cautious in their evaluation of patients at risk . 
 aneurysm of the ascending thoracic aorta ( ata ) most often results from cystic medial degeneration.2 ) atherosclerosis is an infrequent cause of aneurysm of ata , in contrast to that of the descending thoracic aorta ( dta ) and abdominal aorta , where atherosclerosis plays an important role in development of aa.2 ) meanwhile , thoracic aas ( taas ) and abdominal aas ( aaas ) share common risk factors , such as age and hypertension 
 . therefore , elderly male subjects with hypertension might be at high risk for both taa and aaa . 
 efforts to detect high risk populations for coronary heart disease ( chd ) have been widely applied and coronary calcium measurement using non - contrast computed tomography ( ct ) is the method of choice for assessing cardiovascular risk in asymptomatic subjects.6 ) screening for aaa using ultrasound has been recommended in elderly men who are current or past smokers,7 ) but the screening strategy for taa in elderly subjects is not well - established , even though it is a fatal disease.8)9 ) the more widespread applications of cardiac ct and thoracic ct for cardiovascular risk stratification and lung cancer screening have made it possible to measure thoracic aortic diameter using non - contrast ct , resulting in several successful case reports.10)11 ) the purpose of the current study was to use non - contrast ct in a prospective manner to explore the prevalence of aas and determine whether screening for chd and aas is necessary in a population with multiple risk factors for chd in a korean population . 
 five hundreds and two consecutive korean male hypertensive patients without history of aa were prospectively enrolled from june , 2012 to april , 2013 at the outpatient clinic of severance cardiovascular hospital ( seoul , korea ) . 
 subjects were eligible if they were 65 years old and had provided signed informed consent . of these 542 patients who had done non - contrast ct of whole aorta , 15 patients ( 2.8% ) with a history of aortic valve surgery and 30 patients ( 5.5% ) with a suboptimal ct image were excluded . 
 most of the suboptimal images were those with errors in raw ct data storage , which caused problems of analysis in the sagittal and coronal planes using reconstructed images . 
 serum calcium , phosphorus , total cholesterol , low density lipoprotein - cholesterol ( ldl - c ) , high density lipoprotein - cholesterol ( hdl - c ) , triglycerides and serum creatinine levels were measured after a minimum 12-hour fasting period . 
 hypertension was defined as systolic blood pressure 140 mm hg and/or diastolic blood pressure as 90 mm hg or treatment with antihypertensive agents . 
 diabetes was defined as treatment with hypoglycemic agents or insulin , or fasting glucose 126 mg / dl . 
 dyslipidemia was defined as any of the following : total cholesterol 240 mg / dl , ldl - c 130 mg / dl , hdl - c 40 mg / dl , triglyceride 150 mg / dl or treatment with lipid lowering agents . 
 all examinations were performed using an aquilion one 320-row ct system ( toshiba medical systems , otawara , japan ) . 
 dual scanograms were used for planning the examination and determining the anatomical range to be covered . 
 multiple volumes were placed to cover the entire aorta from above the aortic arch to the aortic bifurcation . 
 patients underwent aorta ct with a prospective electrocardiography - gating wide - volume protocol ( from four to six volumes , according to body height ) . 
 the tube voltage was 120 kv and the effective tube current was adjusted using the adaptive iterative dose reduction three dimensional algorithm . 
 the resulting four to six individual volume data sets were automatically stitched together immediately after reconstruction to generate one ct data set of the whole aorta . 
 all data were reconstructed using a standard soft - tissue and lung kernel ( fc43 ) . 
 maximal ascending aorta diameter ( atamax ) was measured in the axial plane from just above the aortic root to the aortic arch perpendicular to the aortic axis . 
 similarly , maximal descending thoracic aorta diameter ( dtamax ) was measured at the dta distal from the aortic arch to the diaphragm level in the same axis ( fig . 
 1 ) . atamax and dtamax were confirmed in the sagittal and coronal planes using reconstructed images ( fig . 
 maximal thoracic aorta diameter ( tamax ) was defined as the largest diameter among atamax , dtamax and maximal diameter of the aortic arch . 
 maximal abdominal aortic diameter ( aamax ) was defined as the maximal diameter of the abdominal aorta from the diaphragm to the first slice superior to the aortic bifurcation . 
 taa was defined as tamax 40 mm and aaa was defined as aamax 30 mm.7)12 ) distribution of relevant variables was reported either as a percentage or as the meanstandard deviation . 
 a binary logistic regression analysis was used to identify risk factors associated with taa and aaa . 
 variables with a p<0.2 in univariate analysis were included in the multiple logistic regression model . 
 collinearity among explanatory variables was identified using variance inflation factor ( vif ) , and highly correlated variables ( vif>5 ) were excluded . 
 five hundreds and two consecutive korean male hypertensive patients without history of aa were prospectively enrolled from june , 2012 to april , 2013 at the outpatient clinic of severance cardiovascular hospital ( seoul , korea ) . 
 subjects were eligible if they were 65 years old and had provided signed informed consent . of these 542 patients who had done non - contrast ct of whole aorta , 15 patients ( 2.8% ) with a history of aortic valve surgery and 30 patients ( 5.5% ) with a suboptimal ct image were excluded . 
 most of the suboptimal images were those with errors in raw ct data storage , which caused problems of analysis in the sagittal and coronal planes using reconstructed images . 
 height , weight , and blood pressure were measured during their visit . serum calcium , phosphorus , total cholesterol , low density lipoprotein - cholesterol ( ldl - c ) , high density lipoprotein - cholesterol ( hdl - c ) , 
 hypertension was defined as systolic blood pressure 140 mm hg and/or diastolic blood pressure as 90 mm hg or treatment with antihypertensive agents . 
 diabetes was defined as treatment with hypoglycemic agents or insulin , or fasting glucose 126 mg / dl . 
 dyslipidemia was defined as any of the following : total cholesterol 240 mg / dl , ldl - c 130 mg / dl , hdl - c 40 mg / dl , triglyceride 150 mg / dl or treatment with lipid lowering agents . 
 all examinations were performed using an aquilion one 320-row ct system ( toshiba medical systems , otawara , japan ) . 
 dual scanograms were used for planning the examination and determining the anatomical range to be covered . 
 multiple volumes were placed to cover the entire aorta from above the aortic arch to the aortic bifurcation . 
 patients underwent aorta ct with a prospective electrocardiography - gating wide - volume protocol ( from four to six volumes , according to body height ) . 
 the tube voltage was 120 kv and the effective tube current was adjusted using the adaptive iterative dose reduction three dimensional algorithm . 
 the resulting four to six individual volume data sets were automatically stitched together immediately after reconstruction to generate one ct data set of the whole aorta . 
 all data were reconstructed using a standard soft - tissue and lung kernel ( fc43 ) . 
 maximal ascending aorta diameter ( atamax ) was measured in the axial plane from just above the aortic root to the aortic arch perpendicular to the aortic axis . 
 similarly , maximal descending thoracic aorta diameter ( dtamax ) was measured at the dta distal from the aortic arch to the diaphragm level in the same axis ( fig . 
 1 ) . atamax and dtamax were confirmed in the sagittal and coronal planes using reconstructed images ( fig . 
 maximal thoracic aorta diameter ( tamax ) was defined as the largest diameter among atamax , dtamax and maximal diameter of the aortic arch . 
 maximal abdominal aortic diameter ( aamax ) was defined as the maximal diameter of the abdominal aorta from the diaphragm to the first slice superior to the aortic bifurcation . 
 taa was defined as tamax 40 mm and aaa was defined as aamax 30 mm.7)12 ) 
 distribution of relevant variables was reported either as a percentage or as the meanstandard deviation . 
 a binary logistic regression analysis was used to identify risk factors associated with taa and aaa . 
 variables with a p<0.2 in univariate analysis were included in the multiple logistic regression model . 
 collinearity among explanatory variables was identified using variance inflation factor ( vif ) , and highly correlated variables ( vif>5 ) were excluded . 
 patient demographics and clinical characteristics are shown in table 1 . the mean age was 73 years . 
 diabetes , dyslipidemia and coronary artery disease were observed in 171 patients ( 34.5% ) , 318 patients ( 64.1% ) and 256 patients ( 51.6% ) , respectively . 
 three hundred and sixty - four patients ( 73.4% ) had a history of smoking . 
 mean diameter of atamax , dtamax , tamax , and aamax was 38.44.0 mm , 28.53.0 mm , 38.94.0 , and 23.05.5 mm , respectively . 
 the prevalence of taa and aaa was 36.5% ( 181/496 ) and 6.0% ( 30/496 ) , respectively . 
 there were 16 patients ( 3.2% ) who had taa and aaa simultaneously . however , prevalence of aa suitable for an indication for intervention according to current guidelines ( tamax 55 mm and aamax 60 mm)2 ) was very low in this study . 
 those who were indicated for intervention were seen only in one case of the taa group and one case of the aaa group . 
 prevalence of taa was 64.6% in patients with aaa and 40.0% in patients without aaa . 
 patients with aaa had a higher occurrence of taa compared with those without aaa ( p=0.010 ) ( fig . 
 there was no significant difference in prevalence of taa between nonsmokers and those who had a history of smoking or were current smokers . 
 in contrast , prevalence of aaa was significantly higher in current / past smokers compared to non - smokers ( p=0.046 ) . 
 of the 365 current / past smokers , aaa was found in 27 patients ( 7.4% ) , but of the 124 non - smokers , aaa was observed in only three patients ( 2.4% ) , and there were no nonsmokers with a large aaa ( > 40 mm ) . 
 determinants for taa , aaa and both taa and aaa are shown in table 2 . in the multivariate logistic regression analysis , 
 determinants for taa were age { odds ratio ( or ) 1.059 , 95% confidence interval ( ci ) 1.018 - 1.101 , p=0.005 } , absence of dyslipidemia ( or 0.621 , 95% ci 0.418 - 0.923 , p=0.018 ) , body surface area ( or 11.92 , 95% ci 2.787 - 50.97 , p=0.001 ) , diastolic blood pressure ( or 1.029 , 95% ci 1.009 - 1.049 , p=0.004 ) and presence of aaa ( or 3.070 , 95% ci 1.398 - 6.754 , p=0.005 ) . 
 in contrast , aaa was independently associated with presence of dysplipidemia ( or 2.792 , 95% ci 1.091 - 7.143 , p=0.032 ) , current / past smokers ( or 4.074 , 95% ci 1.160 - 14.31 , p=0.028 ) and presence of taa ( or 3.367 , 95% ci 1.550 - 7.313 , p=0.002 ) . 
 aortic size is critical to key decisions regarding management of aas.12 ) this study evaluated the entire aorta diameter , from the thoracic aorta to the abdomen aorta just above the iliac bifurcation , using axial images of non - contrast ct scans in a population with multiple risk factors for both chd and aa . 
 the study population included high - risk individuals , but blood pressure and serum lipids were well controlled in most patients , as indicated by blood pressure and laboratory test results . nevertheless , prevalence of taa was considerably high and taa was associated with various clinical factors including older age , larger bsa and higher diastolic blood pressure . 
 interestingly , taa was associated with the absence of dyslipidemia , while aaa was significantly associated with the presence of dyslipidemia . 
 one possible explanation might be that the etiology of aas differs between ascending and descending aorta.1 ) that is , above the ligamentumarteriosum the disease is non - atherosclerotic , while below the ligamentumarteriosum arteriosclerosis is abundant.1 ) therefore , taa might not be associated with dyslipidemia . 
 another explanation for the conflicting results might be that the mean values of total cholesterol , ldl - c and hdl - c were almost within normal limits , suggesting the serum lipids were well controlled in most patients . 
 in addition , since the current study was performed at a tertiary referral hospital , many of the patients were taking statins for the primary or secondary prevention for coronary artery disease , not for the treatment of dyslipidemia . 
 therefore , effects of high lipids on atherosclerosis might be minimized in the studied population and result in conflicting outcomes . 
 therefore , further studies are needed to clarify the relationship among taa , aaa , dyslipidemia , serum cholesterol and statin therapy . 
 thoracic aortic aneurysm was more prevalent in patients with aaa compared to those without aaa , even after adjusting for multiple confounding factors . 
 in addition , more than one - third of the study subjects had a taa , and it was remarkably more prevalent than aaa among high - risk subjects in the current study , in contrast to previous reports that aaa is more prevalent than taa.1 ) taa prevalence continuously increases,13 ) and is thought to be related to increased age and hypertension in the general population . 
 however , most taas had a diameter < 50 mm , which is not large enough to indicate a need for immediate intervention . therefore , the clinical implications of the high prevalence of taa need further evaluation , since taas are known to be an indolent process and to grow very slowly , at approximately 0.1 cm per year.9)14 ) future studies should establish a method for aortic screening for taa and strategies for conservative as well as surgical management in elderly hypertensive patients . 
 previous studies consistently demonstrated a relationship between smoking and aaa.11 ) the present study , which involved a high - risk population of asian males , found that aaa was independently associated with smoking history in addition to dyslipidemia . 
 moreover , prevalence of aaa was 2.4% among nonsmokers , which is very low compared to previous studies involving caucasians.15)16 ) in addition , none of the nonsmokers had a large aaa ( > 40 mm ) . in population - based screening studies in japan , 
 aaa was rarely encountered17)18 ) and the prevalence of aaa was also reportedly low in chinese populations , even in patients with severe coronary artery disease.19 ) this suggests that race influences aaa prevalence and that asians generally have a low prevalence of aaa . 
 the present study also reported a low prevalence of aaa in an asian population and reinforced the impact of smoking on aaa . 
 these results high - light the need to clearly define the usefulness of routine screening for aaa among asian non - smokers . 
 the primary limitation of this study is that a ct scan with axial images could not properly evaluate the very proximal portion of the ascending aorta including the aortic root . 
 in addition , even in the sagittal and coronal planes , aortic root diameter could not be accurately measured using non - contrast ct due to the complexity of the structures surrounding the aortic root . therefore , different imaging modalities are needed to accurately detect aortic root aneurysms . 
 this study was performed in a population of elderly asian males with hypertension at high risk for cardiovascular disease . 
 therefore , the results of the current study can not be applied to females , other ethnicities , or groups with a low - risk of cardiovascular disease . 
 the prevalence of aa was significant and taa was more prevalent than aaa in elderly korean males with hypertension who had multiple risk factors for chd and aa . 
 future research should establish distinct screening strategies for taa and aaa according to risk factors and ethnicity . 
 the primary limitation of this study is that a ct scan with axial images could not properly evaluate the very proximal portion of the ascending aorta including the aortic root . 
 in addition , even in the sagittal and coronal planes , aortic root diameter could not be accurately measured using non - contrast ct due to the complexity of the structures surrounding the aortic root . 
 this study was performed in a population of elderly asian males with hypertension at high risk for cardiovascular disease . 
 therefore , the results of the current study can not be applied to females , other ethnicities , or groups with a low - risk of cardiovascular disease . 
 the prevalence of aa was significant and taa was more prevalent than aaa in elderly korean males with hypertension who had multiple risk factors for chd and aa . 
 future research should establish distinct screening strategies for taa and aaa according to risk factors and ethnicity .","background and objectivesscreening strategies for aortic aneurysm ( aa ) according to risk factors and ethnicity are controversial . 
 this study explored the prevalence of aa and determined whether screening is necessary in a population of multiple risk factors.subjects and methodsfrom june , 2012 to april , 2013 , 542 consecutive elderly ( 65 years ) male hypertensive patients without a history of aa were prospectively enrolled . after excluding 15 patients ( 2.8% ) with aortic valve surgery , 30 patients ( 5.5% ) with suboptimal computed tomography ( ct ) images , the remaining 496 patients ( age 735 years ) comprised the study population . 
 maximal diameters of the thoracic and abdominal aorta were measured using non - contrast ct.resultsthe prevalence of thoracic aa ( taa , diameter 40 mm ) and abdominal aa ( aaa , diameter 30 mm ) was 36.5% ( 181/496 ) and 6.0% ( 30/496 ) , respectively . in the multivariate logistic regression analysis , 
 determinants for taa were age { odds ratio ( or ) 1.059 , 95% confidence interval ( ci ) 1.018 - 1.101 , p=0.005 } , dyslipidemia ( or 0.621 , 95% ci 0.418 - 0.923 , p=0.018 ) , body surface area ( or 11.92 , 95% ci 2.787 - 50.97 , p=0.001 ) , diastolic blood pressure ( or 1.029 , 95% ci 1.009 - 1.049 , p=0.004 ) and aaa ( or 3.070 , 95% ci 1.398 - 6.754 , p=0.005 ) . 
 in contrast , aaa was independently associated with dysplipidemia ( or 2.792 , 95% ci 1.091 - 7.143 , p=0.032 ) , current / past smokerfs ( or 4.074 , 95% ci 1.160 - 14.31 , p=0.028 ) , and taa ( or 3.367 , 95% ci 1.550 - 7.313 , p=0.002).conclusionthe prevalence of aa was significant and taa was more prevalent than aaa in elderly korean males with hypertension . 
 future research should establish distinct screening strategies for taa and aaa according to risk factors and ethnicity .",pubmed
"in developed countries , endometrial cancer morbidity has exceeded that of cervical cancer to become the most common invasive malignancy of the female genital tract . 
 early screening methods cervical cancer have made major progress because of the improvement of cytologic preparation methods and unified evaluation system in the 1950s . 
 drawing from the successful experience of cervical carcinoma screening , we are pressed for an effective approach to screening endometrial carcinoma and its precursor to reduce new cases and deaths . 
 endometrial complex hyperplasia with atypia and endometrial intraepithelial neoplasia ( ein ) are considered as precursors of type i endometrioid adenocarcinoma , and endometrial glandular dysplasia is the precursor of type ii carcinoma . a certain period of time for these lesions developing to carcinoma 
 traditionally , endometrial samples for histologic analysis can be obtained from d & c or under hysteroscope , and these methods are considered to be reliable for evaluating the endometrial condition . to date , the adequacy of specimens obtained using the sap-1 device compared to specimens obtained by d & c or hysteroscopic biopsy , which is better for evaluating the condition of the endometrium , is unknown . 
 the aim of this study was to investigate whether liquid - based surepath preparation for endometrial cytology test ( ect ) can maintain the three - dimensional structure of the endometrium . 
 histology is the gold standard for evaluating the accuracy , sensitivity , specificity , positive prediction value ( ppv ) , and negative prediction value ( npv ) of ect . 
 this study was performed from november 2011 to may 2014 at the obstetrics and gynecology department of peking university first hospital and it had been approved by the hospital ethics committee . 
 the risk factors included : ( 1 ) age  40 years ; ( 2 ) intrinsically high estrogen including polycystic ovarian syndrome and ovarian tumors with an abnormal level of estrogen such as granulosa cell tumors ; ( 3 ) extrinsically high estrogen including hormone replacement therapy and postoperative breast cancer patients taking tamoxifen ; ( 4 ) high body mass index ( bmi ) 25 kg / m ; ( 5 ) hypertension , hyperlipidemia and diabetes ; ( 6 ) family history of cancer including hereditary nonpolyposis colon cancer , lynch syndrome , and first - degree relatives with gynecologic tumor ; ( 7 ) history of radiation and smoking ; ( 8) abnormal uterus bleeding ( aub ) , especially postmenopausal vaginal bleeding ; ( 9 ) abnormal endometrium assessed by ultrasound as follows : thickness ( postmenopausal women 4 mm or menopausal women 20 mm ) , occupation disease of the uterine cavity or heterogeneous . while patients with following conditions were excluded out of this study : ( 1 ) ultrasound scanning suggested uterus cavity was distorted by multiple uterine myomas or adenomyoma ; ( 2 ) patients with intra - uterine device . 
 they provided written informed consent and underwent an ect using the direct sampler sap-1 device ( saipujiuzhou corporation , beijing , china ) [ figure 1 ] . 
 this device was patented and received permission to use it in our clinic in china . the sap-1 sampling device . 
 it consists of a flexible latex loop with spines on the side and a smooth tip to prevent injury to the myometrium . 
 there is an outer protective sheath outside the loop to prevent contamination from cervical and vaginal cells . 
 it is easy to operate and can be used in an outpatient setting , at health examination centers and in community hospitals . 
 steps for obtaining samples using the sap-1 device the loop with the specimen was then immersed in the surepath cell preservation container ( bd diagnostic , burlington , nc , usa ) to release the cells . 
 the 10 ml specimen was transferred into centrifuge tubes with a density reagent ( bd diagnostic , burlington , nc , usa ) to remove blood and mucus . 
 after a two - stage centrifugation at 1000 rpm for 2 min 15 s and then 2000 rpm for 10 min 15 s ( rotina 46s , hettich corporation , german ) , the centrifuge tube was put into the surepath semi - automated slide processor and stained using papanicolaou . 
 the cytological smears were evaluated by two independent gynecological cytologists who were blinded to the study procedures . 
 based on a previously - published diagnostic system , the cytological results were subdivided into four categories as follows : negative for epithelial lesions , benign endometrium , atypical endometrial cell , and suspected for malignant [ figures 3a  d and 4a  d ] . 
 endometrial tissue samples were fixed in neutral buffered formalin , embedded in paraffin , and stained with hematoxylin and eosin . 
 two gynecological pathologists independently assessed the slides , based on the world health organization diagnostic criteria and ein . 
 if a normal or benign endometrium was given , d & c or hysteroscopic pathology was regarded as a final result , we occasionally encountered a situation as cell features fall short of the criteria of simple / complex hyperplasia with atypia , we should carefully evaluated these lesions to determine whether or not a diagnosis of ein could be made . 
 ( a ) negative for endometrial lesions : long , straight tube - shaped cell clumps with a small amount of stromal cells on the margin is the most common type of cell clumps in the proliferative endometrium , observed using a low - power microscope ( papanicolaou stain , 20 ) ; ( b ) benign endometrium : dilated and branched cell clumps are always seen . 
 the contour of the cell clumps is smooth and occasionally a few stromal cells can be observed ( papanicolaou stain , 40 ) ; ( c ) atypical endometrial cell : double - layer or folded irregular cell clumps are observed ( papanicolaou stain , 20 ) ; ( d ) suspected endometrial carcinoma : papillo - shaped bordered cell clumps with atypical cells can be observed ( papanicolaou stain , 100 ) . 
 ( a ) negative for endometrial lesion : regularly arranged and mono - layer endometrial cells with an oval or round nucleus . 
 the spaces between nuclei are regular , and the chromatin in endometrial cells is delicate ( papanicolaou stain , 100 ) ; ( b ) benign endometrium : crowded cells arranged into a single layer with delicate chromatin and a small nucleolus ( papanicolaou stain , 100 ) ; ( c ) atypical endometrial cell : the spaces in atypical cells are heterogeneous . 
 the chromatin is coarse ( papanicolaou stain , 100 ) ; ( d ) suspected carcinoma : variable size cells with obviously round nucleoli . 
 the contingency table chi - square test was used to compare the adequacy of specimens collected using sap-1 with those collected via d & c and hysteroscopic biopsy . 
 negative for epithelial lesions and benign endometrium were considered negative , while atypical endometrial cells and suspected for malignant were considered positive . 
 histopathologic results were the gold standard . a four - fold table was created to calculate the accuracy , sensitive , specificity , ppv , and npv . 
 the contingency table chi - square test was used to compare the adequacy of specimens collected using sap-1 with those collected via d & c and hysteroscopic biopsy . 
 negative for epithelial lesions and benign endometrium were considered negative , while atypical endometrial cells and suspected for malignant were considered positive . 
 histopathologic results were the gold standard . a four - fold table was created to calculate the accuracy , sensitive , specificity , ppv , and npv . 
 the current study comprised of 1514 patients with endometrial carcinoma risk factors underwent sap-1 sampling on an outpatient basis , and 375 of these women also underwent d & c or hysteroscopy . 
 characteristics including age distribution , menstrual status , and patients symptoms and signs are presented in table 1 . 
 most of the patients were over 40 years of age in both the cytology and histopathology groups ( 91.2% and 90.7% , respectively ) . 
 the percentage of postmenopausal women was 69% and 63.5% , respectively , in the two groups . among 1514 cases with cytological specimens , 576 ( 38% ) patients had aub , and 910 ( 60.1% ) had  4 mm thickness endometrium measured by ultrasound . 
 169 ( 45.1% ) out of 375 patients with histopathology had aub , and 245 patients ( 65.3% ) had  4 mm thickness endometrium . 
 patient characteristics aub : abnormal uterus bleeding ; some patients were not examined by ultrasound , and their endometrial thickness is missing . as presented in table 2 , 1458 patients ( 96.3% ) had adequate specimens for cytology out of the 1514 patients sampled using sap-1 , while 285 cases ( 76% ) had adequate specimens for pathology out of the 375 patients who underwent d & c or hysteroscopic biopsy . 
 there were 56 ( 3.7% ) cytology and 90 ( 24% ) biopsies that were inadequate . 
 sap-1 can provide more sufficient materials for cytology than d & c or hysteroscopic biopsy for histology ( p < 0.01 ) . 
 adequate and inadequate specimens obtained using cytology and biopsy of the 469 cytology specimens from premenopausal women , 452 patients ( 96.4% ) had an adequate specimen , and 17 patients ( 3.6% ) had an inadequate specimen . while in 137 cases of premenopausal women obtained biopsy specimens , 132 patients ( 96.4% ) had an adequate biopsy , and only 5 patients ( 3.6% ) had an inadequate biopsy . 
 there was no difference ( p = 0.919 ) between cytology and biopsy in premenopausal women . 
 however , 1006 specimens ( 86.3% ) were adequate , and 37 specimens ( 3.7% ) were inadequate out of the 1045 cytology samples obtained using sap-1 in postmenopausal women . in 238 biopsy specimens obtained from postmenopausal women , 153 biopsy specimens ( 64.3% ) were adequate , and 85 specimens ( 35.7% ) were inadequate . 
 it was easier to collect cytology specimens than histology specimens ( p < 0.05 ) . 
 patients with both cytologic and histologic results n = 254 patients ; negative and benign were served as negative ; atypia and carcinoma were as positive . of the 254 patients who had both of cytology and histology , 205 ( 80.7% ) 
 11 ( 4.3% ) patients were evaluated as positive by cytology , but they were confirmed as negative or benign lesions using histology . there were 28 patients who were positive for atypical cells , as determined by both cytology and histology . 
 there were 10 ( 3.9% ) patients who were diagnosed as negative or benign using cytology , while their occult lesions were discovered upon subsequent hysterectomy . 
 the accuracy of cytology for detecting endometrial precursor and carcinoma was estimated at 91.7% , sensitivity at 73.6% , specificity at 94.9% , ppv at 71.9% , and npv at 95.3% . 
 in past decades , endometrial carcinoma has become the most prevalent gynecologic malignancy in developed cities such as beijing , shanghai , and guangzhou in china . 
 the increased morbidity of endometrial carcinoma is attributed to changes in lifestyle , such as a lack of exercise and increasing fat intake lead to obesity and high bmi . 
 obesity triggered several pathways including hormonal imbalance and hyperactive proliferative pathways to involve in pathogenesis of endometrial carcinoma . 
 the effective procedure for reducing morbidity and mortality in endometrial carcinoma is to screen certain groups with risk factors and design a reliable tool suitable for a mass screening program . 
 dilation and curettage have been used clinically for many years , and originally it was intended to serve as a screening tool for endometrial carcinoma , but researchers realized that specimen adequacy and diagnosis accuracy were unsatisfactory . 
 yarandi et al . reported that the accuracy of d & c was 40.5% , sensitivity 40.5% , specificity 72.3% , ppv 77.1% and npv 25.1% , and in 52.7% of the patients , d & c failed to detect intrauterine disorders , especially focal lesions of the endometrium . 
 moreover , most patients complained of pain and severe discomfort during the operation , and they had persistent bleeding for several days after the procedure . in mossa 
 's study , the patients undergoing d & c had higher pain scores compared with those who underwent brush cytology . 
 currently , several sampling devices were used to take endometrium cytology samples from patients ; samplers include endoflower , tao brush , and endocyte . for tao brush sampling , 
 estimated that out of 519 patients , the samples of biopsy obtained using the tao brush was inadequate in 361 patients ( 39% ) , and samples obtained using the endoflower were inadequate in 15 patients ( 2% ) . 
 in addition , buccoliero et al . showed the same results in another study that compared the endoflower to biopsy in patients receiving tamoxifen . 
 sap-1 , a direct endometrial sampler device , is especially designed for minimal pain during sampling , it meets the requirements for endometrial samplers , which include : ( 1 ) avoiding contamination from the endocervix and vagina ; ( 2 ) procuring an adequate representative sample of the entire endometrium to detect focal lesions ; and ( 3 ) the procedure should be safe , easy to use , and well - tolerated by the patients . 
 the device has to be noninvasive or minimally invasive , cost - effective , and user - friendly to be accepted by primary care physicians and patients for repeated tests 
 . a soft loop can be flexible within the endometrial cavity , and the spindles can easily obtain adequate endometrial cells with minimal chance of injuring myometrium and bleeding . 
 we are the first to evaluate the adequacy of its specimens compared with d & c and hysteroscopic biopsy . in the current study , 
 adequate specimens obtained using the sap-1 sampler ( 96.3% ) are superior to those obtained using d & c ( 76% ) . 
 in addition , most patients experienced no pain during sap-1 sampling , claiming that they had the same feeling as the insertion of a cytobrush for cervical sampling . 
 our previously - proposed screening strategy involved women of 40 years of age or older , or postmenopausal women as the target screening population . in buccoliero 
 's study , of 107 asymptomatic postmenopausal women with a thin endometrium ( < 4 mm ) , a biopsy obtained sufficient material for the diagnosis in only 24% of the cases . 
 our study also shows that there are no differences in specimen adequacy between the sap-1 sampler and d & c in premenopausal women . 
 however , for postmenopausal women , the sap-1 sampler provides more adequate specimens than does d & c or hysteroscopic biopsy . 
 conventional smears have not been widely applied in endometrial carcinoma screening attributes of overlapping cells and a heavy background . 
 in addition , the three - dimensional structure is an important ect reference to evaluate the condition of the endometrium 
 . requirements for a stable fixation system and preparation technique include : ( 1 ) preserving the morphology of endometrial epithelial and stromal cells and the endometrial glandular structure ; and ( 2 ) eliminating obscuring factors such as excess red blood cells , mucus , overlapping cells , and inflammatory cells . the 95% alcohol fixation and conventional cytology can not earn a place in endometrial cancer screen due to regression cells and high background levels , such as excess blood , mucus , overlapping cells , and inflammatory cells . according to previous japanese publications , 
 conventional cytology was initially used to examine the endometrial lesion , with a sensitivity about 78% , specificity of 95% , ppv of 56% , and npv of 98% . 
 transvaginal ultrasonography was previously thought to be an efficient tool because of conventional cytology 's low accuracy . compared with a conventional smear , 
 thin - layer cytology provided more cell clumps and a clearer background , as shown by norimatsu et al . to improve the diagnostic accuracy of endometrial cytology , 
 remondi et al . used the endoflower sampler and the thinprep preparation to evaluate 768 postmenopausal women , and found an accuracy of 93.6% , sensitivity of 92% , specificity of 95% , ppv of 73% , and npv of 99% . 
 the sensitivity , specificity , ppv , and npv for the uterobrush were 88.9% , 100% , 100% and 98.9% , respectively . 
 direct uterine sampling with the tao brush sampler using a liquid - based preparation method for detection of endometrial cancer and atypical hyperplasia , the sensitivity and specificity were 95% and 66% . 
 the results of the current study demonstrate that the sap-1 sampler combined with the liquid - based surepath preparation method is a reliable diagnostic method . 
 the overall accuracy for detecting endometrial cancer , complex hyperplasia with atypia and ein was 92.4% , with a sensitivity of 73% , a specificity of 95.8% , a ppv of 75% , and a npv of 95.3% . 
 almost all patients enrolled into this study received the ect for screening , so women with positive cytologic results or persistent bleeding underwent hysteroscopic biopsy or d & c. in the current study , patients with carcinoma that was subsequently confirmed by histology were initially diagnosed with atypical endometrial cells or suspected carcinoma using cytology . 
 the main reasons that the accuracy of this study was lower than that in previously studies are as follows : patients with positive results including atypical cells underwent hysteroscopic biopsy and d & c , and some of them were subsequently diagnosed as normal or benign . 
 in addition , other methods , including enlarging the sample quantity to collect more information to correctly distinguish focal atypical endometrial lesions from benign lesions , should be investigated using immunohistochemistry and biochemistry to improve the accuracy of endometrial cytology . in conclusion 
 , the sap-1 sampler combined with surepath preparation may become a reliable method for screening endometrial carcinoma and its precursor , especially in postmenopausal and asymptomatic women . 
 if this screening procedure can be used in the high risk - factor women , some unnecessary d & c may be avoided , and asymptomatic women with the precursor may benefit from early detection and management .","background : the aim of this study was to compare specimen adequacy of sap-1 provided for cytology with that of dilation and curettage ( d & c ) or hysteroscopy for histology , and evaluate the accuracy of combining endometrium sampling by sap-1 and liquid - based cytology using surepath preparation for screening endometrial carcinoma and its precursor.methods:endometrial specimens from women ( n = 1514 ) with risk factors were obtained using an sap-1 device for cytological analysis ; histological samples were obtained from 375 of these women who underwent d & c or hysteroscopy . 
 cytological specimens were prepared to liquid - based smear using surepath technology and stained by papanicolaou . 
 histological samples were processed in routine pathology and stained by hematoxylin and eosin.results:adequate specimens for cytology were obtained from 1458/1541 patients ( 96.3% ) , while adequate samples for pathology were obtained from 285/375 patients ( 76% ) . however , for postmenopausal women , 1006 of 1045 cytology ( 86.3% ) were adequate , 153 of 238 histology ( 64.3% ) were adequate , it was easier to collect cytological specimens than histological specimens ( p < 0.05 ) . 
 the accuracy of endometrial cytology for detecting endometrial carcinoma and its precursor was 92.4% ( sensitivity , 73% ; specificity , 95.8% ; positive predictive value , 75% ; and negative predictive value , 95.3%).conclusions : endometrial cytology using sap-1 sampling and surepath preparation may be a reliable approach for screening patients with endometrial carcinoma and its precursor .",pubmed
"breast cancer is the most common cancer worldwide among women , with nearly 1.7 million new cases in 2012 . 
 the highest incidence rates are in europe and north america , and by country are highest in belgium , denmark , and france.1 breast cancer mortality has decreased in some parts of the world , with screening mammography associated with an up - to-50% reduction in mortality rate.2 although there has been controversy about mammogram screening , it remains the most commonly performed procedure for breast cancer detection.3,4 the results of the digital mammography imaging screening trial ( dmist ) proved digital mammography ( dm ) was as efficacious as screen - film mammography ( sfm).5 as such , most of the mammography units in the us are now digital . 
 dm is less likely to be lost or damaged , and has slightly higher sensitivity than sfm.5 however , despite evidence from dmist that dm offers some improved benefit in women with dense breast tissue , this remains a limitation of conventional mammography in general.5,6 breast cancers will have the same density as surrounding tissue , and can be obscured by overlapping tissue.7,8 with tumors potentially obscured by overlapping tissue in two - dimensional ( 2-d ) conventional mammography , a 3-d procedure , such as tomosynthesis , may help solve this problem.7 also , there is hope that the use of intravascular contrast material with dm will take advantage of malignant neovascularity to enhance tumor detection . 
 this article reflects on these technologies in mammographic imaging , current uses , and potential patient impact . 
 previous reviews of tomosynthesis913 have summarized technical developments and the promising early results of small digital breast tomosynthesis ( dbt)-reader studies using selected tomosynthesis cases . 
 we add in this review newer prospective population - screening trials from oslo , italy , and australia , which provided a larger amount of data and more definitive results acquired from clinical settings.1417 the newest developments in solving the challenge of radiation exposure in tomosynthesis are presented . 
 furthermore , this review will address a wider audience than radiologists , to inform those of any medical specialty hoping to learn about developments in mammography . 
 sfm has been in use for over 30 years , and in the last 10 years has been replaced in most practices in the us by full - field dm.18 this advance enables viewing and manipulation of dm images on computer workstations . 
 with these steps separated , each can be potentially optimized.19 dm is similar to sfm in that both are performed with two views of each breast in screening mammography , a compromise between keeping radiation levels low yet imaging the majority of the breast tissue . 
 2-d  is a common abbreviation for two - view dm.9 radiation dose is a concern , given that mammography is a screening examination performed on millions of women potentially every year . 
 the dm systems used in the dmist trial were shown to have had a 22% lower mean glandular dose than sfm per acquired view.20 dose parameters reported for dmist were for state - of - the - art equipment used during the trial , and many of the digital systems have been refined since , and performance may have improved.2023 a specific digital system using slot scanning with a photon - counting detector that eliminates stray or scatter radiation and improves noise reduction and image quality has been found to reduce doses by 40%60%,24,25 and has shown the lowest mean glandular dose in clinical screening settings in europe.26 indeed , it was found in a dose survey of the irish breast - screening program that the lower dose resulted from the use of dm systems , and the photon - counting system accounted for 25% of mammography units in ireland.27 clinical performance for this system is comparable to other dm systems in screening,28 and has not been shown to be inferior in radiologist performance.25 one limitation of both sfm and dm is false positives ( recall for additional imaging ) . 
 women have been recalled for additional imaging from the earliest days of mammogram screening . since screening mammography 
 is performed on normal asymptomatic women , minimizing harm is important.3,4 the false - positive rate of mammography is one of the recurrent criticisms of mammography.3,4 in the us , up to 10% of screened women are recalled for further mammogram views , ultrasound imaging , and/or biopsies , and most of these women do not have cancer.4,5,29 screening mammography can not fully assess all abnormalities on the basis of the two routine views . 
 recalls are necessary , because a possible lesion needs to be distinguished from a true lesion , but recalls can also lead to false - positive biopsies . 
 these evaluations are typically time - consuming for the radiologist and patient , and stressful and inconvenient for the patient . 
 nonetheless , recall improves breast cancer detection , and a balance is sought between increased sensitivity and improved specificity.29 recall rates in the us are about twofold higher than in europe , with guidelines in the us at overall < 10% , compared to guidelines in europe of 3%7% ( < 5%).20 if the additional views for evaluation of abnormal screening mammograms could be reduced , this would be advantageous to patients , referring physicians , and radiologists . 
 sensitivity on average is about 70%.5 the problem of false - positive mammogram findings and missed lesions is caused by the overlapping tissue in 2-d images.7,8 abnormalities may be hidden in dense fibroglandular tissue , and the malignant features of cancer may be obscured . 
 the american college of radiology breast imaging reporting and data system describes four breast parenchymal patterns used in mammography reports , with 
 fatty  having the least amount of fibroglandular tissue and  extremely dense  having the most ( figure 1).30 the number of women with dense breast tissue is not insignificant . over half of women younger than 
 50 years have what is considered dense breast tissue , and a third of women over 50 years.31 in women with the densest breasts , the sensitivity of mammography may be as low as 30%48%,32,33 and has been found to be a major risk factor for interval cancers.32 women with mammographic density in 75% or more of the breast have been shown to have an increased risk of breast cancer , either detected by screening or less than 12 months after a negative screening mammogram ( interval cancer),34 and the risk was found to be greater in younger women.34 it is not known to what extent the increased risk of interval cancer is due to masking of cancer from dense tissue or to rapid growth of tumors in dense tissue.32,35,36 
 dbt also uses x - rays like conventional mammography , but creates image slices of a tissue volume . 
 the slices are thin , and each is seen as a 1 mm plane in sharp focus , with the tissue above and below a plane appearing out of focus . 
 the thin slices decrease the problem of confusing overlapping tissue.10 dbt is a mammography - based technique using breast compression similar to 2-d dm , but obtains multiple low - dose images using a moving x - ray tube head rotating across the breast in an arc of 15%50% ( figure 2).9,10,37 the time varies from about 5 to 25 seconds depending on manufacturer differences.10 the image data are used to reconstruct thin tissue slices , such as are seen in computed tomography scans.38 typically , the two conventional views ( craniocaudal and mediolateral oblique ) are done of each breast of a single breast compression per view . 
 the same anatomy of conventional mammograms is more clearly defined with dbt , with the breast anatomy separated into different tissue planes ( figure 3 ) . 
 dbt is therefore called quasi-3-d technology because it differentiates tissues in slices.37,38 total radiation exposure varies by breast density and thickness , but overall dbt radiation exposure is approximately the same as that of sfm or dm.9,13,39 since the system is a modified dm unit , dbt images can be obtained in any of the usual mammogram views or planes.10 compression of the breast for tomosynthesis is similar to that of conventional mammography.10 compression maximizes image quality , decreases patient motion , and decreases radiation dose , but is somewhat painful.40 however , since the problem of tissue overlap is improved by dbt , some reduction of compression is possible , improving patient comfort.40 the feasibility of using tomosynthesis was studied over 10 years ago,38 with us food and drug administration ( fda ) approval for a commercial system obtained in 2012.41 dbt can be used for screening ( two views , asymptomatic patients ) and diagnostic ( multiple views in symptomatic patients ) mammography.41,42 it has all the advantages of dm , such as fewer artifacts , consistent quality , and digital image processing.9,10 there is emerging evidence that adding dbt to standard mammography increases mammogram accuracy.12,13,43 limited studies in cancer - enriched groups demonstrated the potential to decrease recall rates and increase cancer detection.9,10,13,43,44 studies were done by acquiring both conventional mammogram views and at the same time dbt views . 
 these were done by exposing the patient twice , and thus resulted in a radiation dose about twice that of mammography alone , but nonetheless below the limits set by the mammography quality standards act ( mqsa).15,39,45 a recent population - based prospective clinical trial ( oslo trial ) studied the 50- to 69-year age group with biennial screening , in a single institution with a single group of radiologists , and 12,631 women participating . as in some of the early small studies , the partial results reported 
 that by integrating 2-d and 3-d ( ie , dm + dbt ) mammography screening , there was a 40% higher invasive cancer - detection rate compared to dm screening alone.15 another population - based prospective study has reported partial results of 7,292 screened women who similarly had dbt added to dm ( storm [ screening with tomosynthesis or mammography ] ) , and reported that cancer detection rose 34% and false - positive recalls could decrease by 17% without missing any cancers.45 the additional cancers detected were found in all breast densities , from fatty to dense . 
 the partial results of these clinical trials were still considered small and not adequate to determine which subgroups would benefit most by adding dbt to dm.46 subsequent additional analyses by subgroups reported that centers with high rates of false positives would benefit most from dbt , and that greater accuracy results from adding dbt to dm than from double - reading by dm alone.14,16,17 although using dm with dbt doubles the radiation dose in screening , it has been considered necessary for detection and characterization of calcifications , a potential sign of ductal carcinoma in situ , and additionally to enable orientation of dbt findings when comparing with prior mammograms.47,48 the research studies are promising , indicating that dbt is addressing some of the long - standing criticisms of conventional mammography . 
 the addition of dbt to dm ( ie , standard 2-d mammography ) improves the accuracy of interpretation of mammograms.13,15 as discussed earlier , findings questioned on conventional mammography , such as possible masses , are subsequently found on additional imaging to be overlapping glandular tissue . 
 these structures are more easily recognized to be normal on dbt , because only one thin plane of tissue is seen , diminishing confusing overlap . 
 common benign masses , such as cysts and fibroadenomas , may be more evident as benign masses , because the margins are better seen.7,37 the dbt data suggest that not only is there a reduction in recall rate with dbt49 but also that dbt can more clearly depict benign findings than routine screening views ( such as lymph nodes or skin calcifications ) , diminishing the need for recall for additional imaging . reducing recall rates 
 diminishes the so - called potential harm of recalling normal patients and decreasing the number of false - positive biopsies . in all of the early and most recent studies in both europe and the us , 
 the recall rate for most radiologists was reduced when adding dbt to dm , despite the already much lower recall rates in europe compared to the us.15,29,37,45,49 dbt appears to increase cancer detection , and the additional cancers detected are invasive malignancies missed by conventional mammography.15 ductal carcinoma in situ , a currently a controversial histologic abnormality versus an early breast cancer , is not more frequent with dbt.15 although dbt will help reveal more cancers in women , it will not detect all cancers.50 dbt shows promise in demonstrating the same or higher accuracy as digital spot - compression mammography.51 therefore , dbt alone can also eliminate the need for additional mammogram images in many cases of abnormal mammograms , as well as send patients directly to ultrasound without further mammography . 
 dbt also may decrease the necessity to perform extra mammogram views to identify the exact location of a breast lesion . 
 radiation dose and patient comfort would benefit if supplementary mammogram views were no longer necessary . 
 also , with the overall need for compression decreased for tomosynthesis , some studies found that dbt can be done with more patient satisfaction , because up to 50% less compression than dm can potentially be applied , with no loss in diagnostic capability.40 for all these reasons , as well as time savings , dbt benefits the patient and radiologist , with improved efficiency in detection and characterizing abnormalities . 
 this also translates into less stress for the patient when there is a decrease in the amount of additional imaging necessary for determining when a lesion is benign versus suspicious . 
 the radiation dose for the combined examination of conventional mammography and dbt is slightly more than twice the dose of mammography alone , because combining both dbt and dm delivers double the radiation dose to the breast . 
 although this is lower than mqsa limits , radiation has to be considered with the possibility of screening millions of women each year with double the radiation dose.39,52 to solve this problem , synthetically reconstructed 2-d images can be made from the dbt data , eliminating the need for double exposures.53 this eliminates the need to do an exposure for a dm , and also drops the radiation dose to that of conventional mammography . 
 the most recent studies of these synthesized images demonstrated similar accuracy as those of conventional dm , and fda approval was obtained in the us for the vendor hologic.48,54 therefore , potentially there will no longer be a need for radiation exposure to obtain 2-d images , and the dose for dbt will fall down to that of conventional mammography.37,54 radiologist - interpretation time is longer for dm + dbt than for dm alone.15 this is because a routine mammogram is four images , whereas a dbt can be 200 images or more per patient . 
 longer interpretation times may be offset by the decreased recall rates and decreased need for additional views.37 it is also uncertain how dbt use would be similar or different among radiologists with varying practice patterns and expertise.37 the cost of a dbt system is substantially greater than that of a dm system , and also has the expense of digital storage for the large file sizes of dbt . 
 however , compared with breast magnetic resonance imaging ( mri ) , dbt is a relatively low - cost innovation . 
 in addition , if dbt reduces recall rates , cost savings may result from fewer biopsies and recalls . at present , dbt is undergoing further technological changes and is a focus of clinical research trials under way or nearing completion . 
 large population studies are in progress , and in addition to those already mentioned , there is the malm trial , a uk trial , and a yale study.12 a clinical trial is planned in north america by the american college of radiology imaging network to compare dm to dbt ( t - mist [ tomosynthesis mammographic imaging screening trial ] ) in a multiple - vendor , multisite study . 
 its true performance may become more obvious when it has been integrated into such additional population screenings . incorporating dbt with other modalities 
 is also being studied , as well as contrast - enhanced tomosynthesis , that could provide an mri - like study while being simpler to perform and of much lower cost . automated breast ultrasound with dbt is also evolving , which may be very promising for screening dense breasts . 
 computer - assisted detection , used routinely in conventional mammography to increase sensitivity , is not yet incorporated into dbt but appears feasible,55 and may assist in improving detection of subtle lesions.37 
 investigators have evaluated the combination of dm with intravenous contrast enhancement to obtain more functional information from the neovascularity found in malignant tumors . 
 two methods described have been temporal contrast - enhanced dm ( cedm ) and dual - energy cedm . with cedm 
 , a dm unit has been modified to maximize the sensitivity of the unit to low concentrations of iodine contrast by increasing the voltage . 
 a mask image is obtained , and following the injection of contrast , sequential or temporal images are then obtained.56 the examination takes approximately 15 minutes , and the total dose is similar to a conventional single - view mammogram . in dual - energy cedm , 
 the dm unit is adapted to generate high energy exposures for energies above the energy absorption of iodine ( k - edge ) , taking advantage of the differences in x - ray attenuation through materials of different composition.56 contrast is injected with the breast in compression , and two paired exposures are obtained : one set at low energy and one at high energy . 
 this technique tends to limit problems with patient motion , as the patient is not in compression as long as with cedm , and two views of each breast are obtained and combined to enhance contrast - uptake areas.56 the use of contrast medium takes advantage of mapping abnormal blood flow , as does mri . 
 early evaluation of the feasibility of the use of contrast medium with dm revealed 80% of pathologically proven breast carcinomas were enhanced,57,58 with excellent correlation between the size of enhancement and the histologic size of tumors.58 this technique may be particularly useful in patients with dense breast tissue.5759 high sensitivity has also been found with contrast - enhanced dual - energy techniques , with sensitivities between 86%96%,6062 as well as with contrast use with dbt.63,64 promising results evaluating the feasibility of contrast - enhanced dbt has been shown in one small study.64 jochelson et al demonstrated dual - energy cedm was comparable to mri for the depiction of the index tumor in patients with known breast cancer , with each identifying 96% of cancers , and dual - energy cedm depicted additional cancers in the breast with better specificity.62 potential clinical applications of cedm include not only detecting mammographically occult cancers in dense breast tissue , but also like mri help to determine the local extent of disease , or assessment of residual or recurrent disease . 
 the potential advantages over mri are shorter examination time and ability to perform this exam at the same time as dm , image registration allowing easy comparison to conventional mammography , and preoperative guidance for biopsy if only seen with mammography.56 
 further developments in optimizing imaging parameters and continued improvements in decreasing radiation exposure are ongoing in dm . 
 dbt is proving to address some of the limitations of conventional mammography , with improved cancer detection , fewer false positives , and lower recall rates . 
 the true performance of dbt remains to be determined when the results of more large - population trials have concluded .","despite controversy regarding mammography s efficacy , it continues to be the most commonly used breast cancer - screening modality . with the development of digital mammography 
 , some improved benefit has been shown in women with dense breast tissue . 
 however , the density of breast tissue continues to limit the sensitivity of conventional mammography . 
 we discuss the development of some derivative digital technologies , primarily digital breast tomosynthesis , and their strengths , weaknesses , and potential patient impact .",pubmed
"over - the - counter ( otc ) cough and cold medications have long been criticized for lack of evidence supporting their efficacy . 
 this study demonstrates that diphenhydramine indeed inhibits cough reflex sensitivity.diphenhydramine , a commonly - used first - generation antihistamine in the us , is rightfully also classified as an antitussive ( cough suppressant ) in the united states food and drug administration ( fda ) otc monograph . over - the - counter 
 ( otc ) cough and cold medications have long been criticized for lack of evidence supporting their efficacy . 
 diphenhydramine , a commonly - used first - generation antihistamine in the us , is rightfully also classified as an antitussive ( cough suppressant ) in the united states food and drug administration ( fda ) otc monograph . 
 cough is the most common complaint for which patients in the united states seek medical attention . 
 although billions of dollars are spent annually on non - prescription , otc cough preparations , scant evidence exists supporting the efficacy of these drugs as antitussives . 
 three non - narcotic oral agents are listed as antitussives in the united states food and drug administration ( fda ) final monograph for otc antitussive drug products : chlophedianol , dextromethorphan , and the first - generation h1-antihistamine , diphenhydramine . 
 chlophedianol was first introduced as an antitussive in germany in the 1950s , but very little published literature supports its efficacy . 
 dextromethorphan , also available since the 1950s , is the most commonly used otc antitussive in the united states and many other areas of the world . although published studies exist demonstrating the ability of dextromethorphan to inhibit experimentally induced cough in healthy volunteers and to diminish pathological cough , most of these investigations are decades old and lack the more robust objective and subjective end points employed in current antitussive trials . 
 diphenhydramine has been shown to inhibit citric acid - induced cough in healthy volunteers , as well as to diminish pathological chronic cough relative to placebo . 
 furthermore , the guidelines of the american college of chest physicians ( accp ) recommend the combination of a first - generation h1-antihistamine and a decongestant as the treatment of choice for chronic cough due to upper airway cough syndrome ( formerly known as postnasal drip syndrome ) and acute cough due to the common cold , though this recommendation is based largely on a vast body of clinical experience and expert opinion , in the absence of adequately powered , prospective , randomized , controlled clinical trials . 
 acute cough due to viral upper respiratory tract infection ( uri ; common cold ) accounts for the majority of the approximately 27 million cough - related health care provider office visits annually in the united states , as well as the significant cost of otc products purchased to suppress troublesome cough . yet , to the authors knowledge , none of the three fda - approved otc antitussive products ( chlophedianol , dextromethorphan , and diphenhydramine ) has ever been shown to inhibit experimentally induced cough in subjects with acute cough due to uri . 
 capsaicin , the pungent extract of red peppers , has been shown in over three decades of clinical experience , to induce cough in a safe , dose - dependent and reproducible manner . 
 thus , capsaicin cough challenge testing has become an important tool in clinical research , allowing for the accurate measurement of the effect of a pharmacological intervention on the sensitivity of the cough reflex [ 9 , 10 ] . the standard end point measured in capsaicin cough challenge testing 
 is the concentration of capsaicin inducing 5 or more coughs ( c5 ) . in healthy volunteers , 
 this end point has been demonstrated to be highly reproducible , in the short - term ( 20  min to 14  days ) and long term ( months to years ) . 
 recently , it has been demonstrated that cough reflex sensitivity , though transiently enhanced during acute uri , remains stable in the first week of illness . 
 thus , in a clinical trial , any changes in cough reflex sensitivity observed during this period may be attributed to the pharmacological intervention being studied , rather than to spontaneous variation in cough reflex sensitivity . 
 to evaluate the effect of a diphenhydramine - containing , multicomponent syrup , compared with a dextromethorphan - containing syrup and placebo , on cough reflex sensitivity in otherwise healthy subjects with acute uri . 
 the study protocol was approved by the institutional review board of montefiore medical center , bronx , ny . 
 otherwise healthy adult nonsmokers with the onset of symptoms consistent with acute viral upper respiratory tract infection ( uri ) within the previous 72  h were recruited and enrolled after providing informed consent . 
 the study protocol was approved by the institutional review board of montefiore medical center , bronx , ny . subjects did not have a history of asthma or other pulmonary disease , nor history or symptoms suggestive of gastroesophageal reflux disease . 
 subjects presenting with symptoms suggestive of influenza , such as high fever , myalgias , or severe illness , were excluded . 
 individuals with uri who had taken any cough / cold medicines for their illness were excluded , as were those currently receiving any medication known to affect cough reflex sensitivity , or who had received any such medication within a time frame that would leave the possibility of a lingering effect of a previously - ingested medication on the sensitivity of the cough reflex . upon enrollment 
 , subjects were randomized to receive each of the three study formulations within a 35  days period , in random order , 2  h prior to capsaicin challenge testing , in a double - blind fashion . 
 the study drugs consisted of : diphenhydramine ( 25  mg ) and phenylephrine ( 10  mg ) in a natural cocoa formulation ( dr . cocoa , infirst healthcare , london , uk ) ; dextromethorphan ( 30  mg ) syrup with licorice and sugar water among inactive ingredients ( father john s medicine , oakhurst co. , levittown , ny , usa ) ; and , a solution of dextrose in water . a post - ingestion period of 2  h 
 was chosen to coincide with near - peak blood concentrations of diphenhydramine   and dextromethorphan [ 2 , 13 ] . 
 study drugs were prepared as 20  ml liquid doses in opaque bottles that each subject was instructed to drink , without commenting on the taste or smell of the liquid . 
 the investigators therefore were unaware of the appearance , smell , and other characteristics of the study drugs administered . 
 briefly , subjects inhaled single , vital - capacity breaths of ascending , doubling concentrations ( range 0.491000  m ) of aerosolized capsaicin solution , administered via a compressed air - driven nebulizer controlled by a dosimeter , with 1-min intervals between inhalations , until 5 or more coughs resulted in the 15  s following an inhalation 
 the end point of capsaicin challenge testing is the concentration of capsaicin inducing 5 or more coughs ( c5 ) . to evaluate the effect of each of the three study formulations , differences in log c5 responses 
 were analyzed by 1-way anova for dependent samples combined with tukey s hsd test for pairwise comparisons . 
 twenty - two subjects [ 16 females ; mean age 39.2    13.3(sd ) year ] were recruited and completed the study . 
 sd ) log c5 values observed after administration of each of the study drugs were : 0.57    0.75 after placebo ; 0.80    0.83 after dextromethorphan ; and , 0.97    0.74 after the diphenhydramine - containing formulation ( fig .  1 ) . 
 differences in log c5 responses were analyzed by 1-way anova for dependent samples combined with tukey s hsd test for pairwise comparisons . a significant difference ( p  =  0.0024 ) 
 was established among the groups , with pairwise analysis revealing a significant increase in mean log c5 [ 0.4    0.55(sd ) ; p  <  0.01 ] for the diphenhydramine - containing medication versus placebo , but not for dextromethorphan versus placebo.fig . 
 1values for cough reflex sensitivity to capsaicin ( log c5 ) determined 2  h after ingestion of diphenhydramine ( dph ) , dextromethorphan ( dx ) , and placebo in adult nonsmokers with acute viral upper respiratory tract infection . 
 cough reflex sensitivity was significantly inhibited ( log c5 increased ) after diphenhydramine ( p  <  0.01 ) . 
 error bars represent sem values for cough reflex sensitivity to capsaicin ( log c5 ) determined 2  h after ingestion of diphenhydramine ( dph ) , dextromethorphan ( dx ) , and placebo in adult nonsmokers with acute viral upper respiratory tract infection . 
 cough reflex sensitivity was significantly inhibited ( log c5 increased ) after diphenhydramine ( p  <  0.01 ) . 
 error bars represent sem in terms of side effects , five subjects reported slight drowsiness after ingesting the diphenhydramine - containing formulation , as did one subject after placebo . 
 five subjects reported side effects after ingesting the dextromethorphan syrup : slight drowsiness in two subjects , slight nausea in two subjects , and dry mouth in one subject . 
 in the united states , the majority of otc cough preparations contain dextromethorphan or diphenhydramine as the pharmacologically active component . 
 significant criticism has been directed at the otc cough and cold market because of the lack of adequately performed clinical trials demonstrating these agents to be effective antitussives [ 2.7 ] . 
 to the authors knowledge , this study represents the first demonstration of the ability of diphenhydramine to suppress cough reflex sensitivity in subjects with acute uri . 
 furthermore , the diphenhydramine - containing formulation proved a more effective antitussive than did dextromethorphan , with both agents administered at standard antitussive doses . 
 however , it should be noted that cough reflex sensitivity was measured 2  h after study drug administration , to coincide with near - peak blood concentrations of the agents under investigation . 
 such timing of the cough challenge may not have allowed demonstration of the maximal antitussive effect of dextromethorphan , as a recent study of healthy volunteers found that maximal inhibition of capsaicin cough sensitivity by dextromethorphan was not observed until 6  h after oral administration . 
 the multicomponent diphenhydramine - containing syrup investigated in this study also contains the decongestant phenylephrine at standard otc dose as well as natural cocoa flavoring . to our knowledge , 
 theobromine , a component of cocoa , has been shown to have antitussive effect in healthy volunteers in one previous study , however , the amount of theobromine contained in one dose of the medication evaluated herein is much smaller than that required for cough reflex inhibition . 
 nevertheless , the thickness and cocoa flavor of the diphenhydramine - containing formulation may be contributing to the overall efficacy of the medication by creating a demulcent effect that has been proposed as an important component of the perceived therapeutic effect of cough syrups . 
 the diphenhydramine - containing syrup contained a natural cocoa flavoring ; the dextromethorphan - containing syrup contained licorice and sugar water ; and , the placebo was a dextrose solution . 
 however , we do not feel that the lack of perfect blinding affected our results . 
 subjects knew that during the three study days , they would be receiving two active medications and a placebo . 
 they were unaware , of course , of which flavorings the active and placebo formulations would have . 
 had subjective end points been examined , especially soon after drug administration , then certainly the possibility of a demulcent effect of the various liquids may have contributed to subject perception and experience . 
 however , our study measured only the objective end point of cough reflex sensitivity to capsaicin , 2  h after study drug administration , by which time any local throat sensations and demulcent effects would have dissipated . 
 it is noteworthy that a recent study demonstrated that sweet substances can affect cough reflex sensitivity to capsaicin . 
 thus , our placebo preparation was also sweetened so as to present subjects with sweet liquids on each of the 3  days of testing . 
 although the first - generation antihistamine , diphenhydramine , is classified as an antitussive by the fda and is a component of numerous otc cough and cold preparations , the present study , to our knowledge , contributes the initial evidence demonstrating the ability of this agent to inhibit cough reflex sensitivity in acute pathological cough . 
 further clinical trials are needed to adequately evaluate this and other otc cough and cold products , so as to allow physicians and consumers alike to make informed treatment decisions based on proper scientific data .","background : currently available over - the - counter cough remedies historically have been criticized for lack of scientific evidence supporting their efficacy . 
 although the first - generation antihistamine diphenhydramine is classified as an antitussive by the united states food and drug administration , to the authors knowledge it has never been shown to inhibit cough reflex sensitivity in subjects with pathological cough . 
 objective : to evaluate the effect of diphenhydramine on cough reflex sensitivity . setting : montefiore medical center , an academic medical center in new york city . 
 methods : twenty two subjects with acute viral upper respiratory tract infection ( common cold ) underwent cough reflex sensitivity measurement employing capsaicin challenge on 3  separate days , 2  h after ingesting single doses of study drug ( to coincide with peak blood concentrations ) , administered in randomized , double - blind manner : a multicomponent syrup containing diphenhydramine ( 25  mg ) , phenylephrine ( 10  mg ) , in a natural cocoa formulation ; dextromethorphan ( 30  mg ) syrup ; and , placebo syrup . 
 the standard endpoint of cough challenge was used : concentration of capsaicin inducing 5 coughs ( c5 ) . 
 main outcome measure : effect on cough reflex sensitivity ( c5 ) . 
 results : a significant difference ( p  =  0.0024 ) was established among groups , with pairwise analysis revealing a significant increase in mean log c5 ( 0.4    0.55 ( sd ) ; p  <  0.01 ) for the diphenhydramine - containing medication versus placebo , but not for dextromethorphan versus placebo . conclusions : our results provide the initial evidence of the ability of diphenhydramine to inhibit cough reflex sensitivity in subjects with acute pathological cough . 
 timing of cough reflex sensitivity measurement may not have allowed demonstration of maximal antitussive effect of dextromethorphan .",pubmed
"the global population , especially those in developed countries , is getting older and this trend is predicted to continue in the coming decades [ 1 , 2 ] . 
 some have defined aging as a decreased ability to resist cellular stresses or insults [ 3 , 4 ] , and in fact , aging is one of the most important cardiovascular risk factors for predisposing conditions such as diabetes , hypertension , and hypercholesterolemia . 
 accordingly , the incidence and prevalence of clinical and subclinical cardiovascular diseases increase dramatically with age , making cardiovascular disease the most common cause of death among the elderly . 
 the endothelium has a primary role in adjusting vascular function by the production of nitric oxide ( no ) and other biologically active vasodilator materials   that decrease vascular resistance , inhibit platelet adhesion and aggregation , and decrease vascular smooth muscle cell proliferation . 
 alterations in the control of these processes , a feature of endothelial dysfunction , often leads to atherosclerosis and other vascular disorders   that are accompanied by a proinflammatory , proliferative , and procoagulatory state . the endothelium is ideally placed to bear the brunt of hemodynamic stresses , oxidized lipids , and oxidative radicals , all of which increase their vulnerability to aging . 
 this is true not only in healthy subjects without underlying risk factors , but also in older people   and those with cardiovascular risk factors . 
 indeed , those with cardiovascular risk factor / disease will benefit more . for instance , eight weeks of exercise significantly improve endothelial function , as measured by flow - mediated dilation , in diabetic patients   but not in healthy subjects . 
 although there are clear health benefits of exercise in the elderly , a detailed understanding of the molecular basis underlying these improvements remains incomplete . in this minireview , we discuss some mechanisms thought to be involved in endothelial aging . 
 we focus on the role of oxidative stress and subsequent inflammation and the role of exercise in boosting antioxidative and anti - inflammatory mechanisms . 
 the endothelial cells form a monolayer that lines blood vessels to form an interface between circulating blood and the smooth muscle layer . 
 in addition to its barrier function , the endothelium modulates coagulation , growth , and inflammation throughout the circulatory system 
 . it also contributes to adjusting tissue perfusion by secreting several vasoactive substances , which can be vasoconstrictors ( endothelin-1 , angiotensin ii , thromboxane a2 , etc . ) or vasodilators ( no , prostacyclin , and endothelium - derived hyperpolarizing factor ( edhf ) , etc . ) . among the many vasoactive agents released by the endothelium , no has been characterized in greatest detail . 
 it is released in response to a variety of chemical and physical stimuli to cause vasodilation , such that one of the most common methods for assessing endothelial health is by determining vascular capacity to produce no . 
 no is produced by the catalytic activity of no synthase ( nos ) , which transforms l - arginine to l - citrulline . 
 all isoforms of nos require five cofactors / prosthetic groups : flavin adenine dinucleotide ( fad ) , flavin mononucleotide ( fmn ) , heme , tetrahydrobiopterin ( bh4 ) , and ca / calmodulin . 
 calcium is required for the activation of neuronal nos ( nnos ) and endothelial nos ( enos ) but not for the activity of inducible nos ( inos ) . 
 enos binds to caveolin-1 in endothelial cell caveolae , a subset of specialized lipid domains that form invaginations and so increase intracellular microdomains where organelles and anchoring proteins aggregate . 
 caveolin-1 inhibits enos activity , and this interaction is regulated by ca / calmodulin . upon agonist activation , the increases in intracellular calcium results in ca 
 / calmodulin binding , which then displaces caveolin and reverses its inhibitory interaction with enos . 
 mechanical stimuli such as shear stress and vascular smooth muscle stretch also raise intracellular calcium concentrations . 
 several chemical events such as interaction with ca / calmodulin , heat shock protein 90 , and subsequent association of akt results in enos phosphorylation at ser1177 are involved in no production . 
 released no causes vasorelaxation , which in turn results in increased blood flow and reduced blood pressure , inhibition of platelet adhesion and aggregation , inhibition of leukocyte adhesion , reduction in smooth muscle proliferation , and retardation of atherogenesis . 
 endothelial dysfunction is defined as functional alterations in endothelial physiology characterized by reduction of vasodilator substance output ( in particular no ) and augmentation in endothelium - derived contracting factors . 
 this imbalance leads to a vasoconstrictive , hypercoagulative , proliferative , and proinflammatory state , so favoring atherosclerosis . over time 
 , most humans are exposed to a variety of modifiable cardiovascular risk factors , such as hyperglycemia and insulin resistance , obesity , altered lipid profile , hypertension , and glomerulosclerosis . 
 . bed rest of about 48 hours induces vascular dysfunction , which is then followed by insulin resistance , dyslipidemia , and increased blood pressure . 
 thus , shortening of hospital stays and bed confinement periods are highly beneficial in the elderly . 
 bed rest also increases circulating endothelial cells , possibly due to increased endothelial cell apoptosis resulting from reduced shear stress during bed rest . 
 , as long as the damage is minimal or maintenance systems work properly as in young subjects , the functional capacity of the endothelium is preserved . in cases of severe damage , injured cells are omitted by a poorly described mechanism and replaced by dividing neighboring cells . circulating progenitor endothelial cells also play a role in the repair mechanisms of injured endothelial cells . 
 however , as part of the aging processes , the cumulative effects of stresses coupled with the inevitable metabolic changes that occur with time , there is a decline in the function and repair capacity of the endothelium . 
 since endothelial cells can only undergo a limited number of divisions , they eventually enter a state of senescence , which is an endogenous and hereditary process of biological aging , in which cells are still metabolically active , but express a pro - inflammatory , prooxidative , and proatherogenic phenotype . 
 the cumulative effects of these parameters strongly promote a decline in the functional capacity of endothelium . 
 all forms of cardiovascular disease have an increased prevalence in the elderly , even in those free of cardiovascular risk factors . since there are no changes in endothelium - independent vasodilation in older humans and animals , it is reasonable to suggest that vascular age - dependent endothelial changes largely reflect the release no , although there are also some changes in the production and release of other endothelial derived vasodilators such as prostacyclin and edhf , along with increases in vasoconstrictor prostanoids . 
 there are also several age - related structural changes in endothelium such as increases in the expression of adhesion molecules , permeability , sensitivity to apoptotic stimuli , with decreases in angiogenic and regenerative capacities [ 43 , 44 ] . 
 oxidative stress is an imbalance between production of oxidizing agents , such as free radicals , and opposing antioxidant systems which scavenge or metabolize those reactive agents . 
 free radicals are reactive chemical molecules having a single unpaired electron in an outer orbit . 
 this unstable configuration provides energy which is released through reactions with adjacent molecules such as proteins , lipids , carbohydrates , and nucleic acids . 
 oxygen - free radicals or , more generally , reactive oxygen species ( ros ) , as well as reactive nitrogen species ( rns ) , are products of normal cellular metabolism . 
 oxidative stress interferes with endothelial function in different ways , but the most prominent mechanism is via reduction of no bioavailability , which is the net product of the rate of no production and its degradation by superoxide .   in the case of no generation , reduced vasoconstrictive responses to the nos inhibitor n(g)-monomethyl - l - arginine ( l - nmma ) in older patients   and reduced shear stress - induced no release and vasodilation in older animals   suggest decreased production of no in aged endothelium . in spite of these 
 , direct measurements of enos in aged animals were inconclusive , as raised , fallen , or unchanged levels have been reported [ 4952 ] . 
 the increased production of superoxide anions in the aging vascular wall rapidly inactivates no [ 53 , 54 ] . 
 cyclooxygenase ( cox ) and nadph - oxidase have central roles in ros production [ 47 , 5558 ] . 
 removal of the endothelium or inhibition of nadph - oxidase reduces vascular superoxide generation in the aorta of aged wistar - kyoto rats . 
 superoxide rapidly reacts with no to generate cytotoxic peroxynitrite ( onoo ) , a reaction with several consequences . 
 first , onoo alters the function of biomolecules by protein nitration as well as by causing lipid peroxidation . 
 for example , potassium channels , which hyperpolarize vascular cells and mediate regulate vasorelaxation , are inhibited by nitration [ 60 , 61 ] . 
 second , onoo causes single - strand dna breakage , which in turn activates nuclear enzyme poly(adp - ribose ) polymerase ( parp ) ( a nuclear dna - repair enzyme ) . 
 third , it decreases no bioavailability causing impaired relaxation and inhibition of the antiproliferative effects of no . 
 furthermore , onoo  oxidizes bh4 , an important cofactor for nos , leading to the uncoupling of enos and causing it to produce superoxide instead of no . 
 ros - induced peroxidation of membrane lipids alters the structure and the fluidity of biological membranes , so having global detrimental effects on vascular function . 
 the role of oxidative stress in aged endothelial dysfunction is shown by the ability of vitamin c to restore the impaired vasodilatory response to acetylcholine only in subjects aged 60 years or older . 
 this indicates that oxidative stress is a critical mechanism for endothelial dysfunction only in older subjects . 
 administration of bh4 also improves flow - mediated dilatation ( fmd ) in older sedentary subjects , while having no beneficial effects in young or older trained people . 
 all cells have evolved highly complex enzymatic and nonenzymatic antioxidant systems that act synergistically to defend the body from free radical - induced damage . the most efficient enzymatic antioxidants are glutathione peroxidase , catalase , superoxide dismutase , heme oxygenase-1 ( ho-1 ) , nad(p)h quinone oxidoreductase-1 ( nqo-1 ) , and thioredoxin . 
 nonenzymatic antioxidants include vitamins e and c , thiol antioxidants ( glutathione , thioredoxin ) . 
 attenuation of antioxidant defense mechanisms during the aging process has been proposed in some studies . 
 for instance , reduced concentration of plasma sod , but not cellular sod , occurs in rats . 
 higher production of peroxynitrite causes antioxidant enzyme deactivation , as is the case for manganese sod ( mnsod ) in mitochondria . 
 levels of protein expression and enzymatic activity of glutathione peroxidase 1 ( gpx-1 ) are lower in proangiogenic endothelial progenitor cells ( derived from cultured blood mononuclear cells ) from older subjects . 
 atherosclerosis is an inflammatory disease that is mediated by monocyte - derived macrophages which accumulate in arterial plaques and become activated to release cytokines that cause tissue damage . as evidence accumulates favoring the role of inflammation during the different phases of atherosclerosis 
 , it is likely that markers of inflammation such as high sensitivity c - reactive protein ( hs - crp ) could be increasingly used to provide additional insights on the biological status of atherosclerotic lesions . 
 elevations of crp are considered independent predictors of cardiovascular events and of the outcome of acute coronary syndromes . besides their roles markers of systemic inflammation and as predictors of cardiovascular risk , crp and other inflammatory cytokines also directly trigger vascular dysfunction , possibly by altering calcium channel expression and activity , upregulating of rho - kinase expression and function , increasing ros production , and/or enhancing cox expression . in turn , 
 cox - derived constrictor prostanoid(s ) products cause vascular hypercontractility [ 74 , 75 ] and increased formation of ros . 
 increases in cox - induced ros production and the expression of proinflammatory mediators , such as il-1b , il-6 , tnf- , cox-2 , and inos , occur during aging . 
 the nf-b pathway is a critical component of inflammatory processes activated by oxidative stress [ 33 , 78 ] . 
 nf-b is an ubiquitous transcription factor with multiple roles such as mediating inflammatory responses to a variety of signals , immune function , endothelial cell activation , and control of cell growth [ 7981 ] . 
 nf-b is normally located in the cytoplasm in an inactive form by virtue of binding to a family of inhibitory nf-b ( ib ) proteins . upon cell stimulation by a wide variety of stimuli , signals responsive 
 ikk- and ikk- ( also known as ikk-1 and ikk-2 ) are activated , which results in the phosphorylation of ib and its proteasomal degradation . 
 ib degradation liberates nf-b , allowing it to translocate to the nucleus and induce gene expression of a number of proinflammatory cytokines , such as il-1 , il-6 , tnf- , cox-2 , lipoxygenase , inos , and adhesion molecules ( vcam-1 , icam-1 , pcam , e - selectin ) . 
 aging increases nf-b levels due to activation of ikk/ and degradation of ib . under usual conditions , 
 the activation of nf-b during inflammation is temporary and limited . in aging , however , a chronic and self - perpetuating condition exists . 
 proteins such as tnf- , il-1 , il-6 , and cox-2 that are nf-b induced also activate nf-b production , thus creating a vicious cycle . 
 aging increases plasma levels of tnf- , il-6 , il-1 , crp , and inflammatory blood cells [ 8486 ] . 
 the plasma concentration of il-6 correlates with senile neural atrophy   and inflammatory diseases , such as type 2 diabetes and atherosclerosis . 
 plasma levels of tnf- and il-6 are also predictors of disability and mortality among elderly . 
 the significance of exercise as a modifiable risk factor for cardiovascular disease is widely acknowledged . physical inactivity and poor diet , preceded only by tobacco , 
 are the leading causes of death . the american college of sports medicine , defines exercise as  any and all activity involving generation of force by the activated muscle(s ) that results in disruption of a homeostatic state  . 
 endurance exercise is characterized by prolonged and continuous periods of contractile activity ( high repetition ) against low resistance . 
 resistance exercise ( also termed strength training ) involves short periods of contractile activity ( low repetition ) against a high opposing resistance . 
 sprint exercise consists of short periods of maximal ( intense ) repetitive contractile activity with a low interval and against a low resistance , for example , running 100  m sprint race . 
 however , sprint training can also be performed against high resistance resulting in a combination of resistance and endurance modalities , for example , running with added weights . increased physical activity and fitness , of both men and women , 
 some studies even suggest greater benefits ( up to 50% risk reduction ) for exercise in terms of all - cause mortality and death from cardiovascular disease . 
 follow - up study evaluated the relationship between physical activity and risk of all - cause mortality in a large number of elderly ( 7080 women aged 7075 and 11668 men aged 6583 ) . 
 they found an inverse dose - response relationship between exercise and all - cause mortality . 
 risk reductions were 3050% higher in females than in males in every category of exercise intensity . 
 this study clearly shows that there are clear health benefits from all levels of physical activity . 
 regular aerobic exercise can slow down the age - related losses in endothelial function   supposedly by restoration of no availability consequent to prevention of ros production . 
 aging is associated with a limited capacity of the vasculature to release no , as older subjects show reduced levels of plasma nitrite in response to exercise . the difference in plasma nitrate / nitrite ratio between older and young sedentary subjects 
 heat - stimulated hand and foot skin increased blood flow are higher in trained older subjects compared to sedentary matched controls and are correlated with nitrate / nitrite ratios , suggesting better endothelial function secondary to greater no bioavailability . 
 trained elderly subjects also exhibit higher flow - mediated brachial artery dilation compared to sedentary counterparts . 
 table 1 summarizes the findings of recent clinical studies on the endothelial benefits of exercise in the elderly . 
 exercise training upregulates antioxidant defense mechanisms in several tissues , presumably due to increased levels of oxidative stress that occurs during exercise . 
 exercise - induced production of ros is proposed to evoke specific adaptations such as increasing repair mechanisms for oxidative damage , increasing resistance to oxidative stress , and lowering levels of oxidative damage . on the other hand , excessive production of ros can have detrimental effects . boosting levels of intrinsic antioxidant 
 potential and reduction in lipid peroxidation occur in healthy elderly men after habitual physical activity . 
 a critical role has recently been described for a transcription factor  nuclear factor ( erythroid - derived 2)-like 2 ( nrf2 )  against oxidative stress . 
 normally , nrf2 is located in the cytoplasm and kept dormant by the cytoplasmic repressor kelch - like ech - associated protein 1 ( keap1 ) . a variety of activators , including oxidative free radicals , release , and translocate nrf2 into the nucleus where it regulates the expression of antioxidant enzymes such as nqo-1 , glutathione - s - transferase , glutathione peroxidase , and ho-1 . 
 diminished nrf2 activity contributes to increased oxidative stress and mitochondrial dysfunction leading to endothelial dysfunction , insulin resistance , and abnormal angiogenesis as observed in diabetics . 
 ho-1 , which is mainly induced through the nrf2-keap1 signaling pathway ( also known as heat shock protein 32 ) , is the inducible isoform of heme oxygenase that catalyzes nadph - dependent decomposition of heme to carbon monoxide ( co ) , ferrous iron , and biliverdin . 
 three isoforms of ho have been identified : both ho-2 and ho-3 are 33-kda constitutively expressed isoforms . 
 an important role of ho-1 in the antioxidant defense system arises from an induction of ferritin synthesis that diminishes the cellular pool of free iron   and also from the enhancement of bilirubin levels , which are potent antioxidants . 
 carbon monoxide activates soluble guanylate cyclase , a key enzyme in the cell signaling cascade leading to relaxation of smooth muscle , and thrombocyte disaggregation . 
 ho-1 is a sensitive and reliable marker of oxidative stress   and cytoplasmic expression levels of ho-1 increase in leukocytes of endurance - trained male subjects after a half - marathon run . 
 there is a paradoxical increased expression of ho-1 in a control group of untrained men at rest , suggesting that the downregulation of the baseline expression of ho-1 in athletes reflects an adaptation mechanism to regular exercise training . 
 the direct effect of exercise on nrf2 expression has received much less attention except for a report that exercise increases nuclear levels of nrf2 in the proximal renal tubules of old rats . 
 the increased expression of enos after exercise both in animals and human beings [ 111114 ] also occurs in patients with stable coronary artery disease and chronic heart failure [ 115 , 116 ] . 
 exercise - induced upregulation of vascular enos expression is closely related to the frequency and the intensity of physical forces within the vasculature , especially shear stress . 
 shear stress is the product of all the perpendicular and parallel flow - mediated forces on endothelial cells . 
 the types of these hemodynamic forces , either laminar or oscillatory , greatly impact the function and properties of endothelial cells and also determine the signal transduction pathways that are activated . 
 laminar flow , which is augmented during moderate and intense physical activities , upregulates enos expression  while oscillatory forces , which are associated with hypertension , leads to increased nadph - oxidase activity and augments oxidative stress . 
 the mechanotransduction mechanisms that sense physical forces to cause altered gene expression are not completely described . 
 some reports suggest that activation of inward rectifying k cannels , followed by stimulation of outwardly rectifying cl channels , plays a major role in this process . 
 membrane hyperpolarization , due to inward kcurrents , drives extracellular ca into the cells through two shear stress - dependent ion channels ( p2x purinoceptors and transient receptor potential channels ) . 
 raised intracellular calcium levels lead to a dissociation of caveolae - bound enos and increased production of no . 
 other intracellular events are also thought to mediate increased no production in response to shear stress ; however , the relative importance of these mechanisms is not clear ( table 2 ) 
 . increased no synthesis secondary to amplified shear stress induces extracellular superoxide dismutase ( sod ) expression in a positive feedback manner so as to inhibit the degradation of no by ros . 
 another parallel mechanism that participates in this harmony is the upregulation of enos through exercise - induced ros production , since exercise - induced increases in shear stress stimulates vascular production of ros by an endothelium dependent pathway . 
 superoxides are rapidly converted to h2o2 by sod ; hydrogen peroxide then diffuses through the vascular wall and increases the expression and activity of enos [ 120 , 121 ] . 
 thus , increased expression of sod1 and sod3 ( which facilitate the generation of h2o2 from superoxide ) augments the effect of h2o2 on exercise - induced enos expression . on the other hand , enos expression 
 another putative mechanism is exercise - induced increases in arterial compliance which is mediated by reduction of plasma et-1 concentration as well as the elimination of et-1 mediated vascular tone . 
 twelve weeks of aerobic exercise training increase arterial compliance , while decreasing plasma et-1 levels . 
 moreover , the increase in central arterial compliance observed with et - receptor blockade before the exercise intervention was eliminated after exercise training . 
 these results indicate that endogenous et-1 participates in the beneficial influence of regular aerobic exercise on central arterial compliance . 
 exercise produces a short - term inflammatory response that is accompanied by leukocytosis , increases in oxidative stress and plasma levels of crp . 
 this proinflammatory response is followed by a long - term anti - inflammatory adaptive response . 
 regular exercise reduces crp , il-6 , and tnf- levels while increasing anti - inflammatory substances such as il-4 and il-10 [ 125 , 126 ] . 
 controlling the release and activity of at least two cytokines , tnf- and il-6 , could contribute to the natural protective effects of physical activity . 
 interleukine-6 ( il-6 ) is the first cytokine to be released into the circulation during exercise , and its levels increase in an exponential fashion in response to exercise . 
 contracting skeletal muscles upregulate levels of il-6 mrna   and the transcriptional rate of the il-6 gene is also markedly enhanced by exercise . 
 il-6 acts as both a proinflammatory and anti - inflammatory cytokine : when secreted by t cells and macrophages , il-6 stimulates the immune response and boosts inflammatory reactions , while muscle - produced il-6 exerts anti - inflammatory effects through its inhibitory effects on tnf- and il-1 , and activation of interleukin-1 receptor antagonist ( il-1ra ) and il-10 . 
 il-10 in turn reduces the production of several proinflammatory cytokines , such as tnf- and il-1 . 
 exercise - induced increases in plasma il-6 correlate with the muscle mass involved in exercise activity and the mode , duration , and intensity of exercise , and this is especially the case in older individuals . 
 in addition , starkie et al . report that infusion of recombinant human il-6 ( rhil-6 ) into human subjects simulated exercise induced an il-6 response in the prevention of endotoxin - induced increase in plasma tnf- . 
 exercise also suppresses tnf- production by an il-6 independent pathway , as there are modest decreases in plasma tnf- after exercise in il-6 knockout mice . 
 in addition , il-6 enhances lipid turnover and stimulates lipolysis as well as fat oxidation via activation of amp - activated protein kinase . 
 mice deficient in il-6 ( il6/ ) develop mature onset obesity and have disturbed carbohydrate and lipid metabolism that is partly reversed by il-6 replacement . 
 the lipolytic effect of il-6 on fat metabolism was confirmed in two clinical studies of healthy and diabetic subjects [ 137 , 139 ] . 
 visceral fat is potentially a cause of low - grade systemic inflammation , which in turn leads to insulin resistance , type 2 diabetes , and atherosclerosis . during exercise , il-6 also increases hepatic glucose production . 
 glucose ingestion during exercise reduces il-6 production by muscles , suggesting that il-6 is released by a reduction in glycogen levels during endurance exercise and the consequent adrenergic stimulation of il-6 gene transcription via protein kinase a activation . 
 this cytokine exerts anabolic effects in muscles by inducing protein synthesis and inhibiting protein degradation [ 130 , 142 ] . in some animal studies , 
 this cytokine prevents muscle wasting by attenuating apoptotic dna fragmentation and downregulating tnf - driven apoptotic pathways . in agreement with this , four weeks of exercise reduce the extent of tnf - triggered myocyte apoptosis in old rats [ 144 , 145 ] 
 aging is an important independent risk factor for the development of cardiovascular disease , which is manifest as endothelial dysfunction . 
 a large body of evidence underlines the importance of oxidative stress and inflammation as prominent features of the aging process . reduced no bioavailability and decreased responsiveness to other endothelial - derived vasodilators 
 promote a vasoconstrictive , hypercoagulative , and proliferative state which favors the development of atherosclerosis . 
 vascular oxidative stress is the product of increased reactive oxygen species , such as superoxides , and reduced antioxidant defense . 
 oxidative stress - induced damage promotes a chronic inflammatory state which perpetuates a vicious cycle of endothelial dysfunction . 
 exercise training prevents and restores age - related impairment of endothelial function , possibly by the restoration of no availability consequent to prevention of oxidative stress and alleviating inflammatory processes .","the endothelium plays a critical role in the maintenance of cardiovascular health by producing nitric oxide and other vasoactive materials . 
 aging is associated with a gradual decline in this functional aspect of endothelial regulation of cardiovascular homeostasis . 
 indeed , age is an independent risk factor for cardiovascular diseases and is in part an important factor in the increased exponential mortality rates from vascular disease such as myocardial infarction and stroke that occurs in the ageing population . 
 there are a number of mechanisms suggested to explain age - related endothelial dysfunction . 
 however , recent scientific studies have advanced the notion of oxidative stress and inflammation as the two major risk factors underlying aging and age - related diseases . 
 regular physical activity , known to have a favorable effect on cardiovascular health , can also improve the function of the ageing endothelium by modulating oxidative stress and inflammatory processes , as we discuss in this paper .",pubmed
"axillary lymph node status is the most important prognostic factor in breast carcinoma and prognosis worsens with increasing number of metastatic lymph nodes [ 1 , 2 ] . according to the american joint committee on cancer ( ajcc)/international union against cancer ( uicc ) tumor ( t)-node ( n)-metastasis ( m ) classification , 
 nodal disease is classified in three groups based on the number of axillary metastatic lymph nodes : n1 , 13 metastatic lymph node(s ) , n2 , 49 metastatic lymph nodes and n3 , 10 or more metastatic lymph nodes . however , the number of metastatic lymph nodes depends on the number of removed lymph nodes that are dissected by the surgeon and examined by the pathologist . 
 various studies have shown that the number of metastatic lymph nodes is greater with increasing number of removed lymph nodes [ 412 ] . 
 it is difficult to assess the axillary lymph node status reliably without removing sufficient numbers of lymph nodes depending on the surgeon and/or pathologist . 
 studies conducted in recent years indicate that the ratio of the number of metastatic lymph nodes to the number of removed lymph nodes denoted as lymph node ratio ( lnr ) provide a more useful prognostic information   compared to nodal disease classification according to the number of metastatic lymph nodes [ 9 , 1316 ] . a review on the prognostic value of lnr indicated that the threshold value of clinically significant lnr varies in different studies and emphasized that these studies vary by sampling size and tumor stage . until now 
 , analysis was made mostly in all lymph node - positive patients , without subdivision in n1 , n2 , and n3 disease groups in the lnr studies [ 9 , 13 , 14 , 16 , 1823 ] . 
 lnr threshold value separating the whole series in two prognostic groups with significantly different survival was given as 0.20 [ 18 , 24 ] and 0.25 .   in three series including patients with n2 and n3 disease receiving adjuvant high - dose chemotherapy with stem - cell support , 
 lnr threshold value of prognostic significance was determined as 0.80 by nieto et al .   and schneeweiss et al . , and 0.70 by bolwell et al . . 
 our group determined the lnr threshold value as 0.80 in a previous study including patients with t1,2,3n3m0 disease . in all of the above - mentioned studies patients with higher lnr had significantly worse prognosis compared to those with lower lnr . 
 a striking result from these studies was that the lnr threshold value of prognostic significance was greater in series excluding n1 disease , compared to those including it . 
 based on this , we proposed that identifying a separate lnr threshold value for each n disease group may be useful . 
 fortin et al . recommended axillary radiotherapy for patients with a lnr 0.40 or above in the group with 13 metastatic lymph node(s ) and for patients with a lnr 0.50 or above in the group with 4 or more metastatic lymph nodes among t1-t2 node - positive patients . 
 there is limited number of studies investigating the prognostic value of lnr in patients with only n1 disease [ 2931 ] . in this study 
 , we evaluated the prognostic significance of the number of removed and metastatic lymph nodes and lnr in breast carcinoma patients with 13 axillary metastatic lymph node(s ) . 
 we retrospectively reviewed the file records of women who underwent surgery for breast carcinoma between january 1993 and december 2001 and who were then followed up in sb okmeydan training and research hospital . 
 inclusion criteria for the patients were a histological diagnosis of unilateral invasive breast carcinoma , no previous or concomitant malignant disease , known pathological tumor size ( patients with t4 tumor were not included ) , axillary 13 lymph node(s ) metastasis , no metastasis in ipsilateral internal mammary or supraclavicular lymph nodes and distant site at the time of diagnosis , microscopically tumor - free surgical margins , completion of adjuvant therapy planned according to standard therapy protocols , and a follow - up period of at least five years . 
 a total of 924 patients ( including 174 patients who underwent surgery at the study hospital ) who met these criteria were enrolled in the current study . 
 follow - up data were obtained from file records and , in some patients , through telephone calls . 
 locoregional recurrence was defined as the first site of recurrence involving the chest wall ( local ) or / and ipsilateral axillary , supraclavicular , and internal mammary lymph nodes ( regional ) . 
 first disease recurrence was recorded as distant metastasis if it was either distant metastasis or concurrent distant metastasis and locoregional recurrence . 
 disease - free survival ( dfs ) , locoregional recurrence - free survival ( lrfs ) , and distant metastasis - free survival ( dmfs ) times were defined as the time interval between tumor excision and detection of first disease recurrence , locoregional recurrence or distant metastasis respectively or the date of last follow - up . in 21 patients who developed a second malignancy ( excluding basal cell carcinoma ) , the diagnosis date of the second malignancy 
 was considered as the last followup date . in 9 patients whose death was unrelated to cancer , 
 fifty - five patients developed locoregional recurrence ( including 16 patients who developed axillary recurrence ) , 243 patients developed distant metastasis , and 9 patients developed concomitant locoregional recurrence and distant metastasis ; in patients without disease recurrence median follow - up time was 108.5 months . 
 the fisher exact test was used to compare the axillary recurrence rates of the patient groups . 
 patients were grouped according to the number of removed and metastatic lymph nodes and lnr threshold value . 
 kaplan - meier method was used for calculation and plotting of the dfs , lrfs , and dmfs curves of the patient groups , and the log - rank test was used for the comparison of the survival curves . 
 survival analyses ( for dfs , lrfs , and dmfs ) were performed separately for the whole series and for the patient group having at least 10 lymph nodes removed from the axilla . 
 patients were grouped in two different ways according to the number of lymph nodes removed ( grouping a : 15 , 69 , 1015 and 16 and more removed lymph nodes ; grouping b : 15 and 6 and more removed lymph nodes ) . to determine the lnr threshold value that will separate patients in two prognostic groups of low and high disease recurrence risk with significantly different survival rates , survival analyses were conducted with lnr threshold values between 0.10 and 0.40 for the whole series , and between 0.10 and 0.25 for the group with at least 10 lymph nodes removed , at increments of 0.05 . 
 the lnr that produced the significant survival difference between the groups and gave the highest log - rank x value was considered as the most significant threshold value . 
 in grouping a , dfs was significantly worse in patients with 15 lymph node(s ) removed compared to patients with 69 ( p = 0.016 ) , 1015 ( p = 0.024 ) , and 16 and more ( p = 0.015 ) lymph nodes removed ; there was no significant difference between the dfs of the three groups which had 6 or more lymph nodes removed ( figure 1 ) . in grouping b constructed based on this data , dfs of patients with 15 lymph node(s ) removed was significantly worse than those with 6 or more lymph nodes removed ( p = 0.008 ) . 
 in multivariate cox analysis , grouping b had independent prognostic value ( table 2 ) , whereas grouping a had not . in grouping a , lrfs was significantly worse in patients with 15 lymph node(s ) removed compared to patients with 69 ( p = 0.008 ) , 1015 ( p = 0.002 ) and 16 and more ( p = 0.009 ) lymph nodes removed ; there was no significant difference between the lrfs of the three groups which had 6 or more lymph nodes removed . according to grouping b , 
 lrfs of patients with 15 lymph node(s ) removed was significantly worse than patients with 6 or more lymph nodes removed ( p = 0.001 ) . 
 both groupings had independent prognostic value in cox analysis ( p = 0.005 and p < 0.001 , resp . ) . 
 in terms of dmfs there was no significant difference between patient groups according to either grouping a or b. axillary recurrence was 9.4% ( 8/85 patients ) in patients who had 15 lymph node(s ) removed , while 0.9% ( 8/839 patients ) in patients who had 6 or more lymph nodes removed , and the difference was significant ( p < 0.001 ) . 
 the median value of lnr was 0.143 ( range 0.0261.00 ) for the whole series . 
 the most significant lnr threshold value separating patients in low- and high - risk groups in terms of dfs was 0.20 ( p < 0.001 ) ( figure 2 ) ; this ratio had independent prognostic significance in cox analysis ( p < 0.001 ) . when grouping b , which is based on the number of lymph nodes removed and has independent prognostic significance , 
 was added to this analysis , its prognostic significance was lost ( p = 0.527 ) , while the significance of lnr persisted ( p < 0.001 ) . 
 the most significant lnr threshold value separating patients in two risk groups in terms of lrfs was 0.30 ( p < 0.001 ) ( figure 3 ) ; this ratio had independent prognostic significance in cox analysis ( p < 0.001 ) ( table 3 ) . when groupings a and b were added to this analysis , 
 their prognostic significance was lost ( p = 0.325 and p = 0.190 , resp . ) , while the significance of lnr persisted ( p = 0.004 and p = 0.006 , resp . ) . 
 the most significant threshold value separating patients in two prognostic groups in terms of dmfs was 0.15 ( p < 0.001 ) ( figure 4 ) ; this ratio had independent prognostic significance in cox analysis ( p < 0.001 ) ( table 4 ) . 
 as mentioned above , there was no significant relationship between the number of removed lymph nodes and survival based on any patient grouping for all three types of survival endpoint . 
 dfs was significantly better in patients with 1 positive node compared to those with 3 positive nodes ; there was no significant difference between those with 1 and 2 positive node(s ) or 2 and 3 positive nodes . based on this , when patients were grouped as those with 1 positive node versus 2 - 3 positive nodes , dfs was significantly better for patients with 1 positive node ( p = 0.016 ) ; this grouping had independent prognostic significance in cox analysis ( p = 0.007 ) . 
 there was no significant relationship between the number of metastatic lymph nodes and lrfs . 
 dmfs of patients with 1 positive node was significantly better than those with 3 positive nodes ; there was no significant difference between those with 1 and 2 positive node(s ) or 2 and 3 positive nodes . based on this , when patients were grouped as those with 1 positive node versus 2 - 3 positive nodes , 
 dmfs was significantly better for patients with 1 positive node ( p = 0.022 ) ( figure 5 ) ; this grouping had independent prognostic significance in cox analysis ( p = 0.017 ) . 
 the most significant lnr threshold value to separate patients in low- and high - risk groups in terms of dfs was 0.15 ( p = 0.005 ) ; this ratio had independent prognostic significance in cox analysis ( p = 0.001 ) . 
 when the grouping based on metastatic lymph node number ( 1 positive versus 2 - 3 positives ) was added to this analysis , its prognostic significance was lost ( p = 0.294 ) , while the prognostic value of lnr moved slightly outside of the limit of significance ( p = 0.064 ) . 
 there was no significant lnr threshold value to separate patients in two prognostic groups in terms of lrfs . 
 the most significant lnr threshold value to separate patients in two prognostic groups in terms of dmfs was 0.15 ( p < 0.001 ) ( figure 6 ) ; this ratio had independent prognostic significance in cox analysis ( p < 0.001 ) . 
 when the grouping based on metastatic lymph node number ( 1 positive versus 2 - 3 positives ) was added to this analysis , its prognostic significance was lost ( p = 0.803 ) , while lnr continued to be a significant prognostic factor ( p = 0.010 ) . 
 in our series composed of patients with breast carcinoma having 13 metastatic axillary lymph node(s ) , dfs and lrfs of patients with 15 lymph node(s ) removed from their axilla were significantly worse than those with 6 or more lymph nodes removed , and this grouping had independent prognostic significance for both types of survival outcomes . in patient groups with more than 5 lymph nodes removed , there was no significant relationship between the increased number of removed lymph nodes and dfs or lrfs . 
 studies involving patients with 13 axillary lymph node metastasis indicate significantly better overall survival   and disease - free survival   with increasing number of lymph nodes removed . 
 karlsson et al . determined that in node - positive patients who did not receive radiotherapy , locoregional recurrence decreased significantly with increasing number of nonmetastatic lymph nodes removed and indicated that patients with 13 positive nodes and less than 10 nonmetastatic lymph nodes removed are candidates for postmastectomy radiotherapy , since their 10 year cumulative locoregional recurrence incidence is over 20% .   in a study by schaapveld et al . 
 , overall survival was found to be significantly worse in patients with less than 10 lymph nodes removed compared to patients with 10 or more nodes removed ; however , since overall survival can be deceptive as it includes all deaths along with nonbreast cancer related deaths , relative survival ( the ratio of the overall survival and the expected survival ) was analyzed and the number of removed lymph nodes was shown to be nonsignificant . 
 truong et al . did not observe a significant relationship between the number of lymph nodes removed and locoregional recurrence , distant metastasis , and overall survival rates .   in our series , 
 axillary recurrence rate was significantly higher in patients with 15 lymph node(s ) removed ( 9.4% ) compared to those with 6 or more nodes removed ( 0.9% ) . 
 it is generally accepted that a sufficient axillary dissection to avoid leaving probable metastatic lymph nodes behind is necessary for surgical control of the disease in the axilla and for correct evaluation of the axillary status in node - positive patients . 
 our study results confirm this approach : high rate of axillary recurrence in patients with few lymph nodes removed indicates that actually there may be more than three metastatic lymph nodes in the axilla ( n2 maybe n3 disease ) and that these may have been left in the axilla .   in our study , 
 the most significant lnr threshold value separating patients in low- and high - risk groups with significant survival difference was 0.20 for dfs , 0.30 for lrfs , and 0.15 for dmfs . 
 when grouping b ( with patients grouped according to 15 lymph node(s ) removed versus 6 or more nodes removed ) which had independent prognostic significance was added to these cox analyses conducted for dfs and lrfs , its significance was lost , while the prognostic significance of lnr threshold values persisted . since lnr is a derivative of the number of metastatic lymph nodes and the number of lymph nodes removed , it should not be included in the cox analysis with one of these two parameters . 
 the prognostic significance of lnr is superior to the number of lymph nodes removed for dfs and lrfs , and lnr has independent prognostic significance for dmfs while the number of removed lymph nodes has not ; thus , lnr can be used to separate patients with n1 disease in low- and high - risk groups regardless of the total number of lymph nodes removed . 
 series of patients having 13 positive lymph node(s ) showed that patients with high lnr have worse survival compared to those with low lnr : truong et al . 
 , in their series of patients having 139 node(s ) removed and not receiving radiotherapy , determined the most significant lnr threshold value for locoregional recurrence , distant metastasis , and overall survival as 0.25 , and recommended radiotherapy following mastectomy for patients with lnr > 0.25 . also , in another series of patients having 141 lymph node(s ) removed and not receiving radiotherapy , truong et al . 
 indicated that in terms of locoregional recurrence prediction , lnr was more valuable than the number of metastatic lymph nodes and recommended postmastectomy radiotherapy for patients with lnr > 0.20 , since their 10-year locoregional recurrence risk was above 20% . 
 yildirim and berberoglu , in their series of patients having at least 10 lymph nodes removed and not receiving radiotherapy , determined the most significant lnr threshold value for prediction of locoregional recurrence risk as 0.15 .   in our study , 
 different threshold values for most significant lnr for dfs , lrfs , and dmfs were identified .   if the survival analyses of this study were performed for dfs only , the threshold value of 0.20 would have been higher than the most significant threshold value for dmfs ( 0.15 ) and 167 patients with lnr > 0.150.20 would have been classified as with low risk despite their high risk for distant metastasis . 
 although in daily practice , it may be difficult to determine a different threshold value for each type of disease recurrence , its potential use in planning a more appropriate adjuvant therapy should be taken into consideration . 
 yildirim and berberoglu in their series of all node - positive patients with at least 10 lymph nodes removed from the axilla , identified the optimum lnr threshold value as 0.15 for locoregional recurrence and 0.20 for distant metastasis and determined significantly higher disease recurrence rates in patients having a lnr above these thresholds . in our study , 
 it is generally accepted that at least 10 lymph nodes need to be removed in order to classify nodal disease based on the number of metastatic lymph nodes according to tnm classification [ 8 , 12 , 35 , 36 ] . 
 nodal disease classification can not be done in patients having less than 10 lymph nodes removed , while that was the case with some of the patients in our series . 
 when at least 10 lymph nodes are removed from the axilla and metastasis is found in 13 of them , these patients are classified as with n1 disease by tnm classification , and thus are accepted as a homogenous group . however , our study results suggest that n1 disease does not constitute a homogenous prognostic group : when patients were grouped according to the number of metastatic lymph nodes , dfs and dmfs were significantly better in patients with 1 positive node compared to 2 - 3 positive nodes and this grouping had prognostic significance independent of age , menopausal status , tumor size , histological type , adjuvant chemotherapy , radiotherapy , and hormonal therapy . 
 patients with lnr > 0.15 had significantly worse dfs and dmfs , and lnr had independent prognostic significance according to this threshold value in cox analysis ; when a grouping based on the number of metastatic lymph nodes was added to these analyses , its prognostic significance was lost , while the prognostic significance of lnr persisted though it moved a little outside of the limit of significance for dfs ( p = 0.064 ) . moreover , 
 as also mentioned above , lnr should be included in the cox regression analysis alone , as it is derived from the number of removed and metastatic lymph nodes . according to these results , lnr , which is a more powerful prognostic factor than the number of metastatic lymph nodes , 
 can be used to separate n1 disease patients having at least 10 lymph nodes removed into low- and high - risk prognostic groups for distant metastasis development , thus providing more detailed and useful prognostic information than tnm nodal disease classification .   in patients having 10 or more lymph nodes 
 removed , number of removed and metastatic lymph nodes and lnr did not have prognostic significance for lrfs . 
 this result indirectly suggests that in n1 disease , removal of at least 10 lymph nodes may be sufficient to obtain locoregional control and removal of more lymph nodes may not be related to locoregional recurrence , and hence , n1 disease may be homogeneous in terms of locoregional control . 
 irrespective of the number of lymph nodes removed from the axilla , lnr is a useful tool in separating breast carcinoma patients with 13 metastatic lymph node(s ) into low- and high - risk prognostic groups . 
 thus , lnr may be useful to standardize adjuvant therapy for patients operated in hospitals that use different axillary dissection width and have different median number of removed lymph nodes as well as to draw reliably comparisons between treatment results coming from such different hospitals .","we evaluated the prognostic significance of lymph node ratio ( lnr ) , number of metastatic lymph nodes divided by number of removed nodes in 924 breast carcinoma patients with 13 metastatic axillary lymph node(s ) . 
 the most significant lnr threshold value separating patients in low- and high - risk groups with significant survival difference was 0.20 for disease - free survival ( p < 0.001 ) , 0.30 for locoregional recurrence - free survival ( p < 0.001 ) , and 0.15 for distant metastasis - free survival ( p < 0.001 ) , and the patients with lower lnr had better survival . 
 all three lnr threshold values had independent prognostic significance in cox analysis ( p < 0.001 for all three of them ) . in conclusion 
 , lnr is a useful tool in separating breast carcinoma patients with 13 metastatic lymph node(s ) into low- and high - risk prognostic groups .",pubmed
"the oh functionality is commonly activated by attachment of a good leaving group , e.g. , by conversion into an ester derivative of a strong acid ( tosylate , mesylate , triflate ) , usually employing mitsunobu or appel conditions.1 this classic methodology suffers from poor atom economy and creates large amounts of waste , thus rendering these processes inefficient and unattractive , although remarkable progress towards catalytic processes was made in the past decade.2 in nature , activation of hydroxy groups often proceeds through enzymatic phosphorylation by kinases using activated phosphate ( adenosine triphosphate , atp ) or sulfation catalyzed by mammalian sulfotransferases in conjunction with the respective cofactor , i.e. , 3phosphoadenosine5phosphosulfate ( paps).3 nonspecific bacterial arylsulfotransferases are also able to sulfate hydroxy groups by using organic sulfate donors.4 the requirement for molar equivalents of these cofactors in synthetic applications is problematic from a scaleup perspective . 
 although enzymatic atp recycling is feasible with reasonable efficiency ( up to several hundred ttns),4a,5 paps can not be recycled efficiently so far.4 most importantly , atpdependent kinases and papsdependent sulfotransferases are typically involved in biosynthetic pathways and signal messaging and thus are often highly substratespecific and hence do not allow broad synthetic applications . 
 an efficient alternative to kinases is the use of atpindependent phosphatases using cheap inorganic polyphosphates ( pyrophosphate , ppi ; triphosphate , pppi ; polyphosphoric acid , polyp ) as activated phosphate donor ( pdonor ) , which circumvents the need for cofactor recycling.6 additionally , due to their occurrence in biodegradation pathways , phosphatases usually display a broad substrate spectrum , and inorganic phosphate formed as byproduct is environmentally benign . 
 a major drawback resides in the reversibility of phosphate transfer , causing the undesired hydrolysis of the newly formed phosphate ester , which requires a kinetically tightly controlled system ( figure  1 ) . 
 mechanism of ( a ) competing hydrolysis and ( b ) transphosphorylation involving a covalent phosphoenzyme intermediate catalyzed by phonse.26 
  the use of phosphatases for synthetic applications dates back to the late 1940s , when axelrod and appleyard achieved transphosphorylation using phosphataseenriched extracts and pnitrophenyl phosphate ( pnpp ) or phenolphthalein phosphate as donor.7 later , nordlie identified glucosepyrophosphotransferase activity of e. coli alkaline phosphatase;8 however , the method was applied on a preparativescale only decades later.9 asano et al . 
 applied acid phosphatases from enteric bacteria to the phosphorylation of nucleosides and designed enzyme mutants with reduced hydrolytic activity.10 acid phosphatases from shigella flexneri ( phonsf ) and salmonella enterica ser . 
 typhimurium lt2 ( phonse ) were found to have a broad substrate spectrum11 and were applied in combination with aldolases to synthesize natural and unnatural sugar molecules on a gramscale.12 
  in drug discovery , phosphorylation is a common prodrug strategy to enhance the accessibility of drug candidates within the body by enhancing their polarity and solubility.13 in some cases , the phosphorylated form may be the active species.14 in synthetic chemistry , phosphate di and triesters are widely applied as brnsted acid catalysts for various asymmetric transformations15 and as leaving groups in transitionmetalcatalyzed coupling reactions16 as well as in organolithium17 and alkene synthesis.18 in contrast , phosphate monoesters have attracted so far little attention as tools for functionalgroup modification in classic chemical processes , most likely as a consequence of their insolubility in organic solvents resulting from their negative charge . 
 current chemical routes to phosphate monoesters often proceed via the corresponding phosphate di or triesters and require harsh reagents and conditions leading to hardly separable product mixtures . in this study , we aimed to evaluate a set of phosphatases as potential candidates for synthetic applications . 
 various techniques were investigated to enhance product titers of phosphorylated alcohols and to minimize the thermodynamically favored hydrolytic reaction . 
 enzyme candidates were selected from the family of acid phosphatases , which have been shown to transfer efficiently a phosphate moiety from a highenergy inorganic di / tri / polyphosphate onto various hydroxylated compounds . 
 nonspecific acid phosphatase phonse from salmonella typhimurium and acid phosphatase phonsf from shigella flexneri have been reported to convert various primary and secondary alcohols and sugars11 and were implemented in cascade reactions involving glucose6phosphate19 and dihydroxyacetone phosphate as intermediates.12 acid phosphatase phytase catalyzes the hydrolysis of phytate , a phosphorylated natural cyclitol by liberating inorganic phosphate and is therefore commonly used as animal feedstock additive.20 acid phosphatase aphast from salmonella typhimurium lt221 was selected due to its phosphotransferase activity on alcohols using pnpp as donor.22 piacp from prevotella intermedia 
 23 was selected based on high sequence similarity with robust phocmm developed for nucleoside phosphorylation.24 all enzymes except phytase were overexpressed in e. coli and subsequently purified to homogeneity by using histag or streptag techniques ( figure s1 ) . 
 a common mechanistic feature of phosphatetransferring phosphatases is the formation of a covalent phosphoenzyme intermediate , which undergoes either nucleophilic attack by water ( hydrolysis ) or accepts a suitable nucleophile ( transphosphorylation ) ( figure  1 ) . to this end , most enzymes use histidine as catalytic residue ( phonsf , phonse , piacp and phytase ) except aphast which uses aspartic acid.25 due to the competition between hydrolysis and transphosphorylation in aqueous medium , high substrate loads are required to achieve high product titers , while kinetic control is crucial to keep the phosphorylation / hydrolysis ratio as high as possible . in order to verify the activity of all recombinant proteins , a spectrophotometric assay based on the hydrolysis of pnpp was used . 
 a range of alcohols was investigated , including primary and secondary mono and dihydroxy compounds , as well as cyclic substrates ( table  1 ) . 
 although phytase has been successfully employed in a cascade reaction to convert glycerol into glycerol1phosphate in a nonstereoselective fashion,27 under our assay conditions , the enzyme showed no transphosphorylation activity at ph = 2.5 and 4.2 on any substrate . 
 strong hydrolytic activity on inorganic phosphate donors was still observed , confirming active protein ( data not shown ) . 
 aphast also contains mg ( in addition to the catalytically active nucleophile aspartate21 ) , whose activity is affected by inorganic polyphosphates by metal chelation . 
 although the addition of 10 mm mgcl2 could partly restore the hydrolytic activity on pnpp in the presence of 250 mm ppi ( figure s2 ) , only traces of product and marginal hydrolytic activity were obtained in the transphosphorylation of 1,4butanediol ( 500 mm ) by using 100 mm ppi as donor at ph = 4.2 and 5.5 , independent of the mgcl2 concentration ( 10 , 50 , 100 mm ) ( data not shown ) . 
 this suggests that inorganic oligophosphates ( as opposed to pnpp ) are not ideal phosphate donors for metalcontaining phosphatases , such as aphast . on the contrary , phonsf , phonse and piacp 
 all proved to be efficient catalysts in the phosphorylation of various alcohols ( table  1 ) . 
 substrate scope of phonsf , phonse and piacp in transphosphorylation.[a ] , [ b ] , [ c ] , [ d ] , [ e ] , [ f ] , [ g ] 
  reaction conditions : ph = 4.2 , 50 g  ml enzyme , 1 ml of volume , 1  % dmso as internal standard , 30 c , 600 rpm shaking . 
 product levels were determined by consumption of substrate using hplc with ri detector ( control experiments used as reference ) . 
 phosphorylation of primdiols proceeded to various degrees depending on the chain length : while shortchain ethylene glycol ( 1 ) was selectively monophosphorylated , 1,4butanediol ( 2 ) and the unsaturated ( z ) analog 4 yielded the corresponding mono / bisphosphorylated products in a ratio of 90:10 . extending the chain length even further ( 1,6hexanediol , 3 ) gave 85:15 of mono / diphosphate as determined by lcri chromatography ( see supporting information ) . 
 it is obvious , that the presence of a ( negatively charged ) phosphate group in the proximity of the reacting hydroxy moiety determines whether an ,diol undergoes bisphosphorylation : while shortchain ethylene glycol is exclusively monophosphorylated , longchain 1,6hexanediol yields the bisphosphate in up to 15  % . 
 haloalcohol 5 was also converted : phonse tolerated higher substrate concentrations than phonsf and piacp ( 500 mm vs. 300 mm ) resulting in the formation of 113 mm product in 60 min . to probe the chemoselectivity of the enzymes for o vs. nnucleophiles , 6amino1hexanol ( 6 ) 
 the three enzymes exclusively furnished the ophosphate monoester at very similar product levels ( ca . 
 this was proven by p nmr spectroscopy resulting in a chemical shift typical for signals of phosphorylated primary hydroxy groups ( figure s5).28 in line with this observation , 1,6diaminohexane ( 12 ) was not converted , only pdonor hydrolysis was observed ( data not shown ) , proving that these enzymes do not accept amines in place of alcohols . 
 sterically demanding secondary alcohol 7 was transformed only with phonse , which is in line with the previously observed preference of this enzyme for secalcohols.11 similar observations were made with 1,2propanediol ( 8) , which was exclusively phosphorylated at the primalcohol by phonsf and piacp , while phonse showed reduced selectivity ( ca . 
 90:10 prim / secphosphate , determined by p nmr spectroscopy ; figure s6 ) . in order to elucidate the enantioselectivity of phosphatases , 
 the two enantiomers of 8 were tested separately . in case of phonsf and piacp , similar activities on both enantiomers 
 were observed indicating nonenantioselective phosphorylation . with phonse , a marginal enantiopreference for ( r)8 was detected ( figure s7 ) . 
 to further investigate the enantioselectivity of phonse , both enantiomers of 2butanol ( 9 ) were subjected to screening conditions ; however , no significant difference in the reaction course was observed . 
 furthermore , racemic cyclic transdiol 10 was tested , and beside a moderate amount of monophosphorylated product , no stereoselectivity could be observed ( as determined by measurement of the ee value of remaining 10 by gc after derivatization of both oh functionalities ; see supporting information ) . 
 substrates containing n or snucleophiles or charged groups were not converted ( figure  2 ) , while ppi was still hydrolyzed indicating that the enzymes were still active ( data not shown ) . 
 the absolute chemoselectivity of ophosphorylation vs. n and snucleophiles was corroborated by the nonacceptance of substrates 11 and 12 . 
 mevalonolactone ( rac13 ) and charged substrates , like lactic ( rac14 ) and tartaric acid ( 15 ) ( both enantiomers tested separately ) were not converted . 
 although ophosphod , lserine could be stereoselectively hydrolyzed by a mutant of phonse,29 the wildtype enzyme was not active in the phosphorylation of racserine ( 16 ) . 
 substrates not converted by phonsf , phonse , piacp and phytase [ 300 mm substrate concentration except for rac14 ( 200 mm ) and 17 ( 15 mm ) ] . due to the reversibility of the reaction ( figure  1 ) , kinetic control of the reaction is necessary to ensure optimal product titers by avoiding product depletion by ( reverse ) hydrolysis and to ensure optimal product titers . this may be achieved by adjustment of reaction parameters such as ph or selection of oligophosphate donor.30 therefore , 1,4butanediol ( 2 ) was selected as a model substrate , and enzymes were tested with tri and polyphosphate as alternative to ppi ( table  1 and figures s810 ) . 
 the amount of 4hydroxybutyl phosphate formed could be significantly increased by replacing ppi by high amounts of pppi and polyp in the reaction catalyzed by phonsf and piacp , allowing up to 61  % conversion ( 305 mm product ) with respect to the theoretical maximum . 
 the difference in the slope in product disappearance clearly shows that product hydrolysis is decreased in the presence of tri and polyphosphate compared to ppi . 
 in contrast , these donors had a negative influence on the performance of phonse , where a product maximum was obtained with ppi . moreover , hplc monitoring of the reaction indicated incomplete consumption of polyphosphoric acid . 
 this suggests that this enzyme accepts only short chain oligophosphates as reported for calf intestine alkaline phosphatase.9 
  since phonsf generally delivered the highest product titers with primary alcohols as acceptors , the enzyme was selected for further investigations with 1,4butanediol concerning the effect of ph ( range 3.84.8 ) by using different pdonors ( figure  3 ) . 
 on the other hand , pppi and polyp usually yielded higher product concentrations at lower ph , due to slower product hydrolysis . 
 however , the poorly defined oligomeric composition of the polyphosphate donor renders precise analysis as well as product isolation challenging , and polyp was not investigated further . 
 effect of pdonor type and ph on the phosphorylation of 2 ( product formation over time ) . 
 conditions : 500 mm 
 2 , 250 mm ppi or pppi or 100 g  l polyp , 50 g ml phonsf , 1 ml volume , 1  % dmso as internal standard , 30 c , 600 rpm shaking . 
 the results depicted in figure  3 using phonsf suggest that a high number of transferable highenergy phosphate moieties as well as low ph values are crucial to achieve and maintain high product titers . 
 inspired by this observation , reactions were conducted at variuos ph values using high amounts of ppi and pppi overnight ( table  2 ) . 
 it should be noted that within the incubation time of 16.5 h both transphosphorylation and hydrolysis of product and phosphate donor will occur , and the balance between the two processes is determined by the activity of the enzymes and the ph . 
 although identical amounts of transferable phosphate moieties are available from 500 mm ppi and 250 mm pppi , significant differences in product formation were obtained after 16.5 h reaction time . using 500 mm ppi as a donor , 
 product yields of 2 for phonsf and piacp were similar at the three ph values . using ppi , up to 292 mm product ( up to 58  % conversion ) 
 could be obtained accompanied by nearly total consumption of ppi ( > 90  % ) , and the product / pi ratio was ca . 
 1:2 ( the ideal ratio is 1:1 in the absence of product hydrolysis and complete phosphate transfer ) . 
 however , under these conditions phonse furnished only inorganic monophosphate , indicating total hydrolysis of ppi and product . in the presence of 250 mm pppi the picture changed : the transphosporylation activity at ph = 3.5 of phonsf dropped considerably , that of piacp also dropped , but surprisingly considerable phosphorylating activity of phonse was maintained ( 164 mm , 33  % conversion and 54  % pdonor consumption ) . at higher ph values 
 this enzyme was still active , but only hydrolysis was observed , while phonsf and piacp delivered moderate to good amounts of product at full consumption of the pdonor . at 400 mm pppi and ph = 4.2 
 both phonsf and piacp showed the highest phosphorylating activity resulting in up to 393 mm product ( 79  % conversion , product / pi ratio ca . 
 4:5 ) at 75  % pdonor consumption . amount of product and pi formed in acid phosphatase catalyzed phosphorylation of 2 with ppi or pppi as donor , at three ph values with phonsf , piacp and phons . 
 reaction conditions : 500 mm 
 2 , phosphate donor as indicated , 50 g  ml phonsf , ph as indicated , 1 ml volume , 1  % dmso as internal standard , 30 c , 600 rpm shaking , 16.5 h. ideal ( maximum obtainable ) product / pi ratio is 1:1 with ppi and 2:1 with pppi . 
 cons.donor : consumption of pdonor calculated as follows : ( c 
 prod + c 
 pi)/c 
 theoretical max . 
 , higher pppi concentrations translate into higher product concentration and lower pi concentration , indicating more efficient phosphate transfer due to reduced product hydrolysis . from the data it is clear that using these phosphorylating enzymes in synthesis effectively requires a careful study of the donor concentrations , the type of donor , and the ph ; in batch processes also the time of incubation should be carefully chosen . 
 a long incubation time will result in low yields or near complete hydrolysis of the phosphorylated product . 
 consequently , kinetic control of the phosphorylation is very important , and physical separation of the phosphatase by employing a continuous flowreactor has proven to be an efficient tool to maintain high product levels ( by minimizing hydrolysis ) and scaleup the reaction.12c,31 under optimized conditions , the contact time between enzyme and substrate(s ) is controlled by the flow rate to ensure that ( rapid ) phosphorylation is complete , but ( slow ) product hydrolysis can not take place after the product has left the enzymecontaining column reactor . 
 we evaluated several epoxy and aminofunctionalized polymeric beads for the covalent immobilization of phonsf and piacp ( table s3 ) . 
 relizyme ha403/m , an aminofunctionalized resin , resulted in the highest specific activity for both enzymes ( 159 and 215 u  g dry beads for phonsf and piacp , respectively ) . 
 phonsf ( 300 u ) immobilized on relizyme ha ( 300 mg ) was applied to the preparativescale phosphorylation of 2 , yielding 6.86 g ( 22.5 mmol ) of the corresponding barium salt of 4hydroxybutyl phosphate ( 41  % isolated yield , for details see supporting information ) . 
 the enzymatic phosphorylation of primary and secondary alcohols in aqueous medium using cheap inorganic di , tri and polyphosphate as pdonors catalyzed by three acid phosphatases proceeded with absolute chemoselectivity for o vs. n and snucleophiles and with high regioselectivity for prim over sechydroxy groups . 
 the use of mild reaction conditions allowed the formation of phosphate monoesters as sole products , yielding environmentally innocuous inorganic monophosphate and unreacted phosphate donor as byproducts . 
 the thermodynamically favored hydrolysis of phosphate esters could be minimized by controlling various reaction parameters , thus allowing the use of phosphatases in the synthetic direction . 
 scaleup was demonstrated by the synthesis of 4hydroxybutyl phosphate ( 6.86 g , 41  % yield ) using an immobilized enzyme in flow mode . 
 ","abstracta set of phosphatases was evaluated for their potential to catalyze the regio and stereoselective phosphorylation of alcohols using a highenergy inorganic phosphate donor , such as di , tri and polyphosphate . 
 parameters such as type and amount of phosphate donor and ph of the reaction were investigated in order to minimize the thermodynamically favored hydrolysis of the phosphate donor and the formed phosphate ester . diols were monophosphorylated with high selectivities . this biocatalytic phosphorylation method provides selectively activated and/or protected synthetic intermediates for further chemical and/or enzymatic transformations and is applicable to a large scale ( 6.86 g ) in a flow setup with immobilized phosphatase .",pubmed
"various non - native biological invaders have been noted for centuries , such as the phylloxera ( a homopteran ) that destroyed grapevines and devastated the wine industry in europe in the 1860s   and the chestnut blight that entered north america in the early 20th century and virtually eliminated a dominant tree species . by 1957 , charles elton produced a series of bbc radio broadcasts about the general phenomenon of non - native invasions   and transformed the series into a book for a lay audience the next year . 
 although particular invasions continued to attract great attention , elton 's book did not inspire a broad research thrust on invasions or a widespread public and policy response to their growing number . 
 rather , in the mid-1980s , an international project of the scientific committee on problems of the environment ( scope ) engaged over 100 prominent scientists worldwide , produced several widely read edited volumes ( for example , ) , and triggered an explosion of research on invasions , which soon came to be recognized as one of the great ongoing global environmental changes , along with climate , nutrient cycle enhancement , and land - use changes . 
 this surge of research included many studies of how particular non - native invaders eat native species ( for example , the brown tree snake in guam   and the hemlock woolly adelgid in the eastern us ) , compete with them ( for example , the gray squirrel in great britain , crown vetch and tall fescue in the us midwest , and the  killer alga  in the mediterranean ) , or infect them ( for example , whirling disease of rainbow trout in the us ) and sometimes compete with them while vectoring diseases to them ( for example , signal crayfish in europe   and the gray squirrel in great britain ) . 
 however , two new lines of research have become increasingly prominent , both indicating that impacts of introduced species are more varied and widespread than had previously been thought . 
 first , in contributions to the scope program , peter vitousek   described a different sort of impact . rather than focusing on the population level ( for example , as when the gray squirrel competes with the red squirrel in great britain and transmits squirrel pox ) or the community level ( affecting many species in the community , as when the brown tree snake eats all of the forest bird species in guam ) , he depicted impacts at the ecosystem level : invasions that affected nutrient cycles and other ecosystem - level processes and features ( such as fire cycles or hydrology ) . 
 research on such impacts in a variety of ecosystems has increased greatly , particularly in the new century , showing how a single introduced species can change processes or the physical structure of an ecosystem , or both , sometimes subtly and slowly , but in such a way as to affect a large fraction of the resident species , as in the study by fukami et al .   on impacts 
 the other major new direction of invasion biology is a greatly increased focus on genetics and evolution as increasingly sophisticated and accessible molecular genetic techniques become available . 
 molecular genetic research has increasingly revealed a great number of cases of the  stealth  impact of invaders , hybridization , and introgression with native species ( for example , ) . 
 the phenomenon had previously been detected almost exclusively phenotypically , but many phenotypic responses are sufficiently subtle that they are hidden amidst the normal variation of many natural populations . 
 it is now recognized that substantial introgression can occur even between native species that never hybridize when a newly introduced species constitutes a genetic bridge between them . 
 introgression can also cause substantial evolution of native species , as in the european wildcat , which hybridizes with feral domestic housecats . 
 hybridization between native and introduced species can even produce new , more invasive species ; the best - known example is common cordgrass , spartina anglica , an invader that arose in england from hybridization between native s. maritima and north american s. alterniflora . 
 in addition to showing the great extent of hybridization between introduced and native species , molecular genetic features such as microsatellites , mitochondrial dna , and single - nucleotide polymorphisms have increasingly been used to trace sources and routes of invasions ( for example , ) . 
 a major finding is that introduced populations often combine genes derived from multiple source populations , thus producing novel genetic combinations ( for example , ) . 
 in fact , some introduced populations arising from multiple sources have more genetic variation than any single source population ( for example , ) . 
 also , environmental dna ( edna ) is increasingly used to detect invaders   and played a key role in showing the great advance of asian carp toward the great lakes . 
 the burst of research in the wake of the scope project also revealed an increasing number of cases of  invasional meltdown  , in which two or more introduced species facilitate one another 's establishment or enhance one another 's impact on native species , as when the  honeydew  from introduced scale insects increases introduced ant populations   or when introduced birds disperse seeds of introduced plants . 
 in addition , an increasing number of  lag effects  have been detected , in which full - fledged widespread establishment and impact of an introduced species lags for years behind initial arrival . 
 the reasons for such lags are various , and some remain mysterious , but the commonness of the phenomenon can not be doubted . 
 the many threads of 25 years of increasingly intensive research on biological invasions all lead to the conclusion that problems caused by the phenomenon are numerous , often tremendously important ecologically or economically worldwide , and growing apace with globalization . amidst this plethora of publications , 
 a small but persistent group of critics has assailed the field of invasion biology and much of the invasion management effort on a variety of grounds , eliciting a spirited response . 
 some critics go so far as to call for an end to invasion biology as a scientific discipline [ 3739 ] . 
 the objections fall primarily into five categories : ( 1 ) the problems are overblown , and similar impacts arise from native species ; most introduced species are not problematic . 
 ( 2 ) in some instances , introduced species increase native biodiversity . ( 3 ) the antipathy toward introduced species is xenophobic . 
 ( 4 ) in the face of globalization , we can not substantially slow the spread of invasions ; it is a hopeless battle . 
 ( 5 ) for sentient animals , such as mammals , the methods used to combat invasive species are inhumane ; in any event , individuals of the targeted species have a right to exist . 
 criticism 5 , which is part of a philosophical dispute that is not near resolution , revolves around the issue of the relative weight of rights of individuals and rights of collective entities such as species . 
 consideration of criticism 2which is based on the fact that , in some local biotas , the number of established introduced species exceeds the number of native species locally eliminated ( whether because of introduced species or not)should be tempered by the fact that many complete species extinctions have been wholly or partly caused by introduced species , especially but not exclusively on islands . 
 in addition , in many instances , species populations , though not eliminated , are reduced to a small fraction of their previous numbers . 
 biodiversity in terms of species numbers is not lost , but there is a major functional and demographic change . 
 criticism 3 is an assertion , generally leveled as part of a social construction of the science of invasion biology and management with scant or no regard for the impacts of invasions that are the stated purpose of the disputed activities . 
 the key criticisms from an ecological standpoint are 1 and 4 : which introduced species really cause harm , and can we really do anything about it if they do ? 
 however , modern invasion biology is very new , and the great majority of introduced species have not been studied in their introduced ranges , and so it is not possible to say that they have no significant impact . 
 perhaps the most comprehensive study of this matter   found , for europe , that for only ca . 
 10% of some 11,000 introduced species has impact been documented , but that this is likely to underestimate the number actually causing impacts because of the dearth of research . 
 one of the major recent research thrusts of invasion biology , as noted above , is unraveling various ecosystem impacts , some of which are initially subtle and take a long time to be manifested ( for example , shifts in nutrient cycling ) . 
 .   also found that , of introduced terrestrial vertebrates and freshwater plants and animals , about 30% are known to have harmful impacts . 
 in addition , the lag effect noted above   suggests that it would be foolhardy to wait for an impact to be recognized before acting . by then 
 , the invader may well have spread beyond the point at which eradication or containment is possible . 
 furthermore , although some native species in certain circumstances ( almost always associated with some other anthropogenic environmental change ) do cause impacts of the sort frequently associated with introduced invaders , the relative frequency of such impacts is far less than for non - native species 
 . meanwhile , there is growing recognition of the great extent and variety of impacts of introduced species . 
 the lag effect , the subtlety of some impacts that have been occurring from the outset of the introduction , and the greater propensity for non - native species to cause harm mean that the suggestion   to manage invasions only after impact is evident is ill advised . 
 attempts to identify which species , if introduced , would be likely to become invasive have had some success ( for example , ) , inspiring the construction of several risk assessment tools , especially for plants . 
 the most prominent is the australian weed risk assessment ( awra ) , which has been refined and adapted for use in permitting introductions in many other regions . 
 none of these tools is foolproof ( for example , the awra fails to reject about 10% of known invasive plants in australia ) , and the multiplicity of factors ( including chance ) that affect whether an introduced population survives , spreads , and becomes invasive will certainly limit their accuracy . 
 nevertheless , they have been valuable in preventing some planned introductions that would likely have led to invasions , and the same tools can be used to identify recently introduced , restricted populations that should be prioritized for rapid attempts at eradication or containment . 
 the effort to prevent invasions or to reverse or mitigate their impacts is not hopeless . 
 biosecurity can be very effective , as evidenced by great improvements in new zealand as interdiction procedures became more stringent ( for example , ) . 
 similarly , the technology exists for greatly improved early detection and rapid response ; employing them is a matter of political will . once a population is established , it is possible in some cases to eradicate it , and the technology to do so has improved greatly . 
 increasingly ambitious eradication projects are being undertaken , and they are marked by striking successes ; the current frontiers for vertebrate eradications are attempts to eradicate multiple species simultaneously , attempts to eradicate invasive species on larger islands and even continents , and attempts to eradicate invasive species on islands inhabited by humans ( unpublished data ) . terrestrial plants , insects , and aquatic organisms have been the most refractory targets for eradication . 
 however , there have been striking eradication successes even for these taxa when populations have not yet spread widely beyond the initial introduction site ( for example , ) . 
 when eradication fails or is not attempted , many technologies can be employed to maintain populations of invaders at low densities . 
 the main , widely applied approaches are physical control ( such as pulling weeds by hand ) , mechanical control ( use of machinery ) , chemical control ( use of herbicides and pesticides ) , and biological control ( introduction of natural enemies of a target invader , from its native range ) . 
 each of these methods has succeeded in many instances and failed in others , and sometimes these methods have had undesirable non - target impacts . 
 however , incremental improvements in techniques for all of these methods are leading to higher success rates and fewer non - target impacts . 
 for instance , sterile males have been used in many maintenance management and even eradication programs for insects , especially flies , and pheromones have been used to disrupt lepidopteran mating   and to trap sea lampreys . 
 efficacy of the judas - animal technique , widely used in mammal control and eradication programs , has recently been greatly improved by the use of hormone injections to prolong estrus . 
 autocidal methods using genetic manipulation were proposed in the 1960s and 1970s but not implemented then . today , with the advent of various genetic manipulation techniques , including transgenes , several such projects have advanced to the proof - of - concept stage , particularly for dipterans and fishes , and a similar approach has been proposed for plants . in sum , it is simply untrue that it is futile to fight invasions . 
 a simultaneous and related call for the end of traditional restoration ecology [ 6569 ] as part of a  new world order    accompanied the calls for an end to invasion biology . 
 this is perhaps unsurprising , as a very large component of restoration ecology consists of eliminating or managing non - native invasive species , and the underlying argument of the advocates of abandoning traditional restoration ecology in favor of  novel ecosystems  has been that the overwhelming forces of invasions by non - native species and climate change have rendered obsolete the entire notion of returning an ecosystem to some historical state . among prominent advocates of promoting novel ecosystems and abandoning traditional restoration ecology 
 are several of the critics of invasion biology and management , and authors of articles for lay audiences , such as emma marris   and fred pearce , have vigorously promoted both agendas . 
 the contention that novel ecosystems are so inevitable as to constitute  the new normal    is incorrect on several grounds . for one thing ,  novel ecosystem  is undefined , at least in operational terms . in no instance 
 is it stated what specific criteria show that an ecosystem is now novel or what thresholds it has passed to achieve this status . 
 all ecosystems are constantly changing , even without anthropogenic influences [ 7781 ] , leading pickett et al .   to propose the metaphor of the  flux of nature  to replace  balance of nature  . 
 contrary to claims by advocates of fostering and molding novel ecosystems , modern restoration ecology does not attempt to recreate the past ; rather , the goal is to re - establish the historical trajectory of an ecosystem before anthropogenic influences derailed it . 
 this goal has been endorsed by the united nations convention on biological diversity , the european commission , and major non - governmental organizations such as the international union for the conservation of nature . 
 another problem with the rush to embrace novel ecosystems as a goal is that the sense that much of the earth is rapidly changing to some sort of novel condition is based partly on maps , such as the human global footprint , drawn at far too coarse a scale . 
 the field of restoration ecology is very young and has learned much in a short time from past mistakes . 
 meta - analyses show considerable progress in achieving restoration goals in a variety of ecosystem types , and impressive specific cases abound . 
 there is no evidence that any particular ecosystem can not be restored in the sense of modern restoration ecology ; the impediments are economic and political , not scientific and technological . 
 this does not imply that society should undertake to restore every ecosystem ; each case should be considered by all stakeholders in terms of desirability , cost , and resource availability . 
 policymakers and the general public whose interests they should represent can easily be misled by what they perceive to be the existence of a scientific controversy , and policymakers have used such 
 the drumbeat of claims that the entire enterprise of modern ecological restoration is futile can perniciously influence policy at a time when many nations are undertaking ambitious restoration projects , and the united nations , the three rio conventions , and many governments and large non - governmental organizations have all called for greatly expanded restoration efforts . at the least , the contention that novel ecosystems are inevitable and perhaps desirable encourages any tendency to delay prevention and redress of various harmful environmental impacts rather than to undertake new approaches and to devote new resources to long - term solutions to environmental problems . 
 the same inimical influences on policy are exerted by critics of invasion biology and management . 
 for instance , larson and palmer , in a policy - oriented journal , not only cite  emerging thinking  about novel ecosystems without mention of any of the weaknesses of the concept but cite davis et al .   and schlaepfer et al .   as calling into question the damage caused by invasive species without mentioning that these authors represent a small minority of scientists or citing specific rebuttals of those articles . 
 critics of invasion biology and restoration ecology are not likely to keep these vibrant growing fields from progressing scientifically , but they will be able to slow the transformation of scientific knowledge into actions on the ground . 
 ","invasions by non - native species have caused many extinctions and greatly modified many ecosystems and are among the major anthropogenic global changes transforming the earth . beginning in the mid-1980s , a dramatic burst of research in invasion biology has revealed a plethora of previously unrecognized impacts and laid bare the scope of the phenomenon . 
 similarly , research on various methods of managing invasions has expanded enormously , yielding incremental improvements in traditional methods and the advent of several new approaches , including the use of species - specific genetic and pheromonal methods . 
 this research has advanced the field of restoration ecology , of which invasion management is a key component . amidst this research progress , 
 a group of critics has attempted to cast doubt on the extent of damaging impacts caused by non - native invasive species , the feasibility of counteracting them and restoring ecosystems , and the motives of scientists engaged in such endeavors . 
 the criticisms are misguided but can potentially impede management of this pressing problem .",pubmed
"schwannomas are rare tumors of the nerve sheath that arise from the primary glial cells of the peripheral nervous system . as such 
 although benign and slow growing , schwannomas can cause significant morbidity due to direct involvement of nerves or by local mass effect that can pose particular challenges for treatment . 
 approximately 25 to 45% of all schwannomas are found in the head and neck , where they can originate from virtually any cranial nerve.1 the most common schwannoma is the vestibular schwannoma that arises from the vestibular branch of the eighth cranial nerve and accounts for 51% of all nerve sheath tumors found anywhere in the body.2 this is followed by schwannomas of the trigeminal nerve that comprise up to 8% of all schwannomas.3 by contrast , schwannomas in the nasal cavity or paranasal sinuses  and vidian canal schwannomas in particular  are much rarer . 
 the vidian canal , also referred to as the pterygoid canal , extends from the pterygopalatine fossa to the foramen lacerum in the floor of the sphenoid sinus4 and contains the vidian artery as well as the vidian nerve . 
 the vidian nerve arises from the joining of the greater superficial petrosal nerve and the deep petrosal nerve . 
 the greater superficial petrosal nerve consists of preganglionic parasympathetic fibers from the geniculate ganglion of the facial nerve . 
 the deep petrosal nerve consists of postganglionic sympathetic fibers from the carotid sympathetic plexus.5 
 6 
 7 
 8 the parasympathetic fibers of the vidian nerve synapse in the pterygopalatine ganglion , whereas the sympathetic fibers innervate the lacrimal gland and secretory pathways of the nasopalatine glands.5 
 6 
  schwannomas of the vidian canal can present with a variety of clinical symptoms . 
 damage to the vidian nerve itself can result in decreased or absent lacrimation , corneal desiccation as well as dryness of the nasal mucosa , and cluster headaches.5 additionally , vidian schwannomas have been reported to compress the adjacent internal carotid artery or bulge through the lateral wall of the sphenoid sinus to expand into the cavernous sinus.6 
 9 
  to date , only 4 cases of vidian nerve schwannomas and 11 cases of histologically unconfirmed peri - vidian tumors have been reported in the literature.10 
 11 
 12 
 13 
 14 
 15 here we present a case of biopsy - proven schwannoma of the vidian canal . 
 we discuss the patient 's initial presentation , imaging characteristics , histologic findings , and treatment for this unusual tumor and review the other reported cases of vidian canal schwannomas in the literature . 
 the patient is a 49-year - old woman with a 24-year history of multiple sclerosis ( ms ) who presented for follow - up evaluation after an acute flare of ms . in the past , 
 her ms flares had been relatively infrequent , with motor and/or peripheral sensory deficits that were well controlled with short - term courses of steroids . during her most recent flare , she experienced numbness of her left head , arm , and leg , with full resolution of symptoms after steroid treatment . 
 a routine surveillance brain magnetic resonance imaging ( mri ) was performed to evaluate for ms progression . 
 mri showed minimal progression of disease compared with imaging from 8 years prior , with extensive t2 , fluid - attenuated inversion recovery , and proton - density prolonged signal in the periventricular , pericallosal , and subcortical white matter consistent with ms . 
 incidentally , the mri also revealed a 19    13    10  mm ovoid mass along the inferior margin of the right cavernous sinus that was not present on previous mris . 
 the lesion was well circumscribed and homogeneously enhancing , with t1 hypointense and t2 hyperintense signal . 
 a repeat dedicated mri was performed that demonstrated the lesion originated within an expanded vidian canal ( fig . 
 a sinus computed tomography scan also showed bony erosion of an expanded vidian canal ( fig . 
 the patient noted some mild nasal congestion in the mornings but was otherwise asymptomatic without any facial pain or paresthesias , abnormal lacrimation , or dry eye symptoms . 
 physical examination was similarly unremarkable with a normal nasal endoscopy and no focal neurologic deficits . 
 the mass was easily identified within a noticeably expanded vidian canal along the lateral floor of the sphenoid sinus ( fig . 
 the canal was opened along the floor of the sphenoid sinus to uncover the mass and a biopsy was obtained . 
 the patient 's recovery was unremarkable , and she had no problems with dry eye or nasal irritation postoperatively . 
 two weeks after surgery , her endoscopic examination showed a well - healed sinus cavity . 
 an ovoid 14    20    13-mm mass ( white arrows ) centered in an enlarged right vidian canal and extending along the inferior margin of the right cavernous sinus . 
 the mass has ( a ) bright t1 signal post contrast , coronal view , and ( b ) bright t2 signal with fat saturation . 
 preoperative sinus computed tomography scan without contrast demonstrating bony erosion of the right vidian canal ( red arrow ) . 
 the mass ( black arrow ) is visible protruding from the right vidian canal , located at the floor of the sphenoid sinus . 
 options of endoscopic surgical resection , single - fraction stereotactic radiosurgery ( srs ) , and conventional fractionated radiotherapy using stereotactic radiotherapy ( srt ) were reviewed with the patient , and she decided to pursue treatment with radiation . given the benign histology , a background of ms that could confer additional sensitivity to radiation , and the patient 's young age , the patient decided to pursue srt to minimize late normal tissue injury risks . 
 schwannomas of the vidian canal are an extremely rare type of intracranial tumor with variable presentation . symptoms of vidian schwannomas can result from compression of surrounding structures or extension into surrounding areas that can cause headaches , facial pain and paresthesias , and facial muscle paralysis . 
 dysfunction of the vidian nerve itself may cause nasal dryness , decreased lacrimation , and corneal desiccation . here 
 we present an unusual case of a vidian nerve schwannoma incidentally identified in a patient who was asymptomatic from the lesion . to date , four documented cases of biopsy - proven vidian nerve schwannomas have appeared in the literature . reported cases include both male and female patients , ranging in age from 13 to 78 years , with both unilateral and bilateral lesions described.10 
 11 
 12 
 13 
 14 
 15 presenting symptoms included headache and facial palsy , oculomotor palsy and cerebrospinal fluid leak , unilateral hearing loss secondary to obstructive serous otitis media , and facial paresthesias . 
 complete surgical resection was pursued for all four cases through both open and endonasal approaches , although one patient with bilateral vidian nerve schwannomas had only partial resection of one lesion due to difficulties with surgical access.13 all patients had significantly improved or stable symptoms , and the two patients with the most severe symptoms  one with oculomotor palsy and ptosis and the other with headache and facial nerve palsy  demonstrated the most dramatic postoperative improvement with near to complete resolution of symptoms.10 
 13 
  consistent with these cases , the traditional and curative management of peripheral nerve tumors is surgical ; recent advancements in transnasal endoscopic skull base approaches frequently enable complete resection . 
 radiation therapy by either srs or multiple fraction radiotherapy is also an alternative option for treatment . 
 conservative management through observation with serial imaging is also reasonable when surgical resection is either not feasible or involves a disproportionate risk of postsurgical morbidity and/or mortality . 
 the choice of treatment modality depends on patient choice , functional status , presenting symptoms , tumor location , tumor volume , and  in rare instances  malignant potential . 
 the ultimate goal of both surgery and radiotherapy is to maximize tumor control while also optimizing preservation of surrounding structures . in this case , the patient decided to pursue treatment with radiation therapy . 
 conservative management was not considered , given the significant increase in tumor volume during the eight - year interval of mri surveillance and the presence of bone erosion . given the rare presentation of a mass located in the vidian canal , the decision was made to confirm the diagnosis with a biopsy prior to treatment . 
 the choice between single - fraction srs or multiple - fraction radiation was made based on size and proximity of the tumor to potential radiation - sensitive structures . 
 minimizing the risk of adverse effects from radiation was the determinant for pursuing fractionated radiation . in recent years 
 , radiation therapy has become a common alternative to surgical resection of intracranial schwannomas.16 
 17 
 18 
 19 
 20 
 21 options include either single - fraction srs or conventional daily low - dose fractionation over approximately five weeks . 
 srs is a well - tolerated procedure , typically completed during a single session of irradiation and far more convenient for the patient . 
 srs has become a widely accepted treatment option for vestibular schwannomas and has a high success rate of durable tumor control with often partial reduction in tumor volume , with progression - free survival of 93% at 5 years and 92% at 10 years.22 fractionated radiation has similar results and is chosen over srs when there is a concern that the size of target exceeds safe tolerance to srs , when there are critical radiation - sensitive structures in adjacent areas that may not be able to tolerate the single high - dose collateral exposure , or when there are underlying concerns of the patient to suggest a higher risk of an adverse event . compared with surgery 
 , radiation therapy has been reported in several studies to result in decreased mortality and fewer treatment - related complications for vestibular schwannoma including hearing loss and facial weakness.22 
 23 
 24 
 25 recent advances in imaging and radiation delivery offer more conformal treatment options such as srt  which combines both the superior accuracy of target localization of a stereotactic system and reduction in the size of the radiation treatment fields  and fractionated treatment , a radiobiologically safer means of delivering radiation when there are concerns for late effects of radiation decades after treatment completion . 
 schwannomas of the vidian canal are extremely rare tumors and may present with symptoms of headaches , facial pain , facial muscle paralysis , decreased lacrimation , and nasal dryness or as an incidental finding . 
 when a vidian canal tumor is identified , endoscopic biopsy can be used to confirm a histopathologically benign diagnosis before considering treatment options .","background  schwannomas of the vidian canal are an extremely rare type of intracranial tumor that can have variable clinical presentations including headache , facial pain , facial muscle paralysis , decreased lacrimation , or nasal dryness . 
 we present an atypical case of an incidentally identified asymptomatic vidian canal schwannoma . 
 case description  a 49-year - old woman with a history of multiple sclerosis presented for routine surveillance magnetic resonance imaging that detected an ovoid mass originating in the vidian canal . given the unusual location of the lesion , an endoscopic endonasal biopsy was performed and confirmed the diagnosis of a vidian canal schwannoma , for which the patient chose to receive fractionated radiation therapy . 
 conclusion  when a vidian canal tumor is identified , endoscopic endonasal biopsy can be used to confirm the diagnosis before pursuing either surgical or radiotherapy treatment . in particular , 
 fractionated radiation therapy offers a radiobiologically safe means of delivering radiation when there is concern for late radiation - related side effects following treatment completion .",pubmed
"posterior transpedicular pedicle screw fixation is widely used for obtaining internal fixation for management of the unstable spine mainly caused by trauma , and burst fractures comprise 1020% of such injuries.1 the aims of surgery in these fracture cases include decompression of the neural components , fracture reduction , and providing a stable fixation until arthrodesis is achieved . 
 transpedicular short - segment fixation became popular after the introduction of transpedicular screws by roy - camille23 and the internal fixator by dick.4 this approach includes pedicle screw fixation one vertebra above and one vertebra below the fracture . 
 short - segment spinal instrumentation has been beneficial in the management of thoracolumbar spinal fractures for better correction of kyphotic deformity , greater initial stability , early painless mobilization , and indirect decompression of the spinal canal.57 despite the advantages of this approach , it is also associated with loss of reduction and instrumentation failure in some cases.49 in an attempt to achieve stiffer short - segment constructs , some surgeons add pedicle screws at the fractured vertebra . 
 these screws can be termed  intermediate screws .  according to several biomechanical and clinical studies , monoaxial or polyaxial pedicle screws inserted at the level of fractured vertebrae , along with the segmental fixation of burst fractures , improved biomechanical stability of the construct.1012 in the past 20 years , the design and implantation techniques of the pedicle screw systems have been modified to reduce the rate of pedicle screw breakage and to facilitate easy application of the connecting rod without undue stress on the construct.1316 the polyaxial head coupling of the pedicle screw was found to reduce the compression bending strength at the screw  rod mount , in comparison with a monoaxial screw design.1516 based on research findings , use of the intermediate screw in conjunction with short - segment fixation for fracture reduction and kyphosis correction carries the additional advantages of construct stabilization , lordosis restoration , and preventing screw breakage.10 however , there have been few reports regarding short - segment fixation , which have compared the biomechanical performance of polyaxial versus monoaxial pedicle screws with or without an intermediate screw at the fractured vertebra . 
 this study utilized a calf experimental model of an unstable burst fracture to compare the stiffness of a short - segment pedicle fixation using either monoaxial or polyaxial pedicle screws placed at the level of fracture . 
 twelve fresh - frozen calf lumbar specimens ( l1l5 ) were obtained from a regional slaughterhouse . 
 all specimens were wrapped in doubled plastic bags and stored frozen at 20c . before biomechanical testing 
 , the specimens were thawed at room temperature in a humidity - controlled environment for 8 h. the soft tissues were removed by dissection , leaving the ligaments , facet joint capsules , and intervertebral discs intact . 
 the end vertebrae were trimmed , fixed with plain screws , and embedded in liquid self - curing denture base material and denture base resin ( type ii ) . 
 each pot included a 10-mm screw secured to a metal plate that was incorporated in the resin cast . 
 this screw was located at the level of the middle column of the vertebrae and was used as an anchor for the testing machine . 
 the resin was left to cure for 30 min . to avoid the influence of air exposure on biomechanical behavior , 
 handling experimental material routinely used in in vitro biomechanical investigation in this manner , does not alter the material characteristics of the bone and soft tissues . 
 after the intact spine was tested for flexibility , an unstable burst fracture was created in the l3 vertebra using the pre - injury and the dropped - mass technique . 
 a 2-mm drill bit was used to create holes in the l3 vertebral body with v - shaped corpectomy [ figure 1 ] . 
 after the pre - injury model had been created , the specimen was mounted through its pots on a ferric base . 
 the specimen was flexed to approximately 10 and a 10-kg weight was dropped from a height of 0.5 m on a vertical rail , and then from 0.6 , 0.7 , 0.8 m , and so on , until the v - shaped cut closed . 
 after the creation of the l3 vertebral fracture , it was ensured that the pedicle remained intact for further experimentation . 
 ( a ) the v - shaped corpectomy of the l3 vertebra ; ( b ) the dropped - mass technique used after osteotomy to simulate an unstable burst fracture of the l3 vertebra ; ( c ) short - segment pedicle screw construct involving transpedicular fixation of vertebrae one above and one below the fracture site ; ( d ) use of an intermediate screw at the level of the fractured vertebra to test improvement in stability of the construct the specimens were randomly divided into monoaxial pedicle screw ( mps ) group and polyaxial pedicle screw ( pps ) group by random digits table , and six calves were allocated in each group . two kinds of spinal fixations ( i.e. , with or without intermediate screw placement ) were studied in each group [ figure 1 ] . after a fracture was created , a short - segment posterior pedicle screw construct was used to realign and stabilize the spine . 
 the specimens in the mps group were fixated with monoaxial pedicle screws , and polyaxial pedicle screws were used for those in the pps group . 
 the specimens in which an intermediate screw was not applied received a 4-screw ( monoaxial or polyaxial ) construct incorporating one vertebra above and one vertebra below the fracture . the remaining specimens ( mpsi and ppsi ) underwent a 6-screw construct in which two intermediate screws were added to the fractured vertebrae in addition to the 4-screw construct described above [ figure 2 ] . 
 the intermediate screws were of the same length and were inserted to the same depth as the other four screws . 
 we reused the same specimens in mps group and mpsi group , pps group and ppsi group in order to standardize the procedure and avoid any difference amongst the specimens in the same group . 
 x - ray film and photographs . ( a ) the film showing the burst fracture of vertebral body and the preservation of the anterior longitudinal ligament ; ( b ) mechanical testing for lateral bending ( intact specimen ) ; ( c ) mechanical testing for axial rotation ( intact specimen ) ; ( d ) mechanical testing for flexion and extension ( fracture specimen ) ; ( e ) mechanical testing for flexion and extension ( short - segment fixation specimen ) ; ( f ) mechanical testing for flexion and extension ( intermediate screw fixation specimen ) flow chart showing the sequence of events for each specimen . 
 bt = biomechanical testing ; mps = monoaxial pedicle screw ; pps = polyaxial pedicle screw ; mpsi = monoaxial pedicle screws with intermediate pedicle screws fixation ; ppsi = polyaxial pedicle screws with intermediate pedicle screws fixation each specimen was tested for flexion , extension , lateral bending , and axial rotation as an intact spine , as a fractured spine , and as a fractured spine fixed by either a 4-screw or 6-screw construct . 
 all specimens were subjected to 0500 n flexion and axial compression in a displacement - controlled mode at a rate of 5 mm / min on an mts858 ( material testing system , mts ) testing machine . 
 the mts858 testing machine is used to make the specimen move in the mode of flexion , extension , lateral bending , and axial rotation . 
 the ranges of motion ( roms ) of the relative intervertebral motions were automatically recorded by the eagle-4 and motion analysis position capture and measurement system ( micron company , california , usa ) . 
 the camera system recorded the movements of infrared led markers attached to each of the levels , l2l4 . 
 a digitizing probe was used to locate landmarks on each vertebra from which local coordinate systems were established . 
 the local coordinate systems were aligned to the primary anatomic planes ( sagittal , coronal , and axial ) . 
 the relations of the superior vertebral local coordinate system with respect to the inferior vertebral coordinate system were quantified with euler angle transformations . 
 the roms calculated for the 4- and 6-screw constructs were then compared with the values obtained for the intact and fractured specimens . because the 4- and 6-screw construct results in the mps or pps group were compared for the same specimen , paired student 's t - test was used for statistical analyses , using spss 15.0 statistical software ( spss / pc , chicago , il , usa ) . 
 biomechanical data were evaluated using a general linear model procedure for analysis of variance ( anova ) and the student newman 
 the differences in rom between the two groups were evaluated with independent t - tests . 
 the differences in rom within the same group were evaluated with paired t - tests . 
 twelve fresh - frozen calf lumbar specimens ( l1l5 ) were obtained from a regional slaughterhouse . 
 all specimens were wrapped in doubled plastic bags and stored frozen at 20c . before biomechanical testing 
 , the specimens were thawed at room temperature in a humidity - controlled environment for 8 h. the soft tissues were removed by dissection , leaving the ligaments , facet joint capsules , and intervertebral discs intact . 
 the end vertebrae were trimmed , fixed with plain screws , and embedded in liquid self - curing denture base material and denture base resin ( type ii ) . 
 each pot included a 10-mm screw secured to a metal plate that was incorporated in the resin cast . 
 this screw was located at the level of the middle column of the vertebrae and was used as an anchor for the testing machine . 
 the resin was left to cure for 30 min . to avoid the influence of air exposure on biomechanical behavior , 
 handling experimental material routinely used in in vitro biomechanical investigation in this manner , does not alter the material characteristics of the bone and soft tissues . 
 after the intact spine was tested for flexibility , an unstable burst fracture was created in the l3 vertebra using the pre - injury and the dropped - mass technique . 
 a 2-mm drill bit was used to create holes in the l3 vertebral body with v - shaped corpectomy [ figure 1 ] . 
 after the pre - injury model had been created , the specimen was mounted through its pots on a ferric base . 
 the specimen was flexed to approximately 10 and a 10-kg weight was dropped from a height of 0.5 m on a vertical rail , and then from 0.6 , 0.7 , 0.8 m , and so on , until the v - shaped cut closed . after the creation of the l3 vertebral fracture , it was ensured that the pedicle remained intact for further experimentation . 
 ( a ) the v - shaped corpectomy of the l3 vertebra ; ( b ) the dropped - mass technique used after osteotomy to simulate an unstable burst fracture of the l3 vertebra ; ( c ) short - segment pedicle screw construct involving transpedicular fixation of vertebrae one above and one below the fracture site ; ( d ) use of an intermediate screw at the level of the fractured vertebra to test improvement in stability of the construct 
 the specimens were randomly divided into monoaxial pedicle screw ( mps ) group and polyaxial pedicle screw ( pps ) group by random digits table , and six calves were allocated in each group . two kinds of spinal fixations ( i.e. , with or without intermediate screw placement ) were studied in each group [ figure 1 ] . after a fracture was created , a short - segment posterior pedicle screw construct was used to realign and stabilize the spine . 
 the specimens in the mps group were fixated with monoaxial pedicle screws , and polyaxial pedicle screws were used for those in the pps group . 
 the specimens in which an intermediate screw was not applied received a 4-screw ( monoaxial or polyaxial ) construct incorporating one vertebra above and one vertebra below the fracture . the remaining specimens ( mpsi and ppsi ) underwent a 6-screw construct in which two intermediate screws were added to the fractured vertebrae in addition to the 4-screw construct described above [ figure 2 ] . 
 the intermediate screws were of the same length and were inserted to the same depth as the other four screws . 
 we reused the same specimens in mps group and mpsi group , pps group and ppsi group in order to standardize the procedure and avoid any difference amongst the specimens in the same group . 
 a ) the film showing the burst fracture of vertebral body and the preservation of the anterior longitudinal ligament ; ( b ) mechanical testing for lateral bending ( intact specimen ) ; ( c ) mechanical testing for axial rotation ( intact specimen ) ; ( d ) mechanical testing for flexion and extension ( fracture specimen ) ; ( e ) mechanical testing for flexion and extension ( short - segment fixation specimen ) ; ( f ) mechanical testing for flexion and extension ( intermediate screw fixation specimen ) flow chart showing the sequence of events for each specimen . 
 bt = biomechanical testing ; mps = monoaxial pedicle screw ; pps = polyaxial pedicle screw ; mpsi = monoaxial pedicle screws with intermediate pedicle screws fixation ; ppsi = polyaxial pedicle screws with intermediate pedicle screws fixation 
 each specimen was tested for flexion , extension , lateral bending , and axial rotation as an intact spine , as a fractured spine , and as a fractured spine fixed by either a 4-screw or 6-screw construct . 
 all specimens were subjected to 0500 n flexion and axial compression in a displacement - controlled mode at a rate of 5 mm / min on an mts858 ( material testing system , mts ) testing machine . 
 the mts858 testing machine is used to make the specimen move in the mode of flexion , extension , lateral bending , and axial rotation . 
 the ranges of motion ( roms ) of the relative intervertebral motions were automatically recorded by the eagle-4 and motion analysis position capture and measurement system ( micron company , california , usa ) . 
 the camera system recorded the movements of infrared led markers attached to each of the levels , l2l4 . 
 a digitizing probe was used to locate landmarks on each vertebra from which local coordinate systems were established . 
 the local coordinate systems were aligned to the primary anatomic planes ( sagittal , coronal , and axial ) . 
 the relations of the superior vertebral local coordinate system with respect to the inferior vertebral coordinate system were quantified with euler angle transformations . 
 the roms calculated for the 4- and 6-screw constructs were then compared with the values obtained for the intact and fractured specimens . because the 4- and 6-screw construct results in the mps or pps group were compared for the same specimen , paired student 's t - test was used for statistical analyses , using spss 15.0 statistical software ( spss / pc , chicago , il , usa ) . 
 biomechanical data were evaluated using a general linear model procedure for analysis of variance ( anova ) and the student newman  keuls post hoc test in each group ( mps or pps ) . 
 the differences in rom between the two groups were evaluated with independent t - tests . 
 the differences in rom within the same group were evaluated with paired t - tests . 
 the rom results for l2l4 in all directions for the mps and pps groups are summarized in table 1 . 
 the addition of an intermediate screw decreased the rom of the l2l4 segments in both the mps and pps groups . 
 a smaller rom due to the use of an intermediate screw represents enhanced stability of the construct , in contrast to a larger rom that was observed in the standard short - segment construct . 
 compared to the short - segment transpedicular fixation group , the addition of intermediate screws provided a smaller rom in flexion , extension , lateral bending , and torsion in the mps group ( p = 0.001 , 0.006 , 0.077 , and 0.000 , respectively ) , and in flexion , extension , lateral bending , and torsion in the pps group ( p = 0.000 , 0.000 , 0.017 , 0.000 , respectively ) . 
 there was neither a difference in the rom in the spines of the two groups before injury , nor a difference in rom between the mpsi and ppsi groups ( p > 0.05 ) . 
 however , when using the intermediate screw in injured spines , the stiffness in the mpsi and ppsi groups increased significantly as compared with the intact spine ( p < 0.05 ) . 
 there was a significant difference between mps and pps in flexion ( p < 0.01 ) and extension ( p < 0.05 ) [ figure 4 ] . 
 rom in various planes of the intact , fracture model , and postinternal fixation spine within the same group ( n = 6 ;  ; mean  standard deviation ) bar graphs showing the rom compared with intact spine for ( a ) flexion , ( b ) extension , ( c ) lateral bending , and ( d ) axial rotation loading . 
 mps = monoaxial pedicle screw ; pps = polyaxial pedicle screw ; mpsi = monoaxial pedicle screws with intermediate pedicle screws fixation ; ppsi = polyaxial pedicle screws with intermediate pedicle screws fixation . * p , 0.05 for significant difference between groups 
 management options for unstable burst fractures include anterior , posterior , or combined ( circumferential ) fixation . 
 many surgeons believe that anterior column reconstruction is critical in correcting kyphotic deformity and reestablishing the anterior load bearing capacity of the vertebral component . short - segment posterior spinal instrumentation using pedicle screws remains the standard method for the fixation of thoracic and lumbar fractures , with acceptable results . 
 the advantages of this technique include less surgical dissection , blood loss , and time in surgery , and as a result , decreased perioperative morbidity . 
 however , loss of reduction and instrumentation failure associated with this technique is well described in the literature.159 these failures have been attributed to poor bone quality , inadequate anterior column support , and insufficient points of fixation . 
 the biomechanical properties of monoaxial pedicle screws have been widely reported.1720 segmental fixation with additional monoaxial pedicle screws at the level of the fracture increases construct stiffness and shields the fractured vertebral body from anterior loads . 
 furthermore , this additional point of fixation allows for a 3-point reduction maneuver , analogous to that used for reduction of long bone fractures . in a recent prospective randomized study , 
 the efficacy of the fracture - level screw combination in achieving and maintaining correction for the treatment of unstable thoracolumbar burst fractures was evaluated.21 the authors of that study concluded that reinforcement with a fracture - level screw could help provide and maintain improved kyphosis correction . 
 another mid - term clinical study demonstrated superior results with the fracture - level screw combination , supporting the evidence of other reports.22 interestingly , in most of these studies , the pedicle screws used were monoaxial , despite the current trend toward using polyaxial screws in short - segment fracture fixation.101221 on review of the literature , we found that there was a paucity of data , both clinical and biomechanical , on the use of polyaxial screw as an intermediate in unstable burst fractures , and thus this biomechanical study was instituted . 
 the current study showed that when short - segment constructs contained intermediate screws , flexion , extension , and torsion decreased in both the mpsi and ppsi groups , compared to the respective constructs ( mps and pps ) without this reinforcement . 
 the short - segment construct in the pps group had significantly greater rom in flexion and extension when compared with the mps group . 
 the addition of intermediate screws at the level of a burst fracture significantly increased the stiffness of the short - segment pedicle fixation , irrespective of whether monoaxial or polyaxial pedicle screws were used . 
 however , this increase was more significant for the mpsi construct , as the ppsi already had significantly increased stiffness as compared to the mpsi . these results may be because there is a better coupling between the polyaxial screw heads and the connecting rod . 
 thus , there is less torsion at the coupling site and consequently less stress on the entire construct . 
 the intermediate screw provides a 3-point fixation of the fractured segment . when using monoaxial screws , the addition of an intermediate forces the surgeon to bend the connecting rod to accommodate the additional screw . 
 polyaxial screws , on the other hand , facilitate the installation of the connecting rod , and their biomechanical properties have been reported in several studies.141623 stanford et al.15 suggested that the rod 
 screw link design of the polyaxial screw reduces its static compressive bending yield strength as compared with the fixed screw designs . 
 liu et al.24 pointed out that there were significant differences between the monoaxial screws and polyaxial screws in the bending stiffness , yield load , yield torque , and torsional stiffness in static tests ( p < 0.05 ) . 
 shepard et al.16 suggested that polyaxial screws do not significantly decrease the stiffness of the construct . on the contrary , 
 the polyaxial constructs create more security by permitting better contact and holding strength between the screw head and the rod . 
 when there is the combined effect of bending loads and shear force on the rods , there would be higher resistance to rotational slippage between the rod and the screw head . the limitations of study are that the data were pertinent to results immediately after surgery , but do not take into account early bone resorption or the cyclical loading effect which have long - term impacts on outcome . 
 the flexibility of the construct was tested on a burst fracture model that was created in a controlled and reproducible manner by the pre - injury and dropped - mass technique . nevertheless , the pattern and nature of a spinal burst fracture in clinic is more variable and unpredictable . 
 anekstein et al.12 hypothesized that the true mechanical effect of the intermediate screws is less predictable in in vivo settings . 
 only a prospective clinical study can show the true practical significance of the addition of intermediate screws . 
 although not human , calf spine has been used before as a valid biomechanical model.2527 calf is a tetrapod ; its anatomical characteristics and common fracture site are different from those of the human , and because the calf lumbar spines ( l1l5 ) can be easily dissected from the other vertebrae of calf , we chose the l1l5 calf lumbar spines and made the l3 vertebral fracture instead of the t12 or l1 which are the most common fracture sites in humans . 
 calf spine segments are presumed to have higher bone mineral density than human spines , and therefore have different biomechanical behavior . 
 nevertheless , the use of an animal model allows for smaller inter - specimen differences , as the human spine has been shown to have significant variability in bone mineral density that might potentially affect the test results.2829 as this study is comparative , the authors favored the animal model to that of the human cadaver spine . 
 it can be argued that the number of cases in each group was less in the present study to reach a meaningful conclusion . 
 however , in the previous biomechanical studies,183031 the number of the cases used in the experiment was usually six or seven . in the present study 
 , we used 12 cases and 6 cases in each group and we believe that these numbers have brought forward meaningful results . 
 another disadvantage of this study is that there was no comparison with long - segment fixations and application of the cross link , which have been shown to have better biomechanical stability than the short segment in managing unstable burst fractures . in conclusion 
 , the addition of intermediate screws at the level of a burst fracture significantly increases the stability of a short - segment pedicle fixation , using either monoaxial or polyaxial pedicle screws . 
 however , the short - segment mps fixation group was significantly more stable in flexion and extension than the short - segment pps fixation group . 
 we believe that this study provides a biomechanical rationale for using an intermediate screw at the level of fractured vertebrae with posterior short - segment fixation , especially when using monoaxial screws for fixing the unstable burst fractures of the lumbar region .","background : use of a pedicle screw at the level of fracture , also known as an intermediate screw , has been shown to improve clinical results in managing lumbar fracture , but there is a paucity of biomechanical studies to support the claim . 
 the aim of this study was to evaluate the effect of adding intermediate pedicle screws at the level of a fracture on the stiffness of a short - segment pedicle fixation using monoaxial or polyaxial screws and to compare the strength of monoaxial and polyaxial screws in the calf spine fracture model.materials and methods : flexibility of 12 fresh - frozen calf lumbar spine specimens was evaluated in all planes . 
 an unstable burst fracture model was created at the level of l3 by the pre - injury and dropped - mass technique . 
 the specimens were randomly divided into monoaxial pedicle screw ( mps ) and polyaxial pedicle screw ( pps ) groups . 
 flexibility was retested without and with intermediate screws ( mpsi and ppsi ) placed at the level of fracture in addition to standard screws placed at l2 and l4.results:the addition of intermediate screws significantly increased the stability of the constructs , as measured by a decreased range of motion ( rom ) in flexion , extension , and lateral bending in both mps and pps groups ( p < 0.05 ) . 
 there was neither any significant difference in the rom in the spines of the two groups before injury , nor a difference in the rom between the mpsi and ppsi groups ( p > 0.05 ) , but there was a significant difference between mps and pps in flexion and extension in the short - segment fixation group ( p < 0.05).conclusions : the addition of intermediate screws at the level of a burst fracture significantly increased the stability of short - segment pedicle screw fixation in both the mps and pps groups . 
 however , in short - segment fixation group , monoaxial pedicle screw exhibited more stability in flexion and extension than the polyaxial pedicle screw .",pubmed
"the discovery of electrochemical self - assembly 
 ( esa ) of nanostructured 
 hybrid thin films of inorganic semiconductors and organic dyes has 
 opened a new synthetic route for obtaining photoanode material for 
 dye - sensitized solar cells ( dsscs ) . 
 this method bypasses the high - temperature sintering that is typically 
 needed for nanoparticulate tio2 electrodes , which prevents 
 the use of conductive plastic substrates . 
 we have previously demonstrated 
 the use of esa with the zno structures for the construction of photoanodes . 
 minor addition of water - soluble dye molecules with substituents 
 for anchoring to zno surface brought about a significant impact on 
 the crystal growth for evolution of various nanostructures and alteration 
 of the crystallographic orientation of the film . 
 importantly , the 
 coprecipitation of dye molecules does not necessarily deteriorate 
 the crystallinity of zno . in the case of zno / eosin 
 y ( ey ) hybrid thin 
 films , vertically aligned interconnected and crystalline nanowires 
 of zno are formed . the space between the zno nanowire 
 is filled with aggregates of ey . 
 the solid ey can be completely removed 
 by soaking the hybrid film in a mild alkaline such as dilute koh aq . 
 thus , prepared zno thin films , 
 consisting of large single - crystalline grains with internal porous 
 structure , to be called  porous crystal zno  , have large 
 surface area for adsorption of photosensitizer dye in a large amount 
 and high crystallinity for efficient transport of electrons ; therefore , 
 it can achieve high incident photon to current conversion efficiency 
 ( ipce ) of around 90% . with the dssc application 
 in mind , it would be highly interesting 
 to find a compatible system for fabricating sensitized photocathodes . 
 recently , powar et al . 
 have succeeded in achieving 1.3% conversion 
 efficiency for a dssc in an inverted structure employing a porous 
 p - nio electrode sensitized with a specially designed dye to achieve 
 a high rectification with kinetically reversible co(ii / iii ) ethylene 
 diamine complex electrolyte . 
 we have been 
 seeking to perform cathodic electrodeposition of cuscn , a p - type , 
 wide - bandgap semiconductor , into highly crystallized thin films at 
 low temperature . 
 cuscn has been frequently used as a hole conductor in nanostructured 
 solar cells and also as a sensitized photocathode in combination with hole - injecting 
 dyes . 
 therefore , esa of cuscn - based hybrid thin films could 
 serve nicely as a counterpart to the well - established zno - based hybrid 
 systems . 
 our first attempt for esa of cuscn hybrid system was 
 to employ 
 dye molecules having soft lewis basic ncs groups . on the basis of the hard and soft , acid and base 
 ( hsab ) principle , choice of dyes equipped with hard lewis basic carboxylate 
 is suited for binding to hard lewis acidic ti(iv ) sites of tio2 or zn(ii ) sites of zno . because cu(i ) of cuscn is a typical 
 soft lewis acid , the soft sulfur atom of the ncs should be 
 a good match . as such 
 , dyes having ncs groups , the popular 
 n3 dye ( ru(dcbpy)2(ncs)2 , dcbpy = 2,2-bipyridine-4,4-dicarboxylic 
 acid ) and fltc ( fluorescein isothiocyanate ) were added to the bath 
 for electrodeposition of cuscn . 
 as expected , colored cuscn thin films 
 incorporating n3 and fltc were obtained , while ey added for comparison 
 did not cause any change for the formation of cuscn , which is of course 
 normally colorless . 
 the addition of n3 and fltc also brought about 
 the evolution of characteristic nanostructure of cuscn , clearly indicating 
 their action as structure directing agents ( sdas ) . 
 the electrodeposited 
 cuscn / dye hybrid thin films exhibited dye - sensitized photocathodic 
 properties , evincing the intimate chemistry between cuscn and dyes 
 having soft basic anchors , which appear to be the key for hole injection 
 from the dye excited state . following the success with the above - mentioned 
 strategy , the idea 
 further developed to try substituting cu(i ) of cuscn with cationic 
 dyes instead of substituting ncs with dyes having 
 ncs group . among commercially readily available dyes , we have 
 employed rhodamine b ( rb ) , which is cationic because of the presence 
 of an ammonium group . 
 this strategy indeed showed positive results 
 as we have seen a clear effect of rb as an sda for formation of nanostructure 
 and as a cause of changes in crystallographic orientation of cuscn . 
 however , it was not possible to extract the loaded 
 rb molecules by any kind of post treatment to obtain highly porous 
 structure as in the case of the zno / ey hybrid system . because the 
 mechanism of cuscn electrodeposition was poorly understood at the 
 time of the previous work , the conditions were not properly chosen 
 to achieve hybrid thin films with composition similar to that of zno / ey , 
 for which ey could occupy close to half of the volume of the precipitated 
 solid . in this work , 
 we present a method for esa of hybrid cuscn / rb 
 nanoporous 
 films in which the rb sda may be nearly completely removed . by varying 
 the relative concentration of cu and scn 
 , we can change the crystal orientation of the resulting nanostructure 
 with respect to the substrate . 
 we explore several preliminary dyes 
 and show photoinduced hole transfer to the cuscn ; in addition , we 
 tested the best systems in photoelectrochemical studies to check the 
 dye - sensitized photocathodic properties . at this stage , 
 we still do 
 not have a good combination of dye and electrolyte for cuscn . 
 however , 
 the recent progress with the photocathodic dssc presents a new opportunity 
 to improve the efficiency of dye - sensitized cathode systems that can 
 eventually be combined with existing sensitized anodes for realization 
 of a tandem solar cell to achieve high efficiencies in very simple 
 structures . 
 inorganic chemicals such as lithium thiocyanate dihydrate ( liscn2h2o , kishida ) , copper(ii ) perchlorate hexahydrate ( cu(clo4)26h2o , sigma - aldrich ) , and lithium 
 perchlorate ( liclo4 , sigma - aldrich ) and organic solvents 
 such as n , n - dimethylacetamide ( dma , wako ) and ethanol 
 ( wako ) were of special grade and used as purchased . 
 fluorine - doped tin oxide ( fto ) coated 
 transparent conductive glass ( sheet resistance , 78 /sq , 
 1.1 mm thick soda lime glass , asahi - du , asahi glass ) was used as the 
 substrate to electrodeposit the cuscn thin films . 
 milli - q ultrapure 
 water ( > 18 m ) was used throughout the experiments . 
 the fto glass was cut into 25  25 mm , degreased 
 by ultrasonically cleaning in detergent and acetone , and finally rinsed 
 with water . 
 it was mounted into a specially designed electrode holder 
 for a homemade rotating disc electrode ( rde ) system . 
 the active area of the fto glass electrode was regulated 
 to a 22 mm diameter circle concentric to the rotating electrode holder 
 by applying a masking tape ( nitto denko n-300 ) . 
 the use of a rde system 
 to introduce laminar flow is essential for obtaining cuscn thin films 
 with perfect homogeneity and with a high reproducibility because the 
 reaction occurs under the mass transport limited regime . 
 the fto glass rde that serves as a working electrode is placed with 
 the active surface down and in the center of the electrochemical cell 
 of a cylindrical shape , equipped with a water - jacket for temperature 
 control ( 25 c ) , together with auxiliary electrodes , a pt wire 
 counter and a ag / agcl reference . 
 a mixture of water and ethanol ( 1:3 
 in volume ratio ) was used for preparation of the deposition bath . 
 cu(clo4)2 and liscn were dissolved at 9 and 
 3 or 3 and 9 mm for cu rich or scn rich bath , respectively , while 0.1 m liclo4 was also 
 added as the supporting electrolyte . 
 a small amount of rb up to 1.0 
 mm was added to obtain cuscn / rb hybrid thin films . 
 a hokuto - denko 
 hsv-100 electrochemical system was used for potential control and 
 current monitoring . 
 cathodic electrolysis at + 0.2 v ( vs ag / agcl ) for 
 5 min and with a rotation speed of 500 rpm resulted in cuscn thin 
 films which were washed with water , dried under air at room temperature , 
 and subjected to characterization . 
 vis absorption 
 spectra of the films were measured in 
 transmission on a hitachi u-4000 or a perkinelmer lambda 1050 spectrophotometer . 
 fluorescence spectra of the film samples were measured on a photon 
 technology international quantamaster40 equipped with two double - monochromators 
 and a cooled pmt detector . aside from the cuscn / rb hybrid thin film , 
 cuscn hybrid with fluorescein isothiocyanate ( fltc ) , nile blue ( nb ) 
 , 
 cresyl violet ( cv ) , 3,4,9,10-perylenetetracarboxylic acid 
 diimide ( ptcdi ) , and quinacridone ( qd ) , as well as thin solid films 
 of only these chromophores , were prepared for comparison in the fluorescence 
 study . 
 those of rb , fltc , nb , and cv were prepared by hybrid electrodeposition 
 and spin coating their 1 mm solutions in ethanol on a slide glass , 
 while those of ptcdi and qd were prepared by vacuum evaporation either 
 onto an electrodeposited porous cuscn or on a slide glass . 
 surface 
 morphology of the films was observed by field emission scanning electron 
 microscopy ( fe - sem , hitachi s-4800 or jeol jsm-6700f ) . 
 crystallographic 
 characterization of the film was conducted by measuring x - ray diffraction 
 ( xrd ) patterns on a rigaku rint ultima iii instrument . 
 the amount 
 of rb loaded into the film was determined by dissolving 
 a known area of the film into a known volume of ammonia solution and 
 measuring its absorption spectrum . 
 the amount of electrodeposited 
 cuscn was determined by measuring the amount of cu in 
 the ammonia solution dissolving the film according to the classical 
 colorimetric analysis method , employing 
 sodium n , n - diethyldithiocarbamate 
 trihydrate ( ddtc , wako ) to chelate copper ion for extraction with 
 ethyl acetate which exhibits a strong absorption at 436 nm (  
 = 1.3  10 m  cm ) . 
 comparison with the amount of consumed charge yields faradaic 
 efficiency for the electrodeposition of cuscn . 
 a part of the loaded 
 rb molecules could also be extracted by soaking the film in dma . from 
 comparison of the absorption spectra before and after the dma treatment , 
 the fraction of the extractable rb could be determined . 
 several thin - film 
 samples after the dma treatment were scratched off the substrate . 
 the surface area of powder samples prepared in this manner was determined 
 by brunauer 
 teller ( bet ) analysis of the kr gas 
 sorption measurement conducted on a micromeritics tristar ii 3020 
 system . 
 photoelectrochemical measurements on cuscn thin film 
 electrodes 
 have been performed in a three - electrode setup with a pt wire counter 
 and a saturated calomel reference electrode ( sce ) . 
 porous cuscn thin 
 films were sensitized by soaking them in a 1.0 mm rb aqueous solution 
 for 1 h. a 0.1 m aqueous solution of methylviologen dichloride ( mv , sigma - aldrich ) was used as the electrolyte . 
 the electrode 
 was illuminated from its front side with a visible light generated 
 by a 500 w xe lamp ( ushio ) filtered by uv ( < 420 nm ) and ir ( > 800 
 nm ) cutoff filters ( intensity , 100 mw  cm ) . 
 direct crystallization of -cuscn 
 occurs as a consequence 
 of cathodic reduction of cu in the presence of scn both from aqueous and ethanolic solutions . 
 rhodamine b is commercially provided as chloride 
 and is well - soluble both to water and ethanol . 
 30 m when 0.1 m liclo4 was 
 added to water as a supporting electrolyte because of the salting - out 
 effect , making it difficult to achieve a high loading of rb to cuscn . 
 on the other hand , ethanol dissolved rb too well to hinder its loading 
 to cuscn . 
 therefore , a mixture of water and ethanol ( 25:75 volume 
 ratio ) was chosen as the solvent that provided good solubility of 
 all chemicals and achieved an efficient loading of rb molecules during 
 electrochemical growth of cuscn . 
 our previous studies on electrodeposition 
 of cuscn employing a 
 rotating disk electrode revealed that the growth of cuscn is typically 
 limited by transport of complexes of cu and scn that form in solution , achieving practically 100% faradaic efficiency 
 for the formation of cuscn . in aqueous 
 solutions , only a 1-to-1 complex 
 is formed , while a neutral 1-to-2 complex is also formed and participates in 
 the electrochemical reaction in ethanolic solutions containing excess 
 of scn . 
 thus , the 
 overall reaction is expressed by eq 1 for the 
 aqueous solution and both eqs 1 and 2 operate in the ethanolic solution.12the chronoamperograms measured 
 during the 
 electrodeposition can be seen in figure 1 . 
 following the initial rise of the current , 
 diffusion - limited steady - state 
 current is reached both for the cu rich and scn rich solutions . however , 
 this should reflect the formation 
 of a bis - coordinated cu(ii ) thiocyanato complex which has a diffusion 
 coefficient that is somewhat higher than that of the monocoordinated 
 complex in ethanol as revealed in our electrochemical analysis . 
 the addition of rb to the bath causes a slight 
 decrease of the steady - state current in the scn rich bath , whereas a slight increase of the current was observed 
 in the cu rich bath , which could be caused by the chemical 
 interaction of the complexes with rb molecules . 
 even though the addition 
 of rb caused a slight decrease of faradaic efficiency for the precipitation 
 of cuscn ( vide infra ) , the film thickness increased linearly with 
 the consumed charge ( see figure 1s of supporting 
 information ) . 
 as the current is almost constant , the film thickness 
 can simply be controlled by the time of the electrolysis . 
 however , 
 it should be mentioned that the high addition of rb at 1.0 mm brings 
 about a characteristic change in the initial profile of the current . 
 in such baths , 
 the current stays small in the beginning of the electrolysis 
 then abruptly increases to achieve the steady - state current . 
 the surface 
 of the fto glass substrates was observed by sem before and after the 
 jump of the current ( figure 2 ) . 
 there is apparently 
 no change before the jump as the bare surface of the fto layer is 
 seen . 
 after the jump , tiny cuscn particles are deposited , especially 
 at the bottom of the valleys and along the ridges of the pyramidal 
 fto grains . although the reason for the incubation time is unclear , 
 it is probable that rb molecules are adsorbed on the fto surface , 
 hindering the nucleation of cuscn . 
 chronoamperograms measured during electrodeposition 
 of cuscn and 
 cuscn / rb hybrid thin films onto an fto coated glass rde (  = 
 500 rpm ) at + 0.2 v ( vs ag / agcl ) in ethanol / water ( 75/25 , v / v ) mixed 
 solutions containing cu(clo4)2 and liscn in 
 cu rich ( 9 and 3 mm , respectively ) and scn rich ( 3 and 9 mm , respectively ) compositions ; 0.1 m liclo4 as a supporting electrolyte ; and 0 , 0.1 , or 1.0 mm rb . 
 sem pictures of the surface of the fto glass substrates 
 after electrolysis 
 for 20 s ( a ) and 30 s ( b ) in an ethanol / water ( 75/25 , v / v ) mixed solution 
 containing 3 mm cu(clo4)2 , 9 mm liscn , 0.1 m 
 liclo4 , and 1.0 mm rb . 
 cuscn / rb hybrid thin films were electrodeposited for 5 min 
 both 
 from scn rich and cu rich baths while 
 changing the concentration of rb . 
 the morphology of the pure cuscn 
 film looks totally different for those deposited from scn rich and cu rich baths ( panels a1 and b1 of figure 3 , respectively ) . 
 the former consists of large rounded 
 hexagonal columnar particles , whereas the latter shows assemblies 
 of spiky particles . 
 rhombohedral -cuscn has an elongated hexagonal 
 unit cell with lattice constants of a = b = 3.857  and c = 16.449  . as discussed below , the xrd patterns of the 
 films indicate that films of a1 and b1 are oriented for the c - axis perpendicular with the substrate . 
 the hexagonal faces 
 of the deposits in a1 should thus correspond to the ( 003 ) planes of 
 cuscn , while those faces creating the spikes should probably be of 
 the ( 101 ) planes . 
 such differences can be caused by the change of 
 the stability of the crystal faces by the change of the chemical composition 
 of the bath influencing the dissolution and recrystallization of cuscn 
 during the electrodeposition ( eq 3).3when rb is added to the bath , purple - colored 
 cuscn / rb hybrid thin films were obtained . as the concentration of 
 rb increased , the color of the film got deeper , indicating the increased 
 loading of rb into the film . at the same time , the morphology of the 
 film drastically changed ( panels a2 , a3 , b2 , and b3 of figure 3 ) . 
 as discussed later , the loaded rb 
 molecules can almost completely be extracted by dipping the film shown 
 in a3 in dimethylacetamine ( dma ) , making it highly porous . 
 the shape 
 of the particles differs from sample to sample , especially as recognized 
 from the cross sections , indicating anisotropic crystal growth promoted 
 by rb addition . 
 sem photographs of cuscn and cuscn / rb hybrid thin films 
 electrodeposited 
 from scn rich ( a ) and cu rich ( b ) 
 baths containing 0 ( a1 and b1 ) , 0.3 ( a2 and b2 ) and 1.0 mm ( a3 and 
 b3 ) rb . a1a3 and 
 b1b3 
 correspond to the cross section of a1a3 and b1 b3 , 
 respectively . 
 changes of xrd patterns 
 on rb addition are shown in figure 4 for scn rich and cu rich baths . 
 all these patterns 
 indicate diffraction peaks assigned 
 to -cuscn aside from those originating from sno2 of the fto substrate . the relative intensity of the diffraction 
 peaks of the film samples , however , are different from those of the 
 powder diffraction standard . 
 it is also changes for different rb concentrations 
 and for scn rich ( a ) and cu rich ( b ) 
 baths , indicating changes of their crystallographic orientation . 
 two 
 representative diffraction peaks arising from the ( 003 ) and the ( 101 ) 
 planes are taken to evaluate the orientation change . 
 while the ( 003 ) 
 planes are perpendicular to the c - axis , the ( 101 ) 
 planes are nearly parallel to the c - axis , crossing 
 with the ( 003 ) planes at 78.5 angle because of the elongated 
 unit cell structure . 
 therefore , the relative change of the ( 003 ) and 
 ( 101 ) peak intensities is a good measure for examining how the c - axis of cuscn is oriented with respect to the substrate 
 plane . according to the method described in the literature , 
 the orientation indices ( oi ) are calculated 
 as follows . the intensity factor ( if ) of the standard powder sample 
 is calculated for the respective crystal planes as45taking the intensities indicated in ref ( 23 ) . 
 the if of the film samples are calculated from the ( 003 ) and ( 101 ) peak 
 counts of each measured xrd pattern.67the ratio of the if of the film with respect 
 to that of the powder standard is defined as the oi.89when oi(hkl ) is 
 larger than 1 , the film has a tendency to orient ( hkl ) planes in parallel with the substrate ; when smaller than 1 , just 
 the opposite . 
 pure cuscn thin films electrodeposited without rb show high 
 oi(003 ) , indicating their preferential orientation of the c - axis perpendicular with the substrate , irrespective of 
 the scn rich and cu rich bath compositions . 
 such preferential orientation nicely matches with the arguments for 
 their morphological features described above , namely , the hexagonal 
 facet of the columnar grain in figure 3a1 and 
 the bevels of the spiky grains in figure 3b1 
 corresponding to the ( 003 ) and ( 101 ) planes , respectively . 
 minor addition 
 of rb to the bath then drastically increases the oi(101 ) upon decrease of oi(003 ) , indicating the change of the 
 crystallographic orientation to lay down the c - axis 
 in parallel with the substrate . 
 such preference is most prominent 
 when [ rb ] = 0.3 mm , for both the scn rich and cu rich baths . 
 however , the higher addition of rb abruptly 
 changes the orientation back to the one in which the c - axis is perpendicular with the substrate in the case of the scn rich bath , whereas the oi(101 ) value only 
 moderately decreases for the cu rich bath . 
 consequently , 
 highly porous cuscn thin films with totally different crystallographic 
 orientations , namely , the c - axis perpendicular and 
 parallel with the substrate are obtained for the highest end of the 
 rb addition to the scn rich and cu rich baths , respectively . 
 however , it is obvious that 
 such changes are caused by the difference of the chemical stability 
 of the facets of cuscn crystals in different environments , not only 
 by the scn / cu balance but also by 
 the added rb molecules . 
 xrd patterns of cuscn and cuscn / rb hybrid thin 
 films electrodeposited 
 from scn rich ( a ) and cu rich ( b ) 
 baths containing 0 ( a ) , 0.1 ( b ) , 0.2 ( c ) , 0.3 ( d ) , 0.4 ( e ) , 0.5 ( f ) , 
 0.7 ( g ) , and 1.0 mm ( h ) rb . change of crystallographic orientation of cuscn and cuscn / rb hybrid 
 thin films by changing concentration of rb added to the scn rich and cu rich baths . 
 the amount of rb loaded into cuscn during electrodeposition 
 for 
 300 s from the scn rich and cu rich 
 baths is plotted as a function of rb concentration ( figure 6 ) . 
 the amount of rb precipitated together with cuscn 
 increases as its concentration in the bath increases , although not 
 proportionally but showing a leveling off trend . the loading efficiency 
 in the scn rich bath is clearly higher than that 
 in the cu rich , especially for the low rb concentration 
 range . 
 some properties of the cuscn / rb hybrid thin films electrodeposited 
 from the scn rich bath are summarized in table 1 . 
 change of the amount of rb loaded into cuscn as a function 
 of rb 
 concentration added to the scn rich and cu rich baths . calculated for  = 500 rpm 
 from levich equation by employing the bulk concentration of rb and 
 its diffusion coefficient in water at 25 c ( 3.2  10 cm  s ) . 
 calculated by dividing the amount 
 of rb loaded into the film by the amount of rb transported to the 
 electrode surface in 300 s. calculated by using the formulas 
 weight of cuscn ( 121.6 g  mol ) , the density 
 of cuscn ( 2.84 g  cm ) , the molar weight 
 of rb ( 479.02 g  mol ) and the density of 
 rb solid ( 0.79 g  cm at 20 c ) . 
 the volume of the void was calculated 
 by subtracting the volumes of cuscn and rb from the total volume of 
 the film determined from the film thickness . 
 increase of the concentration of rb in the bath led 
 to a slight 
 decrease of the consumed charge because of the decrease of the steady - state 
 current and the presence of the incubation time for [ rb ] = 1.0 mm . 
 at the same time , faradaic efficiency for the precipitation of cuscn 
 gradually decreased . 
 the film thickness significantly decreased , as 
 seen in the crosssectional sem pictures in figure 3 . while the amount of electrodeposited cuscn decreased , the 
 amount of precipitated rb increased . 
 the change of the film thickness , 
 however , is mostly caused by the change of the porosity as seen in 
 the volume ratio for cuscn , rb , and void for the hybrid film . as the 
 diffusion coefficient of the rb molecule could be found only as measured 
 in water solution , we took this value to calculate the flux of rb 
 for its given concentration under the rotating condition employed 
 in this study . 
 the sticking efficiency of rb was then estimated by 
 calculating the fraction of rb loaded into the film as compared to 
 its maximum amount transported toward the electrode surface during 
 the electrolysis . 
 the highest efficiency of 17.5% was achieved for 
 [ rb ] = 0.1 mm , and that value decreased as the bulk concentration 
 got higher . 
 it is obvious that the precipitation of rb is not under 
 the control of its transport but rather its stability or rate of attaching 
 to cuscn . 
 when the molar amounts of precipitated cuscn and rb are 
 determined , the fraction of the volume occupied by these components 
 in the hybrid film can be calculated by taking their molar weight 
 and density , either taking into account or not taking into account 
 the total volume of the film . 
 the cuscn : rb ratio gives a rough idea 
 up to how much volume of the precipitated solid can be occupied by 
 rb . 
 an approximately 2:1 ratio seems to be reachable for this system , 
 which is equivalent to that for the zno / eosin y hybrid system reported 
 elsewhere . 
 the cuscn : rb : void ratio correlates 
 to the porosity of the film . as shown in the following section , the 
 loaded rb molecules can be removed from the hybrid films with high 
 rb loading , leaving some empty space within the cuscn grains . the 
 voids in the as - deposited films occur from spaces between the cuscn / rb 
 hybrid grains , while that occupied by rb is converted to the tiny 
 pores formed inside cuscn grains after extraction of rb . 
 when the hybrid films are soaked in dimethylacetamide ( dma ) , the 
 films were bleached as the rb molecules were extracted , as seen from 
 the change of the absorption spectra before and after the treatment 
 ( figure 7 ) . 
 for the films with small rb loading , 
 rb molecules could be only partly removed , whereas those with high 
 rb loading allowed almost complete desorption of rb . the film with 
 the highest rb loading , namely , that deposited from the scn rich bath containing 1.0 mm rb , became nearly colorless because 
 97% of the loaded dye was removed as estimated from the absorbance 
 change at the maximum of rb absorption . 
 as discussed above , the extraction 
 of rb should result in tiny nanopores enlarging the surface area of 
 the cuscn films . 
 teller 
 analysis of kr sorption measurements ( figure 8) . the surface area of about 6 m  g is already reached for the film deposited from the cu rich bath without rb . 
 it only moderately increases by hybridization 
 with rb and its extraction for the cu rich baths . on 
 the other hand , that of the film deposited from the scn rich bath results in only about 4 m  g without rb , and it greatly increases up to 12.6 m  g when [ rb ] = 1.0 mm . the roughness factor ( rf ) , defined 
 as the ratio of the actual surface area per projected film area , of 
 about 17 
 is reached as calculated from the amount of electrodeposited 
 cuscn shown in table 1 . 
 considering the small 
 film thickness of 0.8 m , this roughness factor is reasonably 
 high because rf of several hundred can be reached when the thickness 
 is increased to a few tens of micrometers . 
 these thicknesses and rf 
 values are comparable to those of materials typically used for dye - sensitized 
 solar cells . using the reported growth conditions 
 , we were able to 
 extend the growth time and film thickness by roughly a factor of 5 
 before inducing any cracking in the film . 
 transmission absorption 
 spectra of as - electrodeposited cuscn / rb 
 hybrid thin films ( solid lines ) and those after soaking the films 
 for 1 h in dimethylacetamide ( dma ) ( dashed lines ) . 
 the cuscn / rb thin 
 films were electrodeposited from scn rich ( black 
 lines ) and cu rich ( gray lines ) baths containing 0.1 
 ( thin lines ) or 1.0 mm ( thick lines ) of rb . 
 change of specific surface area of cuscn / rb hybrid thin films after 
 extraction of rb by dma treatment as determined by kr sorption measurements . as we have succeeded in obtaining 
 highly porous crystalline cuscn 
 thin films , 
 it is of significant interest to check its usefulness 
 as a dye - sensitized photocathode . 
 however , no stably adsorbed sensitizer 
 dye for efficient hole injection to cuscn has been previously discovered . 
 we first checked fluorescence quenching for several fluorescent dye 
 molecules attached to cuscn , such as fluorescein isothiocyanate ( fltc ) , 
 cresyl violet ( cv ) , nile blue ( nb ) , 3,4,9,10-perylene tetracarboxylic 
 acid diimine ( ptcdi ) , quinacridone ( qc ) , and rb . 
 fltc can be coelectrodeposited 
 with cuscn because of the presence of a soft basic thiocyanate moiety 
 that is expected to act as an anchor to the soft acidic cu(i ) sites 
 of cuscn . 
 the electrodeposited cuscn / fltc 
 hybrid thin film in fact exhibited dye - sensitized photocathodic behavior 
 upon visible light illumination . 
 addition 
 of nb and cv to the deposition bath for cuscn also resulted in formation 
 of hybrid thin films because of their cationic character by the presence 
 of ammonium group , the same as that of rb . 
 ptcdi and qc lack such 
 chemical affinity to cuscn , so they were simply vacuum evaporated 
 on top of the porous cuscn . 
 all of these molecules exhibit fluorescence 
 in solution , but nb and cv actually did not show measurable fluorescence 
 in solid state . whereas no quenching was observed for ptcdi and qc , 
 fluorescence of fltc and rb was strongly quenched for their electrodeposited 
 hybrid thin films . 
 in particular , that of rb was almost totally quenched 
 as shown in figure 9 , while imperfect quenching 
 was observed in the case of the cuscn / fltc hybrid ( see figure 2s of supporting information for the spectra ) . because 
 the valence band edge of cuscn is expected to lie around 5.3 
 ev ( vs vac ) , the highest occupied molecular orbital level of all these 
 dye molecules is deep enough to transfer holes to cuscn from a thermodynamic 
 point of view . 
 it is supposed that the strong chemical affinity of 
 fltc and rb to cuscn is crucial for hole injection to take place actually , 
 unlike ptcdi and qc which are only in physical contact with the surface 
 of cuscn . 
 absorption and fluorescence spectra of electrodeposited cuscn / rb 
 hybrid thin film ( solid lines ) , solid rb thin film prepared by spin 
 coating ( dashed lines ) , and 20 m rb solution in ethanol ( dotted 
 lines ) . 
 the excitation wavelength for the film samples and the solution 
 were 535 and 530 nm , respectively . 
 the fluorescence spectrum of the 
 rb solution is attenuated for better comparison with other spectra 
 because it is far stronger than the other samples . 
 monomeric rb molecules in solution exhibit a sharp 
 absorption 
 peak at 545 nm and a weak shoulder around 505 nm . 
 a strong and sharp 
 fluorescence spectrum occurs like a mirror image of the absorption 
 spectrum , exhibiting a peak at 574 nm and a weak shoulder around 620 
 nm . 
 the solid thin film of rb aggregate shows a characteristic change 
 of the absorption spectrum with a maximum at 579 nm and another distinctive 
 peak at 541 nm . 
 it has been shown that dimerization of rb molecules 
 in solution leads to an increase of the abosorbance of the short side 
 shoulder because of enhanced intermolecular interaction in parallel 
 arrangement of the chromophore , namely , in h - aggregate form . 
 the great enhancement of the blue - side shoulder band to become a 
 clearly distinguished satellite peak for the solid rb film is obviously 
 caused by the strong electronic interaction in the molecular solid 
 in h - aggregate form . 
 the sharp peak at 613 nm should reflect a monomolecularly 
 confined fluorescence band , which is also red - shifted compared to 
 that of the rb monomer solution . on the other hand , 
 the broad character 
 centered at 687 nm should be related to the transition involving the 
 intermolecular band , which exists only in the aggregates . 
 the absorption 
 spectrum of the electrodeposited cuscn / rb hybrid thin film closely 
 resembles that of the solid rb thin film , exhibiting a character for 
 the h - aggregate . 
 however , both peaks are blue - shifted to 536 and 570 
 nm for aggregate and monomer bands , respectively , indicating different 
 environments of the rb molecules which are subjected to chemical interaction 
 with cuscn . 
 because characteristic fluorescence was clearly monitored 
 for rb in the solid state , the total quenching of the fluorescence 
 in the hybrid film should be caused by hole transfer from the exited 
 state of rb to the valence band of cuscn . 
 the electrodes were the pure cuscn with a 
 relatively high surface area , that electrodeposited from cu rich bath without rb , and a highly porous cuscn made by electrodeposition 
 from a scn rich bath containing 1.0 mm rb which 
 then was removed by the dma treatment . 
 thus , we prepared 
 a dye - sensitized photocathode with the rb dye shown to undergo photoinduced 
 hole transfer . 
 however , the lack of a suitable redox electrolyte is 
 a significant problem for studying photocathodic sensitization of 
 cuscn . 
 iodide / triiodide redox electrolyte typically 
 used in dsscs reacts with cuscn to convert it to cui that dissolves 
 rather well in many polar organic solvents . 
 adsorption of rb molecules 
 to cuscn was unfortunately not so stable , as recognized by the fact 
 that the electrolyte solution was clearly colored by desorbed rb both 
 for organic and aqueous solutions . 
 for this reason , it was not useful 
 to use co complex redox systems , which are kinetically highly reversible , 
 because fast recombination from the uncovered surface of cuscn or 
 exposed fto actually killed the voltage under illumination . stably 
 adsorbed highly rectifying dyes are needed for effective use of co 
 redox , which are missing at this stage . 
 thus , we have employed an aqueous solution of methylviologen chloride 
 as the electron acceptor . 
 the electrode was reasonably stable in this 
 electrolyte and exhibited a clear photocathodic action under visible 
 light illumination as shown in figure 10 . 
 pure 
 cuscn film sensitized with rb exhibits about 0.2 v photocurrent onset 
 voltage and 22 a cm photocathodic current ; 
 these values increased to ca . 
 0.3 v and 32 a cm for the porous cuscn electrode prepared by hybrid electrodeposition 
 with rb , extraction of rb , and readsorption of rb . 
 clearly , enlarged 
 surface area made the color of the film more intense than that of 
 the pure cuscn , obviously contributing to the increased current . 
 poorly 
 rectified dark current for pure cuscn ( figure 10a ) can be associated with the the bare fto surface being in contact 
 with the electrolyte , allowing direct charge transfer to methylviologen . because the 
 porous crystalline cuscn ( figure 10b ) covers 
 the fto better than pure cuscn , the dark current is well - rectified 
 and higher voltage is achieved . despite efforts to find a stable dye 
 electrolyte 
 solution combination , the rb dye still desorbs from the surface during 
 measurement , likely contributing to the unusually low photocurrent 
 . i  v curves measured at 
 bulk cuscn ( a ) electrodeposited without rb and porous crystalline 
 cuscn ( b ) electrodeposited with 1.0 mm rb and rb subsequently removed 
 by dma treatment , measured in the dark ( hashed lines ) and under illumination 
 ( solid lines ) with visible light ( 100 mw  cm ) generated by a 500 w xe lamp equipped with uv and ir cutoff filters . 
 the cuscn films were sensitized by readsorbing rb by soaking in a 
 0.5 mm rb aqueous solution for 1 h. the electrolyte was a deaerated 
 aqueous solution of 0.1 m methylviologen chloride . 
 we have developed a method for obtaining a highly 
 porous and nicely 
 crystallized p - cuscn thin film by electrodeposition . by selection 
 of the bath composition , 
 it has become possible to load rb molecules 
 in an amount to occupy as much as 30% of the total volume of precipitated 
 solid , from which the loaded rb could be removed by soaking the film 
 in dma . 
 such a process can now be regarded as a counterpart of the 
 entire technology developed for zno / eosin y hybrid electrodeposition 
 for processinghigh - performance 
 porous crystalline zno - based dye - sensitized photoanodes . 
 porous structures are created by the rb molecules 
 within highly crystallized cuscn . not only the porous nanostructure 
 but also the crystallographic orientation of cuscn could be controlled 
 by the choice of the bath composition . 
 the resulting porous crystalline 
 cuscn thin film has been utilized as dye - sensitized photocathode to 
 prove its usefulness as a counterpart of dye - sensitized zno photoanodes . 
 however , limited surface area as well as the lack of good hole - injecting 
 dye stable on the cuscn surface and suitable redox electrolyte remain 
 as challenges for efficiency improvement of the photocathode . because 
 recent studies have discovered a good sensitizer for p - nio in combination 
 with co complex redox systems , there is 
 a good hope of finding one for p - cuscn . 
 the successfully constructed 
 efficient dye - sensitized cuscn photocathode can be combined with existing 
 dye - sensitized photoanodes to achieve high - efficiency tandem dsscs 
 in a very simple structure sandwiching a common redox electrolyte 
 solution by two photoelectrodes .","nanostructured 
 hybrid thin films of cuscn and rhodamine b ( rb ) 
 are electrochemically self - assembled ( esa ) by cathodic electrolysis 
 in an ethanol / water mixture containing cu2 + , scn , and rb . by selecting the solvent , cu2+/scn ratio , and the concentration of rb , we demonstrate several control 
 parameters in the film formation . 
 high loading of rb into the film 
 has been achieved to reach a cuscn : rb volume ratio of approximately 
 2:1 . 
 the rb solid could almost completely be extracted from the hybrid 
 film by soaking the film in dimethylacetamide ( dma ) , leading to a 
 large increase of the surface area . 
 the crystallographic orientation 
 of the nanostructure with respect to the substrate can be controlled . 
 efficient quenching of fluorescence of rb has been observed for the 
 cuscn / rb hybrid film , implying hole injection from rb excited state 
 to cuscn . 
 photoelectrochemical study on the porous crystalline cuscn 
 obtained after the dma treatment and sensitized with rb revealed sensitized 
 photocathodic action under visible light illumination , indicating 
 the potential usefulness of the porous cuscn electrodes for construction 
 of tandem dye - sensitized solar cells .",pubmed
"the nasopharyngeal bursa originates from the area of communication between the notochord and the foregut , ie , the pharyngeal endoderm.1,2 it is believed to be a remnant of embryonic communication between the notochord and the roof of the pharynx , that normally disappears during the second month of intrauterine fetal development . 
 3 a tornwaldt s cyst develops if the embryonic remnant becomes obstructed ( cystic type ) , and if crusts adhere to the orifice without obstruction , this will form the crust type.4 eagle5 in 1939 indicated that in the crust type the orifice of the bursa is not obstructed despite crust formation . 
 this crust sheds periodically from the nasopharynx , causing an offensive smell and unpleasant taste . 
 the cystic type of nasopharyngeal bursitis is more common and should form part of the differential diagnosis of nasopharyngeal abscesses and cysts . 
 although most patients are symptom - free , some will develop bursitis , a cyst , or an abscess . 
 therefore , the symptoms are of either a space - occupying lesion ( nasal obstruction , eustachian tube obstruction with secretory otitis media , dysphagia , cranial nerve paralysis ) or rhinitis ( choanal discharge , halitosis , pharyngitis , laryngitis , bronchitis , gastritis ) . 
 being located at the posterior wall of the nasopharynx and extending toward the tubercle of the occipital bone , pulsating headache and occipital pain felt at the external occipital tuberculum is often reported . 
 the incidence of nasopharyngeal bursitis is approximately 4% in adults , but it can appear at any age . 
 the peak incidence occurs between 15 and 30 years , with a caucasian predominance and no gender predilection.5,6 nonetheless , most of the published literature comprises selected case reports and describes the cystic type . 
 no single report to the authors knowledge has estimated the true incidence of crust - type nasopharyngeal bursitis or characterized its symptoms . in this clinicoanatomical audit , 
 the aim was to identify the incidence of nasopharyngeal bursa disease , its different types , and its clinical presentation over two years in a single rhinology practice . 
 this was a two - year clinicoanatomical audit of nasopharyngeal bursitis at a rhinology practice in a university unit during 20082009 . 
 all patients diagnosed to have nasopharyngeal bursitis confirmed by endoscopic examination of the nasopharynx and computed tomography scan / magnetic resonance imaging were asked carefully about symptoms of tornwaldt s syndrome . 
 a surgical technique combining an endonasal and transoral approach in the form of disruption of the bursa with electrocauterization was performed to patients presenting with either the cyst type or the crust type . 
 surgical samples of bursitis tissue were fixed in 10% formalin , and paraffin wax blocks were performed . 
 routine hematoxilyn staining of the nasal biopsy was then performed and specimens examined under low and high power fields . 
 representative photos from the slides were taken by a camera attached to software as shown in figure 2 . 
 this was a two - year clinicoanatomical audit of nasopharyngeal bursitis at a rhinology practice in a university unit during 20082009 . 
 all patients diagnosed to have nasopharyngeal bursitis confirmed by endoscopic examination of the nasopharynx and computed tomography scan / magnetic resonance imaging were asked carefully about symptoms of tornwaldt s syndrome . 
 a surgical technique combining an endonasal and transoral approach in the form of disruption of the bursa with electrocauterization was performed to patients presenting with either the cyst type or the crust type . 
 surgical samples of bursitis tissue were fixed in 10% formalin , and paraffin wax blocks were performed . 
 routine hematoxilyn staining of the nasal biopsy was then performed and specimens examined under low and high power fields . 
 representative photos from the slides were taken by a camera attached to software as shown in figure 2 . 
 results of the current audit ( see table ) indicated that six patients ( four males and two females , mean age 54 years ) were diagnosed to have nasopharyngeal bursitis . 
 the incidence was less than 1% of all rhinitis patients seen during the study period . 
 two patients with classical tornwaldt s cyst and one with a fibrosed tornwaldt s cyst were identified , while three patients with crust - type bursitis were diagnosed . 
 the classical cyst type presented with postnasal discharge resulting in hemming , throat irritation , and cough , while the fibrosed type was discovered incidentally in a patient who complained of snoring . on the other hand , the crust type presented with crust expectoration and retching , with or without fetid postnasal discharge , and occasional occipital pain that lasts for a few days , with symptom - free intervals of a few days . for both types of disease , 
 patients who underwent surgery using a combined endoscopic nasal and transoral approach had a complete recovery with no recurrence of symptoms at more than one year postoperatively ( figure 1c ) . 
 interestingly , our audit showed an equal incidence of the cystic and the crust type of the disease . 
 endoscopic examination of the nasopharynx after proper nasal decongestion will clearly show either a cyst or ulcer - like lesion covered with crusts , in the midline of the posterior wall of the nasopharynx ( figures 1a and 1b ) . 
 radiologic investigation ( simple lateral view x - ray , computed tomography scan , and magnetic resonance imaging ) is useful in showing adhesion of the bursa to the cervical vertebrae . 
 although several surgical approaches with good outcomes have been described,69 we believe that the combined endoscopic endonasal and transoral approach with good electrocauterization is a minimally invasive and effective way of treating both types of nasopharyngeal bursitis ( figure 1c ) . 
 nasopharyngeal bursa originate at the interface between the embryologic tissue from which the vertebrae develop . 
 notochord formation is an important change in the embryonic disc that takes place in the third week , and this is used by the embryo as a temporary axial skeleton . during migration of the intraembryonic mesoderm , the node of hansen develops at the cephalic primitive streak , giving rise to the notochord process , in which a small central canal is formed . 
 this canal connects the amniotic cavity and the yolk sac cavity . from this notochordal process , 
 a rod - like solid definitive notochord becomes detached from the endoderm to lie in a position between the ectoderm and endoderm in the midline . 
 this definitive notochord later becomes the permanent vertebral column . in approximately 3%4% of embryos , 
 an invaginated connection remains in the nasopharynx connecting the pharyngeal epithelium with the remnants of the notochord . 
 thus , the lesion is located in the middle of the posterior wall of the nasopharynx and extends to the tubercle of the occipital bone . 
 syndrome10 describes a group of symptoms resulting from inflammation , or cystic or abscess formation . in 1939 
 , eagle5 indicated that the orifice of the bursa is not obstructed in the crust type , despite the crust formation . 
 this crust will shed from the nasopharynx , causing a bad smell and an unpleasant taste . in this audit , 
 three patients presented with retching , expectoration of crusts , and/or fetid postnasal discharge and occipital pain , with no other described symptoms of nasopharyngeal bursitis . 
 the crust reforms quickly , only to shed again every few days with completely symptom - free intervals in between . 
 although most patients with nasopharyngeal bursa remain symptom - free , trauma to the nasopharynx in the form of nasal packing or adenoidectomy may result in obstruction of the bursa orifice and cyst formation . 
 interestingly , our patients developed their symptoms at an advanced age with no history of an initiating event , such as nasal packing or surgical trauma . 
 also , they remained completely symptom - free for years and presented suddenly with disturbing crusts of the nasopharynx . 
 this indicates that the crust type could be a distinct variant of the disease that may develop at any time , and without recognized predisposing factors . 
 the cystic type of nasopharyngeal bursitis in our study was found to have two subtypes . 
 one type is the classical cyst with postnasal discharge , throat irritation , and cough , while the other fibrosed type presented with irritation of the nasopharyngeal area and snoring . 
 histopathologically , a tornwaldt s cyst can be differentiated from a retention lymphoid cyst in that the former appears as an epithelial cyst lined with columnar epithelium on the surface11 while the latter shows lymphoid tissue only . 
 however , the histologic picture of the crust type is not known . in figure 2 
 , we show the histologic appearance of the crust type that demonstrates reactive lymphoid mucosa with necrotic tissue . 
 interestingly , reactive lymphoid mucosa was also demonstrated in biopsies from the adjacent tissues , which may explain the cicatricial streaks around the bursa . 
 this interesting histologic finding presents clinically as rapid formation of crusts with shedding and postnasal drip . in conclusion , 
 the crust type is a recognized form of the disease and may produce no symptoms other than retching and irritative expectoration of crusts . 
 awareness of the crust type of nasopharyngeal bursitis would pick up many missed cases and increase appropriate referrals between generalists , pulmonologists , and otolaryngologists . 
 proper endoscopic nasopharyngeal examination with computed tomography and magnetic resonance imaging remains the best method of clinical assessment . 
 nasopharyngeal bursitis should be differentiated from nasopharyngeal carcinoma , which can mimic the ulcerative nature of nasopharyngeal bursitis , but usually does not form overlying crusts . 
 the lack of symptoms , such as epistaxis and metastatic lymphadenopathy , as well as the characteristic midline anatomic site in the nasopharynx , are all in favor of the diagnosis of crust - type nasopharyngeal bursitis . 
 endoscopic interruption of the bursa with electrocauterization at the base is a minimally invasive technique that would suffice , and allows a better view of the operative field .","nasopharyngeal bursitis is a relatively rare syndrome characterized by a collection of symptoms that multidisciplinary specialists should be aware of . here 
 we present an audit of cases presenting to a rhinology clinic over a two - year period , as well as an overview of the relevant embryology and different clinical presentations of nasopharyngeal bursitis . for 20082009 , 
 six patients were diagnosed to have nasopharyngeal bursitis , including four males and two females , of mean age 54 years . 
 two distinct pathologic types were observed , comprising three patients with classical tornwaldt s cyst and three with crust - type bursitis . 
 this audit highlights the importance of recognition of the crust - type of nasopharyngeal bursitis and its anatomic and clinical features . 
 a combined endonasal and transoral endoscopic approach is a minimally invasive procedure and an effective method of treating both types of the disease . 
 our findings are discussed in relation to the embryology of the disorder , with a clinical emphasis on crust- type nasopharyngeal bursitis .",pubmed
"the incidence of melanoma skin cancer has been increasing over the past few decades [ 13 ] . estimated 76,250 new cases of invasive melanoma were diagnosed in usa in 2012 , with an estimated number of 9,180 that result in death . 
 australia has one of the highest rates of skin cancer in the world . over 1,890 
 the most dangerous characteristic of melanoma is that it can spread widely over the body via the lymphatic vessels and blood vessels . 
 thus , early diagnosis of melanoma is a key factor for the prognosis of the disease . 
 however , dermoscopy   is a noninvasive diagnostic technique that links clinical dermatology and dermatopathology by enabling the visualization of morphological features which are not discernible by examination with the naked eye . 
 there are different techniques , like solar scan , epiluminescence microscopy ( elm ) [ 10 , 11 ] , cross - polarization epiluminescence ( xlm ) , and side transillumination ( tlm ) [ 12 , 13 ] , that can greatly increase the morphological details that are visualized . 
 dermoscopy enables better diagnosis as compared to unaided eye [ 1416 ] with an improvement in diagnostic sensitivity of 1030% . 
 however , it has also been demonstrated that dermoscopy may actually lower the diagnostic accuracy in the hands of inexperienced dermatologists [ 10 , 1820 ] , since this method requires great deal of experience to differentiate skin lesions . as described in [ 9 , 22 ] 
 only experts have arrived at 90% sensitivity and 59% specificity in skin lesion diagnosis , while for less trained doctors these figures show significant drop till around 62%-63% for general practitioners . 
 the main problem is that the diagnosis is highly dependent on subjective judgement and is scarcely reproducible [ 23 , 24 ] . 
 several scoring systems and algorithms such as the abcd - e rule [ 2527 ] , the seven - point checklist [ 2830 ] , three - point checklist , and the menzies method [ 32 , 33 ] have been proposed to improve the diagnostic performance of less experienced clinicians . 
 although this simplification has enabled the development of these diagnostic algorithms with good accuracy , still they showed problems that have not yet been solved . 
 the most important shortcoming is that the purpose for which they were designed was not achieved , because the within- and between - observer concordance is very low , even for expert observers [ 10 , 25 , 34 , 35 ] . despite extensive research in investigating the varied presentations and physical characteristics of melanoma 
 thus , a growing interest has developed in the last two decades in the automated analysis of digitized images obtained by elm techniques to assist clinicians in differentiating early melanoma from benign skin lesions . 
 application of computational intelligence methods helps physicians as well as dermatologists in faster data process to give better and more reliable diagnoses . 
 studies related to the automated classification of pigmented skin lesion images have appeared in the literature as early as 1987 . 
 after some successful experiments on automatic diagnostic systems for melanoma diagnosis [ 3642 ] , utility of machine vision and computerized analysis is getting more important every year . 
 the importance of the topic is patent if we analyse the enormous quantity of research works related with the melanoma diagnosis . 
 numerous computerized diagnostic systems have been reported in the literature where different border detection , feature extraction , selection , and classification algorithms are used . 
 some researchers [ 37 , 4348 ] reviewed and tried to critically examine image analysis techniques for diagnosis of skin cancer and compared diagnostic accuracy of experts dermoscopists with artificial intelligence and computer aided diagnosis . 
 more research , however , is needed to identify and reduce uncertainties in the automatic decision support systems to improve diagnosis accuracy . 
 a comprehensive up - to - date review of automatic diagnostic model for skin lesions is not available . 
 continuous emergence of new classification algorithms and techniques for dermoscopic image analysis in recent years necessitates such a review . 
 this paper describes a standard automatic decision support system which is based on semantic analysis of melanoma images and further classification of characteristic objects commonly found in pigmented skin lesions . 
 the aim of this review is to summarize and compare advanced dermoscopic algorithms used for the classification of skin lesions and discuss important issues affecting the success of classification . a brief and comprehensive review of feature extraction and selection algorithms that are so far being used for extracting various features of malignant melanoma 
 analysis of various papers is performed with respect to several criteria , such as lesion segmentation , feature extraction , size of data sets , classification techniques , and performance measures used in reporting the diagnosis results . 
 this paper will provide a framework that represents a comprehensive guideline for selecting suitable algorithms needed for different steps of automatic diagnostic procedure for ensuring timely diagnosis of skin cancer . 
 performance evaluation measures and model validation details are presented for analysing various algorithms / models and finally concluding comments are provided . 
 predictive models are used in a variety of medical domains for diagnostic and prognostic tasks . 
 the data can be preprocessed and expressed in a set of rules , such as that it is often the case in knowledge - based expert systems , and consequently can serve as training data for statistical and machine learning models . 
 the general approach of developing a cad system for the diagnosis of skin cancer is to find the location of a lesion and also to determine an estimate of the probability of a disease . 
 the first step in this paper was to establish a standard general scheme of a cad system for skin lesions . 
 the inputs to the computer aided system are digital images obtained by elm , with the possibility to add other acquisition system such as ultrasound or confocal microscopy . in the first phase preprocessing of image 
 is done that allows reducing the ill effects and various artifacts like hair that may be present in the dermoscopic images . 
 once the lesion is localized , different chromatic and morphological features can be quantified and used for classification . 
 differentiation of malignant melanoma images demands very fast image processing and feature extraction and classification algorithms . 
 a detailed research is necessary to make the best choice and to set the benchmarks for diagnostic system development and validation . 
 the following section focuses on the description of the major steps that may be involved in skin cancer diagnosis . 
 numerous imaging modalities are under investigation to determine their usefulness in imaging and ascertaining a correct in vivo diagnosis of melanoma . 
 these include total cutaneous photography , dermoscopy , confocal scanning laser microscopy ( cslm ) , ultrasound , magnetic resonance imaging ( mri ) , optical coherence tomography ( oct ) , and multispectral imaging . 
 we have provided here a bird eye view of the currently available cutaneous imaging devices and new frontiers in noninvasive automated diagnosis of melanoma in table 1 . 
 readers may refer to [ 33 , 4952 ] for analysing performance comparison of some of the existing screening techniques . 
 relative to other specialties , dermatologists have been slow to adopt advanced technologic diagnostic aids . 
 sometimes simple elm does not sufficiently increase the diagnostic accuracy in distinguishing pigmented spitz nevus ( psns ) from melanoma . for obviating the problems of qualitative interpretation , methods based on the mathematical analysis of pigmented skin lesions ( psls ) , such as digital dermoscopy analysis ( dda ) and d - elm , 
 efficient image processing techniques must therefore be developed to help physicians in making a diagnosis . 
 the introduction of digital elm and sophisticated image processing software has opened up a new horizon in the evaluation of cutaneous benign and malignant pigmented skin lesions ( psls ) as it enables the observation , storage , and objective evaluation of many parameters . in this paper 
 we have focussed on automatic diagnostic system based on digital dermoscopy images normally collected from different dermoscopy atlases [ 55 , 56 ] or from dermatologists since it is the most widely used . 
 however , we anticipate that multimodal systems that combine different imaging technologies will further improve the ability to detect melanoma at an earlier stage and reduce the trauma of dermatologic diagnosis . 
 the main processing step towards a complete analysis of pigmented skin lesion is to differentiate the lesion from the healthy skin . 
 detection of the lesion is a difficult problem in dermatoscopic images as the transition between the lesion and the surrounding skin is smooth and even for trained dermatologist ; it is a challenge to distinguish accurately . 
 it has been observed that dermoscopy images often contain artifacts such as uneven illumination , dermoscopic gel , black frames , ink markings , rulers , air bubbles , and intrinsic cutaneous features that can affect border detection such as blood vessels , hairs , and skin lines and texture . 
 these artifacts and extraneous elements complicate the border detection procedure , which results in a loss of accuracy as well as an increase in computational time . 
 thus , it requires some preprocessing steps to facilitate the segmentation process by the removal of unwanted objects or artifacts and colour space transformation . 
 everything that might corrupt the image and consequently affect the results of image processing must be localized and then removed , masked , or replaced . 
 many approaches can be used that include image resizing , masking , cropping , hair removal ( or attenuation ) , and conversion from rgb color to intensity grey image . 
 it is meant to facilitate image segmentation by filtering the image and enhancing its important features . 
 the most straightforward way to remove these artifacts is to smooth the image using a general purpose filter such peer group filter ( pgf ) , mean filters , median filter [ 5860 ] , gaussian filters [ 61 , 62 ] , or anisotropic diffusion filters ( adf ) . 
 a major issue with these aforementioned filters is that these filters are originally formulated for scalar images . for 
 vector images one can apply a scalar filter to each channel independently and then combine the results , a strategy referred to as marginal filtering . despite being fast 
 another noteworthy thing is setting mask size proportional to the image size to manage a tradeoff between smoothing of image and blurring of edges . 
 inspite of taking care of all the forementioned things , it is still not guaranteed to get an image free of all artifacts . 
 an alternative strategy for artifact removal is to use specialized methods for each artifact type . 
 many methods have been suggested ; very few [ 6466 ] discussed different aspects of artifacts together , but none of them have discussed all cases of artifacts . 
 for this rationale , we have presented an overview of effective preprocessing methods , namely , color space transformation , color quantization , contrast enhancement , and artifact removal , which are being used for reducing all the possible ill effects present in the dermoscopic images . 
 dermoscopy images are commonly acquired using a digital camera with a dermatoscope attachment . due to the computational simplicity and convenience of scalar ( single channel ) processing , the resulting rgb ( red - green - blue ) color image 
 is often converted to a scalar image using different methods like retaining only the blue channel as lesions are often more prominent in this channel or applying the luminance transformation or karhunen - love ( kl ) transformation and retaining the channel with the highest variance . 
 skin lesions come in a variety of colors but absolute colors are not very useful in segmenting images . 
 normally the analysis is based on changes in color within the lesion or with the surrounding skin particularly color changes belonging to the lesion boundary . 
 therefore , it is quite common to transform the images that are in rgb color coordinates into other color spaces like ciel*a*b * , ciel*u*v * , kl , and hsi ( hue - saturation - intensity ) . 
 typical 24-bit color images have thousands of colors , which are difficult to handle directly . 
 for this reason color quantization is commonly used as a preprocessing step for color image segmentation . 
 the process of color quantization consists of two - phases palette design ( i.e. , selection of a small set of colors that represents the original image colors ) and pixel mapping ( i.e. , assignment of one of the palette colors to each input pixel ) . 
 .   showed that , for skin lesion , the color quantization method should reduce the number of colors in image to 20 for getting precise quantization . 
 one of the factors that complicate the detection of borders in dermoscopy images is insufficient contrast . 
 the contrast of image is enhanced to ensure that edges of the lesion are eminence . 
 gmez et al .   proposed a contrast enhancement method based on independent histogram pursuit ( ihp ) . an easy , yet powerful way to enhance the image contrast 
 another very popular technique is histogram equalization , which alters pixel values to achieve a uniform distribution . 
 homomorphic filtering , fft , and high pass filter can be used to compensate for uneven illumination or specular reflection variations in order to obtain the high contrast lesion images . for the removal of black frames produced in the digitization process , celebi et al . 
 [ 59 , 70 ] proposed an iterative algorithm based on the lightness component of the hsl ( hue - saturation - lightness ) color space . in order to remove air bubbles and dermoscopic gels , adaptive and recursive weighted median filter developed by dehghani tafti and mirsadeghi   can be utilized . 
 a method that can remove bubbles with bright edges was introduced in   where the authors utilized a morphological top - hat operator followed by a radial search procedure . 
 line detection procedure based on the 2d derivatives of gaussian ( dog )   and exemplar - based object removal algorithm   can be used for removing dark lines like ruler marking . in most cases 
 one of the most undesirable elements that are most commonly present in dermatoscopic images is hair . 
 erosion / dilation with straight line segments can efficiently eliminate ( or at least weaken the effect of ) hairs [ 77 , 78 ] . 
 [ 79 , 80 ] suggested a scheme based on a morphological closing operator , while in   they applied to the three components of the l*u*v * uniform color space . 
 however , it is being observed that most of these techniques often leave behind undesirable blurring ; disturb the texture of the tumor ; and result in color bleeding . due to these problems , it is very difficult to use the color diffuse image for further skin tumor differentiation . 
 in contrast , a new artifact removal algorithm that focuses on accurate detection of curvilinear artifacts and pays special attention to lesion structure during the removal stage has been introduced by zhou et al . . 
 this approach effectively removes artifacts such as ruler markings and hair , but it has high computational requirements . to address all these issues abbas et al . 
 abbas et al .   presented a comparative study about hair removal methods which indicate that hair - repairing algorithm based on the fast marching method achieves an accurate result . 
 all the above mentioned strategies are meant to facilitate the segmentation and feature extraction stages which consequently lead to better diagnostic results . 
 segmentation refers to the partitioning of an image into disjoint regions that are homogeneous with respect to a chosen property such as luminance , color , and texture . 
 the goal of segmentation is to simplify and/or change the representation of an image into something that is more meaningful and easier to analyse . 
 some researchers   argued that manual border detection is better than computer - detected borders in order to separate the problems of feature extraction from the problems of automated lesion border detection . 
 however , for the development of automated diagnostic system for skin lesion detection , it is very important to develop automatic segmentation algorithms . 
 as segmentation is a crucial early step in the analysis of lesion images , it has become one of the important areas of research and many algorithms and segmentation techniques are available in the literature . 
 we have briefly provided an overview of various segmentation algorithms being used for dermoscopic image analysis as tabulated in table 2 . 
 several comparative studies [ 59 , 61 , 66 , 88 , 89 ] are also present in the literature which provides performance analysis of several segmentation algorithms . 
 there are several issues that should be kept in mind for selecting a suitable algorithm , for example , scalar versus vector processing , automatic versus semiautomatic , and the number of parameters whose values need to be determined a priori . 
 interested readers may check relevant references to identify a suitable approach for a specific study . 
 it is important to identify the most effective features to extract from melanoma , melanoma in situ and clark nevus lesions , and to find the most effective pattern - classification criteria and algorithms for differentiating those lesions . 
 thus , the next stage of the image analysis process is to extract the important features of the image . 
 the purpose of feature extraction is to reduce the original data set by measuring certain properties , or features , that differentiate one input pattern from another . 
 the feature extraction is performed by measurements on the pixels that represent a segmented object allowing various features to be computed . 
 unfortunately , the feature extraction step is often subject to error . in most of the publications dealing with this topic , 
 many features are extracted to feed a sophisticated classifier , but there is very little discussion about the real meaning of those features and about objective ways to measure them . 
 thus , we investigate this topic in detail to come up with a guideline for future research . 
 different feature extraction methods found in the literature include statistical and model - based and filtering - based methods , among which multichannel filtering is the most efficient and accurate one . 
 various researchers used principal component analysis ( pca ) of a binary mask of the lesion , wavelet packet transform ( wpt ) [ 9094 ] , grey level cooccurrence matrix ( glcm ) [ 61 , 95 ] , fourier power spectrum , gaussian derivative kernels , and decision boundary feature extraction [ 98100 ] in order to reduce data redundancy . 
 some of the typically used filter banks are laws masks , the dyadic gabor filter bank , and wavelet transform . 
 a particular problem in the related literature is that a significant number of studies do not report the details of their feature extraction procedure ; see table 6 . 
 the abcd - e system [ 25 , 26 , 102 ] , 7-point checklist [ 29 , 103 ] , 3-point checklist , pattern analysis , and menzies method   offer alternative approaches in deciding the differentiating features that need to be extracted . according to the conclusion made by johr , 
 the automatic extraction of characteristics that take into account the rule abcd [ 25 , 102 , 106 ] is computationally less expensive than the ones that take into account 7-point checklist [ 29 , 103 ] or the menzies method [ 32 , 107 ] . 
 furthermore , the reliability in the clinical diagnosis is very high for abcd - e rule . 
 so , most of the automated decision support systems also use abcd rule as the base of their feature extraction step . however , abcd is more prone to over classification of atypical melanocytic nevi as melanomas . 
 .   showed , in a comparative study , that menzies method achieved the highest sensitivity , 84.6% , for the diagnosis of melanoma , followed by the 7-point checklist ( 81.4% ) , the abcd rule ( 77.5% ) , pattern analysis ( 68.4% ) , and assessment of a macroscopic image ( 60.9% ) . 
 pattern analysis and assessment of the macroscopic image showed the highest specificity , 85.3% and 85.4% , respectively . 
 so many researchers [ 109114 ] are trying to develop efficient automatic diagnostic systems based on 7-point criteria and pattern analysis . 
 numerous methods for extracting features from clinical skin lesion images have been proposed in the literature as figure 2 illustrated the distribution of features used in dermoscopic studies . 
 several studies have also proven the efficiency of border shape descriptors for the detection of malignant melanoma on both clinical- and computer - based evaluation methods [ 115 , 116 ] . 
 very simple parameters , such as area and perimeter , are extracted in [ 117119 ] . 
 measurements of shape features are also used like fragmentation index [ 120122 ] , thinness ratio / circularity factor [ 61 , 123125 ] asymmetry index [ 77 , 116 , 122 ] , aspect ratio [ 118 , 126 ] , compactness [ 118 , 126 ] , symmetry axis , bulkiness score , irregularity index [ 129 , 130 ] , fractality of borders , convex hull ratio , and skin line pattern . 
 some groups use the sharpness of the transition from the lesion interior to the skin [ 61 , 123 , 125 ] as descriptors of the structure and irregularity of the border . 
 lacunarity   is another measure that can be used to characterize a property of fractals and quantifies aspects of patterns that exhibit changes in structure . 
 color features are mainly statistical parameters calculated from different colour channels , like average value and standard deviation of the rgb [ 120124 ] or hsv colour channels . 
 other color features used in different studies include colour asymmetry , centroidal distance , and luv histogram distance . 
 cotton and claridge   found that all normal skin colours lie on a two - dimensional surface patch within a three- dimensional ( 3d ) colour space ( cie - lms ) . 
 some researchers [ 61 , 117 , 118 , 134 , 135 ] used glcm - based texture features [ 136138 ] like dissimilarity , contrast , energy , maximum probability , correlation , entropy , and so forth . 
 parameters for the description of dermatoscopic structures and elm criteria are difficult to find in the literature . 
 major issues are concerned with the difficulty in relating such information as lesion shape and color to medical structures ( tissues , vessels , etc . ) which experts are more familiar with . 
 some of the dermoscopic feature extraction studies include atypical pigment networks [ 72 , 110 , 139 ] , globules / dots / blotches [ 72 , 140143 ] , streaks , granularity , and blue - white veil [ 87 , 146 ] . 
 it is noteworthy that diagnostic systems based on extraction of critical high level features show an increase in the diagnostic accuracy of computerized dermoscopy image analysis systems . 
 thus , in addition to general features like area , border , shape , and color , these high level features should also be integrated in the automated diagnostic system to gain greater clinical acceptance . 
 some researchers used some unique features for classification , but we know from skin cancer research that a unique feature is not sufficient to diagnose precisely skin cancer and that the combination of different criteria is the key to the early detection of malignant melanoma and other types of skin cancer . 
 the evolution of competing dermoscopic algorithms with variable definitions of specific attributes complicates dermoscopic diagnosis . 
 it is necessary to identify features that are the most reproducible and diagnostically significant and formulate them into a single algorithm . for clinical purposes , 
 similarly , features selection is a critical step for successfully distinguishing between malignant melanoma , benign , and dysplastic nevi . 
 many potential features may be used , but it is important to select a reasonable reduced number of useful features while eliminating redundant , irrelevant , or noisy features . 
 however , it is important to make sure that there may not be loss of significant information . from the classification perspective 
 , there are numerous potential benefits associated with feature selection : ( i ) reduced feature extraction time and storage requirements , ( ii ) reduced classifier complexity for better generalization behaviour , ( iii ) increased prediction accuracy , ( iv ) reduced training and testing times , and ( v ) enhanced data understanding and visualization . 
 there are many methods available for feature selection   which include principle component analysis   and search strategies like sequential forward selection ( sfs ) , sequential backward selection ( sbs ) , plus - l - take - away - r ( pta ( l , r ) ) , floating search methods [ 54 , 151 ] , sequential forward floating selection ( sffs ) , sequential backward floating selection ( sbfs ) ) and fisher score ranking . 
 all these algorithms use stepwise inclusions and exclusions of features into / from the subset of consideration , but they differ in their strategy of applying them . although the floating methods are considered to be more intelligent , they are still suboptimal and even more there is no warranty that they yield better results . 
 in addition to these , some of the filter - based methods include relieff , mutual information - based feature selection ( mifs ) , and correlation - based feature selection ( cfs ) . 
 filter methods are usually very fast and allow one to compare several alternative methods within an optimization framework . 
 it is possible , and also desirable , to use clinical criteria or statistical methods to reduce the number of candidate variables , thus reducing the risk of an overoptimistic model . 
 a particular problem in the related literature is that there is very little number of studies that report the details of their feature selection procedure . 
 normally we do not find details of feature selection procedures that are used for choosing the appropriate features for skin cancer diagnosis . 
 handels et al .   described feature selection as an optimization problem and compared several approaches including heuristic strategies , greedy and genetic algorithms . 
 zagrouba and barhoumi   proposed an accelerated system for melanoma diagnosis based on subset feature selection . 
 the number of features retained by the feature selection algorithm ( k ) is an important parameter . 
 sometimes a small number of features are not likely to discriminate between the classes well . on the other hand , a large number of features might lead to overfitting . 
 green et al .   showed by calculating correlation coefficients that the size of the lesion is the most important feature in their system . 
 they achieve a tremendous reduction to five features starting with 87 features calculated from surface profiles of skin lesions . 
 used sbfs and sffs and showed that the best selection performances were with subset size of between 10 to 15 and performance degrades with subsets size of more than 20 features . on the other hand by inspecting individual sensitivities on the malignant class of several subset sizes , it turns out that an acceptable performance is only achieved with subsets of more than 20 features . while celebi et al . 
 showed by using cfs feature selection algorithm that auc peaks can be obtained with the use of 18 features and inclusion of features beyond this value does not add much to the classifier performance . 
 presented a study particularly based on feature selection for melanoma recognition and showed a strong increase in performance for small subsets followed by a slight increase up to medium sized subsets . 
 larger subsets cause a drop in the recognition rate . ruiz et al .   also confirmed this thing in the evaluation done using sbfs and sffs and showed that minimum error rate was observed using subset of 6 features and a significant increase in classification error rate is observed by using a subset of more than 20 features . by inspecting the overall achieved performances one even could imagine that using 5 to 20 features is enough to get acceptable classification results . 
 the aim of feature selection is to find the optimum number of features to obtain the best achievable performance ( i.e. , recognition rate ) in classification . 
 therefore , the feature selection algorithms should be evaluated to get performance estimation on some standard classifier by applying tenfold cross - validation ( xval ) , that is , repeating feature selection ten times with slightly different data for all algorithms . 
 classification phase of the diagnostic system is the one in charge of making the inferences about the extracted information in the previous phases in order to be able to produce a diagnostic about the input image . 
 there are two different approaches for the classification of dermoscopic images : the first considers only a dichotomous distinction between the two classes ( melanoma and benign ) and assigns class labels 0 or 1 to data item . the second attempts to model p(y | x ) ; this yields not only a class label for a data item , but also a probability of class membership . 
 logistic regression , artificial neural networks , k - nearest neighbours , and decision trees are all members of the second approach , although they vary considerably in building an approximation to p(y | x ) from data . 
 we do not intend in this paper to delve deeply into the technical aspects of all the classification algorithms . 
 however , to make the reader analyse the performance of algorithms that are mostly used for dermoscopic image analysis , we believe that it is helpful to air them briefly . readers who wish to have a detailed description of a specific classification approach should refer to cited references . 
 the k - nearest neighbour classifier [ 161 , 162 ] is a nonparametric method of pattern recognition . for a lesion belonging to the test set ( query vector ) 
 , it is found that the k vectors are the closest to the query vector in the training set . the unclassified sample 
 is then assigned to the class represented by the majority of the k closest neighbours . 
 the most critical requirement of the k - nearest neighbour classifier is to have a training set including enough examples of each class of pigmented lesions to adequately represent the full range of measurements that can be expected from each class . optimizing the procedures of feature selection and weight definition could additionally improve the performance of the k - nearest neighbour classifier . in medicine , 
 most applications use nearest - neighbour algorithms as benchmarks for other machine learning techniques [ 156 , 164 ] . 
 classification based on the k - nearest neighbour algorithm differs from the other methods considered here , as this algorithm uses the data directly for classification , without building a model first [ 162 , 165 ] . 
 the only adjustable parameter in the model is k , the number of nearest neighbours to include in the estimate of class membership , and the value of p(y | x ) is calculated simply as the ratio of members of class y among the k - nearest neighbours of x. by varying k , the model can be made more or less flexible ( small or large values of k , resp . ) . 
 k - nn algorithm permits retrieval and visualization of the  most similar  cases to those at hand . 
 this aspect partly resembles the medical reasoning and allows a dermatologist to directly compare unknown lesions with other known skin lesions . this case - based explanation can provide an advantage in areas where black - box models are inadequate . 
 k - nn can also be used for the evaluation of feature subset selection process because it allows incorporating / eliminating characteristics easily and it has low computational cost . 
 the major drawback of k - nearest neighbour lies in the calculation of the case neighbourhood . 
 thus , it needs to define a metric that measures the distance between data items . in most application areas , 
 it is not clear how to , other than by trial and error , define a metric in such a way that the relative ( but unknown ! ) importance of data components is reflected in the metric . 
 it is popular for its simplicity in constructing , efficient use in decision making , and simple representation , which is easily understood by humans . 
 this algorithm repeatedly splits the data set according to a criterion that maximizes the separation of the data , resulting in a tree - like structure [ 167171 ] . 
 it does this by identifying a variable and a threshold in the domain of this variable that can be used to divide the data set into two groups . 
 the best choice of variable and threshold is the one that minimizes the disparity measures in the resulting groups . 
 the most common criterion employed is information gain ; this means that at each split , the decrease in entropy due to this split is maximized . 
 the estimate of p(y | x ) is the ratio of y class elements over all elements of the leaf node that contains data item x. various modifications of decision trees like adwat and lmt are also used for dermoscopic image classification . 
 advantages and disadvantages of decision trees in medicine have been widely investigated [ 172 , 173 ] . 
 the advantage of decision trees over many of the other methods is that they are not black - box models but can easily be expressed as rules . 
 decision tree classifiers have been preferred to other solutions ( also including ann and svm ) because they are often fast to train and apply and generate easy - to - understand rules . 
 a major disadvantage of decision trees is given by the greedy construction process . in this process at each step , the combination of single best variable and optimal split - point is selected . however , on the other hand if we use multistep look ahead , it considers combinations of variables which may obtain different ( and better ) results . given a large training set , decision tree classifiers , in general , generate complex decision rules that perform well on the training data but do not generalize well to unseen data . in such cases , 
 a further drawback lies in the fact that continuous variables are implicitly discretised by the splitting process , losing information along the way . 
 logistic regression is an algorithm that constructs a separating hyperplane between two data sets , using the logistic function to express distance from the hyperplane as a probability of class membership . 
 although the model is linear in parameters and can thus only calculate linear decision boundaries , it is nevertheless a widely used predictive model in medical applications [ 155 , 175 , 176 ] . the main advantage that this method has over other algorithms is its ease of use ( it is implemented in numerous software packages ) , allowing the interpretation of results as probabilities and variable - selection capability . 
 .   showed in a comparative study that logistic regression performs on about the same level as artificial neural networks and support vector machines , which are both capable of implementing nonlinear separating surfaces . 
 artificial neural network [ 165 , 177180 ] is one of the great vital parts of soft computing . 
 the ann consists of several small processing units ( the artificial neurons ) that are highly interconnected . 
 the supervise ann is an iterative process which requires many presentations of the training set ; the system is said to learn from examples . 
 it has conspicuous capacity to obtain idea from complex data and is used to take out patterns and determine trends that are too difficult to be noticed by humans or any other computer skills . 
 a lot of research is being carried out nowadays on dermoscopic image analysis using anns . the general working mechanism for artificial neural network 
 many of the early implementations required a significant amount of parameter tuning to achieve satisfactory results , a process that needed too much time and expertise for a nonexpert . over the past few years , statistically motivated bayesian methods   and implementations of faster learning algorithms   have allowed nonexperts use to sophisticated methods that require little to no parameter tuning . 
 various neural networks - based clustering techniques and algorithms are being used in this regard   which include back propagation network ( bpn ) , radial basis function network ( rbf ) and extreme learning machine ( elm ) . 
 support vector machines ( svms ) are a machine learning paradigm based on statistical learning theory [ 184 , 185 ] . 
 performances on par with or exceeding that of other machine learning algorithms have been reported in the medical literature . 
 algorithmically , support vector machines build optimal separating boundaries between data sets by solving a constrained quadratic optimization problem . 
 while the basic training algorithm can only construct linear separators , different kernel functions ( i.e. , linear , polynomial , radial basis function , and sigmoid ) can be used to include varying degrees of nonlinearity and flexibility in the model . 
 svms have several advantages over the more classical classifiers such as decision trees and neural networks . 
 therefore , there is no risk of getting stuck at local minima as in the case of back propagation neural networks . 
 svms are based on the structural risk minimization ( srm ) principle which minimizes the upper bound on the generalization error 
 . therefore , svms are less prone to overfitting when compared to algorithms such as back propagation neural networks that implement the erm empirical risk minimization principle . 
 another advantage of svms is that they provide a unified framework in which different learning machine architectures ( e.g. , rbf networks and feed forward neural networks ) can be generated through an appropriate choice of kernel . 
 the disadvantage of support vector machines is that the classification result is purely dichotomous , and no probability of class membership is given . 
 the only difference is that the hidden elements can be independent from the training data and target functions . 
 because of this independence of hidden elements , this feed forward network provides better generalization performance and it can learn much faster as compared to the other conventional algorithms . 
 the important features of extreme learning machine are that even simple math is enough for it . 
 the learning speed is extremely fast . unlike the traditional classic gradient - based learning algorithms which often face several issues like local minima , improper learning rate , and overfitting . 
 this learning algorithm looks much simpler than many other learning algorithms like neural networks and support vector machines . 
 there is very less work being done on the classification of dermoscopic images using extreme learning machine . 
 research work done on extreme learning machine shows that extreme learning machine needs much less training time as compared to popular bp and svm . 
 the prediction accuracy of elm is usually slightly better than bp   and close to svm in many applications . 
 compared with bp and svm , extreme learning machine can be implemented easily since there is no parameter to be tuned except an insensitive parameter l. it should be noted that many nonlinear activation functions can be used in extreme learning machine . 
 extreme learning machine needs more hidden nodes than bp but much less nodes than svm . 
 this implies that extreme learning machine and bp have much shorter response time to unknown data than svm . 
 the papers propose that , for skin lesion classification , three different classification tasks should be used as benchmarks : the dichotomous problem for distinguishing common nevi from dysplastic nevi and melanoma , the dichotomous problem for distinguishing melanoma from common nevi and dysplastic nevi , and the trichotomous problem for correctly distinguishing all the three classes . 
 the two criteria to assess the quality of a classification model are discrimination and calibration . 
 discrimination is a measure of how well the two classes in the data set are separated and calibration is a measure of how close the predictions of a given model are to the real underlying probability based on expert knowledge . 
 some of the common measures of analysing discriminatory power of different methods are reported in this paper as can be noticed in table 3 . 
 accuracy can be used as a single parameter , but if there is imbalance between the classes ( melanoma , benign ) , then accuracy is not a suitable approach of evaluation . 
 a better performance measure in unbalanced domains is the receiver operating characteristic ( roc ) curve . 
 auc is a statistically consistent and a more discriminatory measure than accuracy [ 191 , 192 ] . 
 the log diagnostic odds ratio is also sometimes used in meta - analyses of diagnostic test accuracy studies due to its simplicity ( being approximately normally distributed ) . 
 dclass is a measure to compare different classifiers presented by sboner et al .   that enable giving a simple estimation of how useful one classifier is with respect to another . by using this parameter instead of accuracy , out 
 the comparison between classifiers can be carried in an accurate but intuitive way , avoiding the unbalanced class problem . to provide an unbiased estimate of a model 's discrimination and calibration 
 there are some important considerations like the effect of class imbalance , train / test ratio , and cross - validation . 
 several studies have demonstrated that the accuracy degradation on unbalanced data sets is more severe when the classes overlap significantly [ 190 , 194 , 195 ] which is the case in skin lesion classification . 
 most classifiers focus on learning the large classes which leads to poor classification accuracy for the small classes such as classifying the minority ( melanoma ) samples as majority ( benign ) which implies serious consequences . 
 it has been observed   that as the training - set size increases , the results improve . 
 the effect of train / test ratios on classification accuracy is studied in   and the best classification results were reached with 70/30 train to test ratio . 
 there are two approaches for selecting training and test data : either to separate test and training feature vectors or pick training feature vectors as a subset of the test vectors . 
 a classification result may be overly optimistic if performance can not be measured on a data set not used for model building . in the ideal case , testing on a separate data set will provide an unbiased estimate of generalization error . if the original data set is too small for this approach , the recommended strategy is to use cross - validation   or bootstrapping   to make the best possible use of the limited amount of data . 
 one way is to divide the whole data into n pieces , n  1 pieces used for training , and the last piece as the test set . 
 this process of n - fold cross - validation builds n models ; the numbers reported are the averages over all n test sets . 
 the extreme case of using only one data item for testing is known as leave - one - out cross - validation . 
 bootstrapping is rarely used in the literature for skin lesion case , but it has shown to be superior to cross - validation on many other data sets . the increasing number of electronic data bases containing dermoscopic images has led to an increasing interest in their utilization for building classification models that can  learn  from examples . 
 the need to use data and learning techniques in order to make correct diagnosis requires proper choice of the learning algorithms and of their statistical validation . 
 the problem is difficult given the relative paucity of lesion data and consequently the low quality of training data available and the imbalance between the classes . 
 a variety of statistical and machine learning approaches are used for the classification of dermoscopic images . as illustrated in table 4 , while figure 5 presents the percentage of classification methods as used by existing diagnostic systems in literature . 
 the question of which classification approach is suitable for a specific study is not easy to answer . 
 different classification results may be obtained depending on the classifier(s ) chosen , differences in sample sizes , proportion of melanomas in the sample , and the number of features used for discrimination as can be notice in table 5 . 
 many factors , such as different sources of obtaining dermoscopic images , availability of classification software , time consumption , computational resources , and the number of melanoma and benign images available for training must be taken into account when selecting a classification method for use . 
 very few researchers provided comparisons of different classification algorithms using the same set of images [ 46 , 94 , 126 , 166 , 196 ] . 
 the review of all these comparative studies reveals that mlp gives better performance than bayesian and knn classifiers , while svm with rbf kernel normally outperforms mlp , decision trees , and other statistical methods . 
 the results of an experimental assessment of the different designs can be the basis for choosing one of the classifiers as a final solution to the problem . 
 it had been observed in such design studies that although one of the designs would yield the best performance , the sets of patterns misclassified by the different classifiers would not necessarily overlap . 
 , all the designs , or their subset , are used for decision making by combining their individual opinions to derive a consensus decision . 
 some classifier combination schemes have been devised [ 126 , 193 , 200 ] for dermoscopic images and it has been experimentally demonstrated that some of them consistently outperform a single best classifier 
 . however , there is presently inadequate understanding why some combination schemes are better than others and in what circumstances . 
 such models do not always work well in practice , so it is widely recommended that they need to be validated [ 201 , 202 ] . to be useful 
 , a prognostic index should be clinically credible and accurate and have generality ( i.e. , be validated elsewhere ) , and the study should be described in adequate detail . to gauge the current state of reporting results in the literature , we sampled many papers on dermoscopic images data sets analysis . 
 the paper is proposing some criteria as quality assessment criteria which can be noticed in table 6 . 
 it includes lack of calibration in image acquisition , unspecified method for extracting and selecting variables in the model , and risk of overfitting through too few events per variable . 
 many researchers did not specify the test / train or used uneven number of melanoma and benign images for training which may lead to biased classification . some articles do not report comparisons and cross - validation ; instead they just reported the performance of a single method . 
 it is imperative that these details should be presented in papers as otherwise the validity of the claims in the papers can not be assessed by the reader . 
 when assessing the quality of the results obtained using any diagnostic models , the work should consider the quality of the data set employed in model building , the care with which adjustable model parameters were chosen , and the evaluation criteria used to report the results of the modelling process . 
 this is important in distinguishing between overly optimistic claims ( such as when performance is reported on the training set ) and needlessly pessimistic ones ( when model parameters are chosen in a suboptimal manner ) . 
 apart from all this , in order to judge the performance of an automatic diagnostic model it is important to mention who is going to use that model . 
 if automated diagnostic systems will be used by general practitioners or in pharmacies and shopping centres , these systems should be used with very high sensitivity and reasonably good specificity . 
 that is , it should recognize the greatest number of melanomas in early stage , without misclassifying too many nevi so that unnecessary excision of benign lesions could be avoided . if the target is the expert user , studies should be designed with the aim to help clinicians in distinguishing between benign lesions , dysplastic nevi , and malignant tumors of the skin . 
 an increase in specificity might be the goal for an automated system directed to expert users together with sensitivity at least equal to that achieved by the expert . 
 overall , our objective is to get a classifier with the sensibility and the specificity balanced . 
 it should be noted that the ability to diagnose correctly melanoma is by far the most important property that an automated system must have . 
 the consequence of failure to diagnose correctly a malignant tumor may lead to the eventual death of the patient . on the other hand , 
 if we get a classifier with a high sensibility but a low specificity , it is not going to be useful as a screening method to avoid biopsies ( an invasive technique ) . 
 and , off course , we want a classifier with a high sensibility to avoid false negatives .","image - based computer aided diagnosis systems have significant potential for screening and early detection of malignant melanoma . 
 we review the state of the art in these systems and examine current practices , problems , and prospects of image acquisition , pre - processing , segmentation , feature extraction and selection , and classification of dermoscopic images . 
 this paper reports statistics and results from the most important implementations reported to date . we compared the performance of several classifiers specifically developed for skin lesion diagnosis and discussed the corresponding findings . 
 whenever available , indication of various conditions that affect the technique 's performance is reported . 
 we suggest a framework for comparative assessment of skin cancer diagnostic models and review the results based on these models . 
 the deficiencies in some of the existing studies are highlighted and suggestions for future research are provided .",pubmed
"type 2 diabetes ( t2d ) is a chronic disease characterized by high blood glucose which is induced by both insulin resistance and relative insulin deficiency , and the complications of t2d , such as cardiovascular disease , renal failure and blindness , always bring heavy burden to society . 
 the latest studies from the international diabetes federation ( idf ) reported that currently there were 366 million people with diabetes in the world by 2030 this number is expected to rise to 552 million with diabetes if no action is taken . 
 some 90% of patients with diabetes have t2d , which is always as a result of a combination of lifestyle and genetic factors . 
 peroxisome proliferator - activated receptor  coactivator 1 ( pgc-1 ) gene is originally identified as a coactivator of ppar. it is a multifunctional regulatory factor involved in mitochondrial biosynthesis , -oxidation of fatty acids , beta - cell insulin secretion , hepatic gluconeogenesis , glucose transport in muscle [ 46 ] . 
 our and other genetic association studies showed that the associations of pgc-1 loci with t2d were found in different populations [ 710 ] . 
 recently , it was reported that estrogen - related receptor- ( esrra ) protein binding pgc-1 protein could regulate the expression of mitofusin-2 ( mfn2 ) gene by stimulating the activity of the mfn2 promoter . 
 esrra gene is an orphan nuclear receptor ( nr ) belonging to the nr superfamily , group iii , and it is a vital regulator involved in a wide variety of cell functions such as mitochondrial biogenesis , mitochondrial oxidative metabolism , carbohydrate metabolism , adaptive energy metabolism , and lipid metabolism [ 1214 ] . 
 therefore , we hypothesize that esrra gene may be a candidate gene involved in the pathogenesis of t2d . 
 mfn2 gene maps on chromosome 1p36.22 . 
 mfn2 serve as a mitochondrial fusion protein regulating their morphology and distribution [ 15 , 16 ] . 
 tissues with high energetic requirement , such as skeletal muscle and heart , show high expression of mfn2 gene . 
 some evidences demonstrated that it also played an important role in the oxidative metabolism   and er - mitochondria juxtaposition , and repression of mfn2 gene reduced glucose oxidation , mitochondrial membrane potential , cell respiration , and mitochondrial proton leak . 
 in addition , the mfn2-dependent mechanism of mitochondrial control is disturbed in skeletal muscle in animal or human obesity and in patients with t2d [ 17 , 19 ] , but no study has confirmed that mfn2 gene was associated with t2d . in the present study , we evaluated the associations between the mfn2 and esrra genetic polymorphisms and t2d , and then combining our published data of pgc-1  , we estimated the interactions between genetic variants in the mfn2 , esrra , and pgc-1 on the t2d risk in han chinese . 
 the total of stage 1 populations comprised 1204 unrelated subjects , including 555 patients with t2d and 649 nondiabetic control subjects , ascertained from han chinese . 
 all subjects were randomly enrolled from three top - grade hospitals in beijing in 2010 . 
 total case subjects were diagnosed according to the world health organization criteria ( who 1998 ) . as total control subjects , the age was between 35 and 70 years old , and they had normal fasting plasma glucose levels ( 5.0  0.5  mmol / l ) without diabetic record or t2d family history . 
 the clinical characteristics of stage 2 populations are also summarized in table 1 ; it included 546 unrelated patients with t2d and 419 nodiabetic control subjects and of han chinese origin , as described previously . 
 international hapmap project snp database ( http://www.hapmap.org/ ) was implemented to search for snps in mfn2 and esrra gene . using tagging with an r 
 > 0.8 and minor allele frequency ( maf ) of > 5% and sequencing data in 20 nondiabetic control subjects , we selected 5 tag snps ( rs873458 , rs2878677 , rs2236058 , rs3766742 , rs3766741 ) in mfn2 gene and 3 tag snps ( rs731703 , rs650008 , rs11600990 ) in esrra gene ; they were all located in intron region and could capture 100% of common variations across mfn2 and esrra gene region ( containing 21 hapmap snps ) . 
 7 of 8 snps were successfully genotyped using the taqman method on a bio - rad iq5 system . 
 primers and probes of rs873458c_8861262_10 , rs3766741c_25606040_10 and rs650008 ahwr23 m were purchased from applied biosystems , the others from shanghai genecore biotechnologies ( primers showed in table 2 ) . 
 only one snp ( rs3766742 ) in the mfn2 was genotyped with pcr - restriction fragment - length polymorphism ( pcr - rflp ) method , using the following primers ( 5-agcaggacatgataggttag-3and 5-cacagcttgtcacagtttag-3 ) and pcr tm was 57c , pcr product length is about 900  bp , and restriction enzyme ( hha i ) provided by mbi fermentas . 
 a 10  l aliquot of pcr product was digested overnight at 37 in a 20  l reaction containing 5 units of hha i. after overnight digestion , the products were separated on a 3% agarose gel stained with ethidium bromide . 
 tt genotypes were represented by a dna band with a size of 900  bp , tc genotypes were represented by dna bands with sizes of 900 , 700 , and 200  bp , and cc genotypes were represented by dna bands with sizes of 700 and 200  bp . 
 20 samples randomly selected from the whole sample bank were sequenced for 8 tag snps and the results showed excellent correspondence between sequencing and taqman genotyping or pcr - rflp technique . 
 genotype distributions for all studied snps were tested for hardy - weinberg equilibrium ( hwe ) by chi - square tests and no significant deviation was found in control subjects . 
 allele frequencies and genotype distributions , linkage disequilibrium ( ld ) , and haplotypes were tested using the online software shesis ( http://analysis.bio-x.cn/myanalysis.php/ ) . 
 logistic regression analysis with additive model was used to adjust for sex , age , and body mass index ( bmi ) , and the analysis was performed by spss ( version 16.0 ) . 
 tests for association between genotypes and quantitative traits were performed in control subjects using kruskal - wallis analysis of ranks for traits with nonnormal distribution or , alternatively , anova for normally distribution in spss . 
 we examined the analyses of gene - gene interactions on the risk of t2d using multifactor dimensionality reduction ( mdr ) version 2.0 beta 6 ( http://www.epistasis.org ) . 
 in stage 1 populations , we found that the a allele and c allele frequencies of rs873458 and rs2878677 of mfn2 gene in the diabetic group were lower than in the control group ( p = 0.005 , or = 0.79 , 95% ci= 0.670.93 ; p = 0.01 , or = 0.81 , 95% 
 ci = 0.680.95 ) ( table 3 ) ; it showed a allele and c allele of rs873458 and rs2878677 of mfn2 gene confered protection agasist t2d . after adjusting for age , sex , and body mass index by using analysis of logistic regression with additive model 
 , the results showed that the genotype distributions of rs873458 and rs2878677 of mfn2 gene also were significantly different in case - control subjects ( pc = 0.01 , or = 0.80 , 95% ci = 0.670.95 ; pc = 0.02 , or = 0.83 , 95% ci = 0.710.97 ) ( table 3 ) . replicating the two associated snps in stage 2 populations , data suggested that the c allele of rs2878677 of mfn2 gene in the diabetic group was lower than in the control group ( p = 0.01 , or = 0.79 , 95% ci = 0.650.94 ) ( table 3 ) . and it revealed that there was a borderline association between genotype distributions of rs873458 of mfn2 gene and type 2 diabetes ( adjusted pc= 0.08 , or = 0.84 , 95% ci = 0.691.02 ) ( table 3 ) . 
 additionally , analyses in the combined populations showed that the allele frequencies and genotype distributions of rs873458 and rs2878677 of mfn2 gene had more significant effects on type 2 diabetes susceptibility ( p = 0.002 , or = 0.83 , 95% ci = 0.730.93 ; p = 0.0001 , or = 0.79 , 95% ci = 0.690.89 and adjusted pc = 0.002 , or = 0.82 , 95% ci = 0.720.93 ; pc = 0.0004 , or = 0.81 , 95% ci = 0.720.91 ) ( table 3 ) . 
 no significant association of other snps in mfn2 or esrra gene with type 2 diabetes was found ( see supplementary table 1 in supplementary material available online at doi:10.1155/2012/205752 ) . 
 analyses of association between haplotypes and phenotypes were performed in nondiabetic control subjects , for most patients with t2d included in the present study had medical treatments , which may affect the real parameters . 
 we found that the genotype distributions of snps in mfn2 and esrra gene in control subjects displayed no significant association with quantitative traits of type 2 diabetes including body mass index , diastolic blood pressure , systolic blood pressure , fasting plasma glucose , triglycerides , and glycated haemoglobin a1c ( data not shown ) . 
 we further examined the degree of linkage disequilibrium of snps in mfn2 and esrra gene ( supplementary tables 2 and 3 ) . to test whether haplotypes represent the causal variants better than single snps , we estimated the frequencies of haplotypes between the case - control subjects in stage 1 populations ( all those frequencies < 0.03 were ignored ) . 
 it was showed that a - c - g - t - c and g - t - c - t - c in mfn2 gene were significantly associated with t2d ( p = 0.007 , or = 0.79 , 95% ci = 0.660.94 ; p = 0.009 , or = 1.26 , 95% ci = 1.061.49 ) ( table 4 ) , while the frequencies of haplotypes in esrra gene did not significantly differ in case - control subjects ( data not shown ) . 
 the model was considered to be statistically significant with a testing accuracy > 50% and p < 0.05 via permutation test of 1000 iterations . the rs2878677/rs3766741/rs731703/rs11600990 model ( p < 0.0001 ) in mfn2-esrra from stage 1 populations , the rs2878677/rs3774923/rs7656250 model ( p < 0.0001 ) and the rs2878677/rs3774923/rs7656250/rs13131226 model ( p 
 < 0.0001 ) in mfn2-pgc-1 gene from stage 2 populations ( shown in table 5 ) , data of pgc-1 gene from our previously study , suggest that there are interactions between genetic variants in the mfn2 , esrra , and pgc-1 gene . 
 in the present study , we examined eight common variants in mfn2 and esrra genes and confirmed that g allele of rs873458 and t allele of rs2878677 in mfn2 gene are associated with higher t2d risk compared with a and c allele , respectively . 
 consistently , in haplotypes analyses of mfn2 gene , results show that individuals carrying g - t - c - t - c present increased t2d risk compared with those carrying a - c - g - t - c . because rs873458 and rs2878677 map within intron 2 and 3 , it is difficult for us to shed light on their downstream consequences , given our rudimentary knowledge of the mechanics of gene regulation . in multifactorial disease , 
 most susceptibility variants , mapping outside the coding regions of genes , are assumed to influence transcript regulation rather than gene function . 
 locations of rs873458 and rs2878677 are surrounded by the regions coding the gtpase domain of mfn2 which drive gtp hydrolysis to provide energy for the mitochondrial fusion activity induced by mfn2 [ 16 , 22 ] . 
 in addition , mfn2 gene is the first structural tether identified gene in the molecular basis of er - mitochondria juxtaposition , it has a role in the intercommunication during ca signaling . 
 a study revealed that ablation or silencing of mfn2 gene in mouse embryonic fibroblasts or hela cell created confusion in er morphology and increased distance between the er and mitochondria , hence retarding the mitochondrial ca uptake , which may lead to insulin secretion disorder and insulin resistance . besides 
 in addition , the mfn2-dependent mechanism of mitochondrial control is disturbed in skeletal muscle in animal or human obesity , and in patients with t2d [ 17 , 19 ] . 
 it is possible that the associated snps may be in strong linkage disequilibrium with unidentified causal variant(s ) that may regulate the expression or function of mfn2 gene . 
 although many genomewide association studies ( gwass ) of t2d are now emerging , mfn2 gene is not involved . 
 indeed , the effect sizes of the known , common variants influencing the risk of type 2 diabetes are modest , and the proportion of overall predisposition explained is approximately 5 to 10% for t2d . 
 it has been found that t2d loci identified by linkage analyses and gwas lack the correlation of t2d . 
 differences in genetic background , risk - factor profile , environment , and study design may lead to ethnic differences in susceptibility loci . 
 different allele frequencies of rs873458 and rs2878677 among the hapmap populations indicate that differences in genetic architecture may play a role ( supplementary table 4 ) . 
 esrra is an orphan nuclear receptor , it could regulate a number of downstream genes to control energy balance in animals . 
 esrra gene ko mice showed altered expression of several target genes implicated in the regulation of adipogenesis and energy metabolism . 
 however , in the present study , no association between the phenotypes of t2d , such as bmi , was found . 
 our data also showed that there were three common variants ( rs731703 , rs650008 , rs11600990 ) in esrra in han chinese populations , while only one common variant rs11600990 was found in danish populations . 
 three tag snps could capture 100% of common variations ( maf > 5% ) across esrra gene region , but we can not exclude rare causal genetic polymorphisms in esrra gene , for the rare variations may be responsible for disease [ 2931 ] . 
 pgc-1  is an important transcriptional coactivator involved in the regulation of genes related to energy metabolism . 
 mice lacking pgc-1 developed fasting hepatic steatosis which may lead to lower rates of fatty acid oxidation that might play a causative role to develop insulin resistance [ 33 , 34 ] . 
 in addition , pgc-1 maintained higher number of active mitochondria and oxphos proteins that were reduced in t2d subjects . as it relates to the human genetics , several snps of pgc-1 gene in certain populations 
 the analysis of association between pgc-1 gene and t2d has been published in our previous paper . 
 in addition , it was found that the interactions between genetic variants of the mfn2-esrra and mfn2-pgc-1 in the pathogenesis of type 2 diabetes . 
 moreover , a positive result of association analyses regarding single snp rs2878677 in mfn2 gene indicates mfn2 gene may be the main factor during the interaction and has an important role in the pathogenesis of t2d . 
 it is consistent with the study that it proved the interactions between mfn2 , esrra , and pgc-1 proteins , which revealed the stimulatory effect of pgc-1 on the activity of mfn2 gene needed to integrate the esrra , esrra , and pgc-1 caused a synergic effect on activity of the mfn2 gene expression , and the stimulatory effect of pgc-1 gene on mitochondrial membrane potential can be weakened by mfn2 loss of function . therefore , further studies are needed to explore the functions and interactions of the mfn2 , esrra , and pgc-1 genes .   in most patients , 
 t2d is due to alterations of many genes , each of which has a partial and additive effect . 
 consequently , we hypothesize that these three genes may serve as partial or additive factors leading to t2d , while the intrinsic mechanism is unknown . functional studies , multiplex - gene analyses , and computational biology are needed to further characterization of their interactions in the pathogenesis of t2d . however , some limitations should be noted in the present study . 
 first , the effect of gene - environment interactions was not evaluated ; environmental risk factors may influence the effect estimates . 
 second , given the study samples were relatively small , we need to replicate our finding in additional samples in future studies . 
 , the present analysis shows that mfn2 gene has significant associations with t2d in han chinese . 
 but more studies with different ethnic populations are needed to verify this finding and the intrinsic mechanism is expected to be uncovered .","mfn2 and esrra are candidate genes involved in the pathogenesis of t2d . 
 five tag - snps in mfn2 gene and three in esrra gene were selected and genotyped with taqman or pcr - rflp method in stage 1 populations ( 555 patients with t2d and 649 control subjects ) and stage 2 populations ( 546 patients with t2d versus 419 control subjects ) in han chinese . and combining our published data , we estimated the interactions between genetic variants in the mfn2 , esrra , and pgc-1 genes on the t2d risk using mdr . 
 rs873458 ( g > a ) and rs2878677 ( c > t ) in mfn2 gene were significantly associated with t2d ( p = 0.005 and 0.01 ) in stage 1 populations , and the association of other snps with t2d was not found . in stage 2 populations , we further confirmed the association between rs2878677 and t2d ( p = 0.01 ) . combining the two stage populations , the data supported more significant effect of rs873458 and rs2878677 on t2d risk ( p = 0.003 and 0.0001 ) . 
 a - c - g - t - c and g - t - c - t - c in mfn2 had significant association with t2d ( p = 0.007 and 0.009 ) . 
 the present study also provided the evidence that mfn2 had interactions with pgc-1 ( p < 0.0001 ) or esrra ( p < 0.0001 ) . 
 this study suggested a role of mfn2 polymorphism in the risk of t2d ; however , further studies are needed .",pubmed
"interindividual variations of ex vivo platelet aggregation during clopidogrel therapy have been correlated to reduced exposure of active clopidogrel thiol metabolite and differences in p2y12 receptor occupancy.1 high on - treatment platelet reactivity during therapy with clopidogrel and low platelet inhibition are associated with risk of ischemic events after coronary stenting  in particular , stent thrombosis.15 as compared with extensive metabolizers , carriers of cytochrome p450 2c19 ( cyp2c19 ) nonfunctional alleles have been shown to have reduced platelet inhibition by clopidogrel and are at increased risk of adverse events and stent thrombosis after coronary.59 recent advances in the understanding of clopidogrel metabolism suggest that the inactive prodrug undergoes two steps of bioactivation . 
 first , clopidogrel undergoes oxidation to 2-oxo - clopidogrel by hepatic cyp450 enzymes ( 1a2 , 2b6 , 2c9 , 2c19 , and 3a4/5 isoenzymes ) . 
 then , in the second step of the biotransformation , thiol metabolite is generated.10 a recent study employing sophisticated metabolomic methods suggested a hydrolytic process involving paraoxonase-1 ( pon1 ) as the rate - limiting enzyme.10 a common polymorphism of pon1 ( q192r ) is associated with reduced paraoxonase activity in carriers of the qq ( aa ) haplotype . in two cohorts of patients with coronary stenting , and using a case study design , the investigators found the pon1 ( qq192 ) genotype was associated with increased risk of stent thrombosis.10 a group of patients with a history of prior stent thrombosis but who were not receiving current therapy with clopidogrel were administered a clopidogrel 600 mg loading dose . within this group , reduced plasma activity of pon1 and pon1 ( qq192 ) genotype were associated with reduced active thiol metabolite exposure and reduced platelet inhibition by clopidogrel.10 only one genome - wide association study ( gwas ) has been performed examining the association of common genetic polymorphisms on platelet inhibition.11 this gwas examined platelet aggregation data from 429 generally healthy white subjects of amish descent after administration of a clopidogrel 300 mg loading dose , followed by maintenance clopidogrel dosing of 75 mg daily for 6 days . 
 platelet aggregation was assessed before administration of clopidogrel and again after 6 days of maintenance dosing . 
 the results from the gwas only found single nucleotide polymorphisms ( snps ) on chromosome 10q24 in linkage equilibrium with the cyp2c19 * 2 loss - of - function variant to be significantly associated with clopidogrel platelet inhibition.11 the gwas did not find evidence of a significant association of snps located on or near the pon1 gene on chromosome 7 with variation in platelet inhibition by clopidogrel.11 the aim of the current study was to assess the impact of pon1 ( q192r ) and cyp2c19 * 2 polymorphisms on clopidogrel platelet inhibition , as measured by light transmittance aggregometry and verifynow ( vn ) p2y12 assay , in a north american study population of mixed racial background . 
 the indiana university school of medicine s institutional review board for research approved the study protocols . 
 subjects were eligible to be enrolled if they had established coronary disease and were on dual antiplatelet therapy with clopidogrel and aspirin 81325 mg daily for secondary prevention . 
 subjects were included in this study either if they had been taking clopidogrel 75 mg for at least 14 days prior to enrollment or if they had received a clopidogrel 600 mg loading dose during a percutaneous coronary intervention ( pci ) . 
 subjects were excluded if they had a history of drug or alcohol abuse , bleeding disorder , myelodysplastic or myeloproliferative disorders , chronic liver disease , or current warfarin use . 
 subjects were also excluded if they were pregnant , if the platelet count was less than 150,000/mm , and if there was planned glycoprotein iib / iiia antagonist use during pci . 
 after 14 days of taking clopidogrel 75 mg daily , peripheral venous blood samples were obtained from subjects prior to the next dose of clopidogrel and aspirin to determine on - treatment platelet aggregation . for patients who received clopidogrel 600 mg at the time of a pci 
 , a baseline blood sample was obtained from the arterial access sheath prior to administration of the clopidogrel loading dose and administration of heparin or bivalirudin . 
 peripheral venous blood samples were drawn at 4 and 1624 hours after administration of the clopidogrel loading dose . for subjects who received a clopidogrel 600 mg loading dose , 
 the 16- to 24-hour sample was used to determine the final on - treatment platelet aggregation used for the primary analysis of platelet aggregation among genotypes . 
 all blood samples were directly transferred into vacutainer tubes containing 3.2% sodium citrate and were analyzed within 2 hours . 
 ex vivo platelet function was assessed by light transmittance aggregometry ( lta ) at 37c with an optical lumi- aggregometer ( model 700 with aggro / link 8 software ; chrono - log corporation , havertown , pa ) . 
 platelet - rich and platelet - poor plasma were obtained by differential centrifugation , as previously described.12,13 platelet aggregation in platelet - rich plasma was induced with adenosine diphosphate ( adp ) at 5 , 10 , and 20 
 mol / l . a vn - p2y12 point - of - care assay ( accumetrics , inc , san diego , ca ) 
 was used to assess platelet inhibition in whole blood in subjects during maintenance clopidogrel dosing , as previously described.14 genomic dna was isolated from whole blood with the aid of the qiaamp dna blood midi kit ( qiagen , germantown , md ) . 
 subjects were genotyped for cyp2c19 * 2 ( 681g > a ; rs4244285 ) , cyp2c19 * 3 ( 636g > a ; rs4986893 ) , and pon1 ( q192r ; 575 a > g ; rs662 ) snps using a bio - rad laboratories real - time icycler thermal cycler ( bio - rad laboratories , inc , hercules , ca ) . 
 sequence - specific primers were used to amplify the alleles of interest , along with two allele - specific taqman probes ( applied biosystems , foster city , ca ) . 
 allelic discrimination software ( optical system , v 3.1 ; bio - rad laboratories ) was used to determine individual genotypes . 
 all statistical tests were two - sided , and values are represented as the mean plus or minus the standard deviation , unless otherwise specified . 
 unpaired two - sided student s t - test was used to compare normally distributed continuous data between two groups and for genotype group comparisons using one - way analysis of variance . 
 non - normally distributed continuous data were compared across two groups with the two - sided unpaired wilcoxon test and genotype group comparisons with the kruskal 
 genotypes for cyp2c19 and pon1 were identified and included in univariate and multivariate linear regression , along with clinical variables that are known to affect platelet reactivity and response to clopidogrel and variables with p < 0.1 in univariate analysis ( table 2 ) . 
 the indiana university school of medicine s institutional review board for research approved the study protocols . 
 subjects were eligible to be enrolled if they had established coronary disease and were on dual antiplatelet therapy with clopidogrel and aspirin 81325 mg daily for secondary prevention . 
 subjects were included in this study either if they had been taking clopidogrel 75 mg for at least 14 days prior to enrollment or if they had received a clopidogrel 600 mg loading dose during a percutaneous coronary intervention ( pci ) . 
 subjects were excluded if they had a history of drug or alcohol abuse , bleeding disorder , myelodysplastic or myeloproliferative disorders , chronic liver disease , or current warfarin use . 
 subjects were also excluded if they were pregnant , if the platelet count was less than 150,000/mm , and if there was planned glycoprotein iib / iiia antagonist use during pci . 
 after 14 days of taking clopidogrel 75 mg daily , peripheral venous blood samples were obtained from subjects prior to the next dose of clopidogrel and aspirin to determine on - treatment platelet aggregation . for patients who received clopidogrel 600 mg at the time of a pci 
 , a baseline blood sample was obtained from the arterial access sheath prior to administration of the clopidogrel loading dose and administration of heparin or bivalirudin . 
 peripheral venous blood samples were drawn at 4 and 1624 hours after administration of the clopidogrel loading dose . for subjects who received a clopidogrel 600 mg loading dose , 
 the 16- to 24-hour sample was used to determine the final on - treatment platelet aggregation used for the primary analysis of platelet aggregation among genotypes . 
 all blood samples were directly transferred into vacutainer tubes containing 3.2% sodium citrate and were analyzed within 2 hours . 
 ex vivo platelet function was assessed by light transmittance aggregometry ( lta ) at 37c with an optical lumi- aggregometer ( model 700 with aggro / link 8 software ; chrono - log corporation , havertown , pa ) . 
 platelet - rich and platelet - poor plasma were obtained by differential centrifugation , as previously described.12,13 platelet aggregation in platelet - rich plasma was induced with adenosine diphosphate ( adp ) at 5 , 10 , and 20 
 mol / l . a vn - p2y12 point - of - care assay ( accumetrics , inc , san diego , ca ) 
 was used to assess platelet inhibition in whole blood in subjects during maintenance clopidogrel dosing , as previously described.14 
 genomic dna was isolated from whole blood with the aid of the qiaamp dna blood midi kit ( qiagen , germantown , md ) . 
 subjects were genotyped for cyp2c19 * 2 ( 681g > a ; rs4244285 ) , cyp2c19 * 3 ( 636g > a ; rs4986893 ) , and pon1 ( q192r ; 575 a > g ; rs662 ) snps using a bio - rad laboratories real - time icycler thermal cycler ( bio - rad laboratories , inc , hercules , ca ) . 
 sequence - specific primers were used to amplify the alleles of interest , along with two allele - specific taqman probes ( applied biosystems , foster city , ca ) . 
 allelic discrimination software ( optical system , v 3.1 ; bio - rad laboratories ) was used to determine individual genotypes . 
 all statistical tests were two - sided , and values are represented as the mean plus or minus the standard deviation , unless otherwise specified . 
 unpaired two - sided student s t - test was used to compare normally distributed continuous data between two groups and for genotype group comparisons using one - way analysis of variance . non - normally distributed continuous data were compared across two groups with the two - sided unpaired wilcoxon test and genotype group comparisons with the kruskal 
 genotypes for cyp2c19 and pon1 were identified and included in univariate and multivariate linear regression , along with clinical variables that are known to affect platelet reactivity and response to clopidogrel and variables with p < 0.1 in univariate analysis ( table 2 ) . 
 baseline characteristics of the study subjects are described according to cyp2c19 * 2 and pon1 q192r genotypes in table 1 . 
 overall clinical variables were well balanced among genotype groups . a total of 96 subjects on maintenance clopidogrel therapy for over 14 days and 55 subjects who received a clopidogrel 600 mg loading dose at the time of pci were enrolled . 
 the prevalence of african - americans was significantly different across pon1 q192r genotypes , with a higher prevalence of african - americans in carriers of pon1 qq192 ( table 1 ) . 
 these findings are consistent with the increased prevalence of qq192 genotypes previously described in the african population as compared with populations of european descent.15 among the 151 patients included in this study , 63 ( 42% ) were carriers of the qq192 genotype , 67 ( 44% ) were heterozygous allele carriers ( qr192 ) , and 21 patients ( 14% ) were homozygous rr192 genotype carriers . 
 for the cyp2c19 * 2 allele , 107 ( 71% ) were wild - type homozygous for the * 2 allelic variant ( * 1/*1 ) , 39 ( 26% ) were heterozygous * 2 allele carriers ( * 1/*2 ) , and five patients ( 3% ) were homozygous * 2 allele carriers ( * 2/*2 ) . 
 for both genotype distributions , no significant deviations from the hardy weinberg equilibrium were observed ( p = 0.64 for pon1 q192r ; p = 0.54 for cyp2c19 * 2 genotypes ) . 
 there were no cyp2c19 * 3 alleles detected in the study population , which is consistent with the very rare frequency ( < 1% ) of this loss - of - function allele in caucasians and african- americans . 
 platelet aggregation induced by adp demonstrated wide interindividual variability during therapy with clopidogrel , as previously documented ( table 2 , figures 14).15 pon1 qq192 was not associated with increased platelet aggregation induced by adp at various concentrations during clopidogrel therapy before and after adjustment for 2c19 * 2 , diabetes mellitus , obesity , smoking , and proton pump inhibitor therapy ( table 2 ) . 
 pon1 qq192 was also not associated with increased vn - p2y12 reactivity ; in fact , there was a trend towards lower platelet reactivity in pon1 qq192 individuals . 
 no significant difference in the percentage of platelet inhibition measured by vn was documented among individuals of varying pon1 genotype ( table 2 , figure 2 ) . 
 carriers of 2c19 * 2 alleles had higher platelet aggregation and higher vn - p2y12 reactivity with significantly lower vn platelet inhibition than noncarriers ( table 2 , figure 1 ) . among the subgroup of patients who received a clopidogrel 600 mg loading dose prior to undergoing pci , platelet aggregation measured before and after administration of clopidogrel was not significantly different between subjects of pon1 qq192 and qr192/rr192 genotypes ( figure 3 ) . 
 there was no significant difference in platelet inhibition among homozygous carriers of reduced - function pon1 ( qq192 ) and qr192 and rr192 genotypes at 4 and 1624 hours after administration of a clopidogrel 600 mg loading dose ( figure 3 ) . 
 in the same subgroup of patients who had received a clopidogrel 600 mg loading dose and who had a baseline platelet aggregation measurement , 2c19 * 2 carriers status was associated with significantly lower percentage of platelet inhibition measured by adp 10 
 mol / l , without reaching statistical significance for other concentrations of adp ( figure 4 ) . in the univariate linear regression analysis , only 2c19 * 2 carrier status ( adp 10 mol / l ; p = 0.034 ) , current smoking ( adp 10 mol / l ; p = 0.025 ) , and weight ( adp 10 mol / l ; p = 0.018 ) were significantly associated with adp - induced platelet aggregation . 
 pon1 q192r homozygous reduced - function genotype ( qq192 ) ( adp 10 mol / l ; p = 0.7 ) , diabetes ( adp 10 mol / l ; p = 0.22 ) , and proton pump inhibitor use ( adp 10 mol / l ; p = 0.65 ) were not significantly associated with adp - induced platelet aggregation ( table 2 ) . 
 covariates with p < 0.1 were included in a multivariate regression analysis , as well as variables such as diabetes and proton pump inhibitor use that have previously been established as affecting clopidogrel platelet inhibition . in a multivariate linear regression analysis that included diabetes , smoking , obesity , and proton pump inhibitor use as variables in addition to pon1 q192r and cyp2c19 * 2 genotypes , cyp2c19 * 2 carrier status ( adp 10 mol / l ; p = 0.036 ) , but not pon1 q192r ( adp 10 
 mol / l ; p = 0.89 ) , were associated with significant differences in adp - induced platelet aggregation ( table 2 ) . 
 clopidogrel is transformed into 2-oxo - clopidogrel by cyp450 enzymes and , in a second step , into the active thiol metabolite . 
 10 a recent metabolomic study suggested that pon1 , a serum esterase , may represent the rate - limiting enzyme by hydrolysis of 2-oxo - clopidogrel into thiol clopidogrel . 
 common snps of pon1 have been suggested to affect enzymatic activity of the enzyme . in bouman 
 et als10 study , pon1 activity was significantly reduced in subjects homozygous for the wild - type allele ( pon1 qq192 ) compared with carriers of the mutant allele . 
 in addition , in a group of patients with stent thrombosis and matched controls without stent thrombosis , pon1 qq192 was associated with decreased platelet inhibition by clopidogrel and decreased plasma exposure to active thiol metabolite after a clopidogrel 600 mg loading dose . 
 also , pon1 qq192 genotype was associated with an odds ratio of 3.3 for the occurrence of stent thrombosis as compared with qr192 or rr192 genotypes.10 the only gwas on clopidogrel response had previously not found an association with snps located on or near the pon1 gene , but the platelet assays in that study were performed after 7 days of therapy with clopidogrel.11 the patients included in the gwas were generally healthy white subjects , who had received a clopidogrel 300 mg loading dose followed by 75 mg daily for 6 days prior to analysis of platelet aggregation.11 similar to findings from other investigators since the publication of the study by bouman et al,10 the current study could not document an association of pon1 ( q192r ) genotype with platelet inhibition by clopidogrel in patients of mixed racial background with established coronary artery disease during treatment with clopidogrel.1621 neither platelet inhibition measured by vn - p2y12 assay nor on - treatment platelet aggregation using various doses of adp showed significant differences among genotypes of pon1 q192r . among the group of patients who received a clopidogrel 600 mg loading dose immediately before pci , platelet studies performed before and after administration of 600 mg of clopidogrel showed no significant differences in platelet aggregation among genotypes of pon1 q192r ( figure 3 ) . 
 the results of the current study are consistent with findings of shuldiner et al,11 who did not demonstrate an association of snps in or near the pon1 gene with clopidogrel nonresponse during clopidogrel maintenance dosing , and they confirm the association of 2c19 * 2 polymorphism with reduced platelet inhibition by clopidogrel.22,23 in the current study , cyp2c19 * 2 carrier status was associated with reduced platelet inhibition , as measured by vn - p2y12 assay and lta 10 mol / l , and increased on - treatment platelet reactivity , as measured by both lta and vn - p2y12 assay ( table 2 , figures 1 and 4 ) . 
 cyp2c19 * 2 polymorphism has been demonstrated to be a strong determinant of reduced active clopidogrel metabolite formation , increased on - treatment adp - induced platelet reactivity , and clinical ischemic events after coronary stenting.59,11,22,23 in a meta - analysis investigating the effect of reduced - function 2c19 alleles on recurrent ischemic events in patients receiving clopidogrel after coronary stenting , the presence of one reduced - function allele was associated with a hazard ratio of 2.67 and the presence of two reduced - function alleles was associated with a hazard ratio of 3.97 for the occurrence of stent thrombosis . 
 these findings support the clinical importance of the reduced - function cyp2c19 * 2 polymorphism and clopidogrel nonresponse after coronary stenting.9 although not supported by the findings of this study , that pon1 q192r polymorphisms could affect the generation of active thiol metabolite with administration of 600 mg or greater loading doses ( as suggested by the findings of bouman et al)10 and that it could be associated with small differences in platelet inhibition can not be excluded.10 however , in contrast to bouman et als10 study , several investigators were unable to confirm the association between pon1 q192r genotypes and stent thrombosis in other independent large cohorts of patients undergoing pci who were treated with clopidogrel.1621 limitations of the study include a relatively small sample size and a lack of pharmacokinetic analysis to detect differences in active metabolite concentrations . 
 although considered the gold standard for measuring platelet aggregation and clopidogrel platelet inhibition , lta is subject to operator variability , and on - treatment platelet aggregation may not be a direct reflection of p2y12 receptor inhibition by active clopidogrel metabolites . 
 this study confirms the previously reported impact of reduced - function cyp2c19 * 2 polymorphism on platelet inhibition by clopidogrel . 
 polymorphisms of pon1 q192r appear not to be predictive of clopidogrel response when measured by lta and vn - p2y12 assay in patients with coronary artery disease .","backgroundthe metabolic activation of clopidogrel is a two - step process . 
 it has been suggested that paraoxonase-1 ( pon1 ) is a rate - limiting enzyme in the conversion of 2-oxo- clopidogrel to an active thiol metabolite . 
 conflicting results have been reported in regard to ( 1 ) the association of a common polymorphism of pon1 ( q192r ) with reduced rates of coronary stent thrombosis in patients taking clopidogrel and ( 2 ) its effects on platelet inhibition in patient populations of european descent.methodsblood samples from 151 subjects of mixed racial background with established coronary artery disease and who received clopidogrel were analyzed . 
 platelet aggregation was determined with light transmittance aggregometry and verifynow p2y12 assay . 
 genotyping for cytochrome p450 2c19 ( cyp2c19)*2 and * 3 and pon1 ( q192r ) polymorphisms was performed.resultscarriers of cyp2c19 * 2 alleles exhibited lower levels of platelet inhibition and higher on - treatment platelet aggregation than noncarriers . 
 there was no significant difference in platelet aggregation among pon1 q192r genotypes . 
 homozygous carriers of the wild - type variant of pon1 ( qq192 ) had similar on - treatment platelet reactivity to carriers of increased - function variant alleles during maintenance clopidogrel dosing , as well as after administration of a clopidogrel 600 mg loading dose.conclusioncyp2c19*2 allele is associated with impaired platelet inhibition by clopidogrel and high on - treatment platelet aggregation . 
 pon1 ( q192r ) polymorphism does not appear to be a significant determinant of clopidogrel response .",pubmed
"toxoplasmosis is caused by the obligate intracellular protozoan parasite , toxoplasma gondii , for which preferred primary host is a cat and human may become infected as an intermediate host.1 most people with toxoplasmosis are asymptomatic . 
 however , if a pregnant woman is infected , the parasite can enter the fetal circulation through placenta and may cause congenital toxoplasmosis in the fetus . 
 congenital toxoplasmosis may present with a broad spectrum of clinical manifestations , ranging from mild chorioretinitis to severe central nervous system ( cns ) involvement , including hydrocephalus , mental retardation , seizures and intracerebral calcifications.2,3 seizure is one of the main presenting symptoms in patients with cns infection of congenital acquired toxoplasmosis and many patients with congenital toxoplasmosis took anti - epileptic drugs ( aed ) constantly . 
 however , there have been few reports of detailed description of seizure semiology and besides few reported cases of a patient with congenital toxoplasmosis who was suffering from temporal lobe epilepsy with hippocampal sclerosis ( hs ) , which is one of the most common epileptic syndromes and is considered medically intractable but surgically remediable in any causes.4,5 hereby we described a patient with congenital toxoplasmosis who showed intractable mesial temporal lobe epilepsy . 
 a 34-year - old left handed arabian female was presented with our hospital with medically intractable seizure . 
 she had hydrocephalus and right ventriculoperitoneal ( vp ) shunting was performed at 1 month old . 
 she also complained of decreased visual acuity in both eyes . on past history , she had no history of febrile seizure , metabolic disease or head trauma and her family also had no history of seizure . in a neurological examination , contracture and 
 her mental status was evaluated using mini - mental state examination with the help of a translator and her score was 22 , suggesting memory impairment and inattention . in ophthalmologic evaluation for visual impairment , macular inferior chorioretinal scar in right eye and large gray - whitish retinal necrotic lesion on posterior pole in left eye 
 was found compatible with chorioretinitis which was characteristic finding usually seen in the patients with congenital toxoplasmosis ( fig . 
 test using enzyme linked fluorescent assay , toxoplasma - specific igg test was positive and igm test was negative , suggesting chronic phase of t. gondii infection.6 the patient and her mother reported that her seizures were started with psychic aura such as anxiety initially and then secondarily generalized seizure with right eyeball deviation and head version . in the beginning , her seizures occurred one to three times a month . however the frequency of her seizure were gradually increased over time , and though the dose of aed was increased , the frequency of seizure was not reduced . 
 finally , she had the polytherapy of aeds including levetiracetam , lamotrigine and topiramate , but seizures were still poorly controlled . for further evaluation and management of her seizure , she was referred to our epilepsy clinic . 
 she underwent 3.0 t high resolution brain magnetic resonance imaging ( mri ) . on brain mri , multifocal encephalomalacic change related to vp shunt , 
 left hs and left amygdala hypertrophy , and multifocal parenchymal calcification including left perihippocampal region were observed . 
 the parenchymal calcification was confirmed again by brain computed tomography ( ct ) , which was compatible with congenital toxoplasmosis . 
 video / electroencephalogram ( eeg ) monitoring was performed for 6 days . during video - eeg monitoring , 5 complex partial seizures ( cps ) were observed , and 2 of those 5 cps were led to generalized tonic clonic ( gtc ) seizure . 
 all cpss were started with psychic aura such as anxiety , and dialeptic seizure was followed . in secondary gtc seizures , tonic posture of right arm and leg and head version to right side were accompanied . 
 the rhythmic delta activity was observed initially on left temporal area , and then led to rhythmic theta waves in the same area , spreading into left hemisphere . 
 intermittent slowing in left temporal area was also seen in interictal eeg monitoring and no epileptiform discharges were seen . 
 she underwent ictal and interictal 99m - technetium ( tc ) hexamethylpropyleneamineoxime single photon emission computed tomography ( hmpao - spect ) . 
 ictal hyperperfusion was seen in the bilateral frontotemporal areas , more pronounced on the left side . 
 interictal spect , taken after at least 24 hour seizure freedom , showed hypoperfusion in the left frontotemporal area . 
 substracted ictal spect coregistered to mri ( siscom ) shows the ictal hyperperfusion zone in left mesial temporal areas . 
 brain f - fluorodeoxyglucose positron - emission tomography ( fdg - pet ) demonstrated focal hypermetabolism in left medial temporal cortex compatible with postictal status and hypometabolism in the left fronto - parieto - temporal cortex . 
 wada test was performed to determinate the lateralization of hemispheric speech and memory dominance and it revealed right hemisphere language and memory dominance , predictive of good outcome after left anteromesial temporal resection . considering that her seizure was medically intractable and epileptogenic focus was determined to be in left temporal lobe by presurgical evaluation , we decided to perform epilepsy surgery with left anteromesial temporal resection including calcified lesion to achieve seizure control . 
 this is a case report of medically intractable mesial temporal lobe epilepsy in a patient with congenital toxoplasmosis , diagnosed by toxoplasma - specific serologic test with typical symptoms and signs including chorioretinitis , growth retardation , cerebral palsy and hydrocephalus . in patients with congenital toxoplasmosis 
 , it is well known that infection may invade tissues in central nervous system and can cause diffuse and multifocal parenchymal necrosis forming calcifications , which are main characteristics of this disease . 
 in addition , the distribution and severity of calcifications vary according to the date of maternal infection and also correlate with neurologic signs and symptoms.7,8 in our case , multifocal small parenchymal calcifications including a calcified nodule in left perihippocampal area was found in brain imaging findings , and hs and amygdala hypertrophy on left temporal lobe were also observed . 
 our patient presented predominantly dialeptic seizure accompanied by psychic aura during ictal events , which was a commonly observed finding in tle . 
 tle was confirmed with ictal and interictal eeg findings , and neuroimaging including spect and siscom . 
 hs is the most common finding in mesial temporal lobe epilepsy , closely associated with medically intractable epilepsy . 
 though the cause of hs remains unknown , many hypotheses including febrile seizures , genetic susceptibility or acquired damage such as inflammation , are considered . 
 also , it is well known that recurrent seizures may cause hippocampal neuronal damage in animals similar to hippocampal sclerosis , which may be considered as a mechanism in patients with dual pathology , coexistence of hs with extrahippocampal lesion.5,9 our patient had no history of febrile seizure and no family history of epilepsy , but on brain imaging study , hs and amygdala hypertrophy were demonstrated concomitant with a calcified nodule in perihippocampal region by congenital toxoplasmosis . in this case , the pathogenesis of hs , which may be the cause of the tle , can be considered by several different mechanisms . 
 one of possible mechanism of hs is that seizure due to a calcified nodule constitutes an initial precipitating illness , leading to the development of hs . 
 the kindling phenomenon of epilepsy is known as a process in which repeated low - level stimuli result in seizure development until a plateau is reached . 
 this phenomenon is one of well - known model of epileptogenesis and also has been closely related to mesial temporal lobe epilepsy in respect of inducing progressive neuronal loss in hippocampal formation by repetitive seizure.10,11 this hypothesis has been suggested in several case reports of other disease such as neurocysticercosis . 
 another possible mechanism is that hippocampus may be impacted by the inflammatory reaction and resulting gliosis around the calcified nodule . 
 there was a case report of a patient with neurocysticercosis and intractable mesial temporal lobe epilepsy.12 in that case , calcified mass near the hippocampus and abundant corpora amylacea were found , suggesting that degenerating cysticercus induced inflammatory injury to hippocampus . 
 also , the possibility that a calcified nodule by congenital toxoplasmosis and hs may coexist purely by chance can not be excluded . 
 tle accompanied with hs is usually medically intractable , and hippocampal damage is known to progress in patient with medically intractable tle . 
 so it needs to be very careful when managing with seizure in patients with congenital toxoplasmosis , accompanied with calcification in the perihippocampal area . 
 comprehensive evaluation including video - eeg monitoring , spect , fdg - pet and mri might be needed for defining epilepsy and localizing epileptogenic foci accurately , and be necessary to consider the surgical treatment for managing seizure if possible . in korea , there have been many surveys on seroprevalence of toxoplasmosis , which are in the range from 1.9 to 7.7% in the 198090s.1315 these result was significantly lower than those of the other endemic countries of 3070%.16 however , with increase of the number of domestic cats and change of eating habits to meat preference , seroprevalence of toxoplasmosis in korea has increased to 1325%.17,18 furthermore , considering high prevalence of t. gondii in the stray cats in korea,19,20 these data imply that the number of patients with congenital toxoplasmosis like our case may increase , thus careful prenatal and neonatal monitoring for congenital toxoplasmosis is necessary and essential in korea . in our opinion , the association of congenital toxoplasmosis with mesial temporal lobe epilepsy has been poorly reported so far , and also patients with congenital toxoplasmosis showing seizure who underwent video eeg monitoring and appropriate neuroimaging study were reported rarely . 
 we report a case with medically intractable mesial temporal lobe epilepsy with congenital toxoplasmosis , in which presented both hs and a calcified nodule in perihippocampal area 
 . there may be several hypotheses for the observed hs in patients with congenital toxoplasmosis , and limits to define a cause - effect relationship between the two conditions among several mechanisms . 
 when managing seizure disorder in patients with congenital toxoplasmosis , neurologists should be aware of this association , and the surgical treatment should be considered for managing seizure when patients with congenital toxoplasmosis presented with medically intractable tle and a calcified nodule in perihippocampal area .","toxoplasmosis is a rare disease caused by intracellular protozoan parasite , toxoplasma gondii . 
 though most patients with toxoplasmosis are asymptomatic , congenital toxoplasmosis in the fetus can cause ocular involvement such as chorioretinitis and central nervous system disease including intracerebral calcification , nystagmus , hydrocephalus and microcephaly . also , these brain lesions can cause seizure secondarily . 
 our patient was diagnosed with congenital toxoplasmosis , based on toxoplasma - specific serologic test with typical clinical symptoms , including chorioretinitis , nystagmus , hydrocephalus and cerebral palsy . 
 her brain imaging findings revealed not only the multifocal encephalomalacia , but also multifocal cerebral calcification including intracerebral calcification in left perihippocampal region . 
 her epileptogenic zone was defined as mesial temporal lobe including hippocampus on left side by seizure semiology , electroencephalogram and neuroimaging including single photon emission computed tomography and 18f - fluorodeoxyglucose positron - emission tomography . 
 her seizures were refractory to multiple anti - epileptic drugs . 
 we report a patient with congenital toxoplasmosis who showed intractable mesial temporal lobe epilepsy .",pubmed
"approximately 20 million people ( 10 - 15% of the adult population ) have gallstones and 1 million new patients are diagnosed with cholelithiasis every year . as a result 
 , the treatment of cholelithiasis has become the most costly digestive disease , with an estimated annual cost of more than $ 5 billion . in september , 1992 , 
 the national institute of health convened a consensus development conference to discuss the treatment of cholelithiasis and laparoscopic cholecystectomy . 
 after considerable discussion and review of the current literature , the panel concluded that laparoscopic cholecystectomy offered substantial advantages over the traditional open cholecystectomy with no apparent increase in morbidity or mortality . because of the reduction in pain and disability , the conference concluded that treatment costs would be equal to or slightly less than the cost of open cholecystectomy and that there would be substantial savings to patients and society because of the shortened period of disability . 
 the disability and loss of income following standard open cholecystectomy further increase cost , but these items are generally not measured by traditional appraisal methods . this convalescent period and , consequently , costs have been greatly reduced by laparoscopic cholecystectomy . 
 this example of cost analysis should be requested by business and possibly patients , as they are impacted the most . 
 indeed , charges ( not costs ) for laparoscopic cholecystectomy often surpass those of open cholecystectomy . 
 this is surprising because charges should reflect the costs resulting from increased length - of - stay ( los ) , nursing and hospital support required by open cholecystectomy . 
 the charges for equipment and other technical requirements for laparoscopic procedures probably explain the excess charges noted in laparoscopic cholecystectomies . 
 surgeons at maricopa medical center ( mmc ) identified excessive los and charges by comparing the cholecystectomy drg ( diagnostic related group ) 195 with 16 other hospitals in the region . 
 the remainder were either old patients with complications or young patients with advanced disease and complicated courses . 
 little could be done to reduce charges except to either lessen the conversion rates or costs for laparoscopic cholecystectomy . in order to get 
 a precise estimate of laparoscopic charges , match pair comparisons from the same data base of charges between open cholecystectomy and converted laparoscopic cholecystectomy were accomplished . since the comparisons matched age , sex and similar diagnosis ( ex . : acute vs. chronic cholecystitis , pancreatitis , etc . ) , many confounding variables could be eliminated . 
 this suggested that the only reasons for the increased charges were the charges for laparoscopy . 
 other reasons for conversion , such as intraoperative complications , were eliminated because none of the matched pairs had serious complications or concomitant disease . 
 therefore , this comparison provided a relatively straightforward technique for statistical comparison to determine the charges for laparoscopy . 
 a retrospective review of 70 cholecystectomy patients was conducted from a four - year data base of 633 patients who had undergone cholecystectomy . 
 thirty - five patients with converted laparoscopic cholecystectomies and thirty - five patients with open cholecystectomies were selected from the data by matching for age , sex and similar diagnosis . 
 a paired sample t - test was used for statistical analysis with significance at the p=0.05 level . decision tree and 
 sensitivity analyses were then used to choose the most cost - effective operation and then determine what the limitation of charges would be needed to make laparoscopic cholecystectomy cost - effective . 
 the average age was 37 years ( range = 15 - 70 years ) ( standard deviation [ sd ] = 15.9 ) . 
 the average charge for converted laparoscopic cholecystectomies was $ 19,922 ( sd = $ 7,134 ) , and that of open cholecystectomies , $ 13,042 ( sd = $ 9,715 ) ( p=<0.01 ) . 
 the amount attributed to laparoscopy was $ 6,880 ( $ 19,922 - $ 13,042 ) . 
 decision tree analysis showed that , regardless of the rate of conversion , open cholecystectomy was more cost - effective . 
 however , if charges could be reduced to certain levels , depending on the conversion rate , laparoscopic cholecystectomy would become cost - effective . 
 steiner reviewed surgical rates and operative mortality for open and laparoscopic cholecystectomy in maryland and found the overall mortality rate had been reduced by 33% . 
 however , the total number of cholecystectomy - related deaths had not fallen because of a 28% increase in the rate of cholecystectomies . 
 now that the technique has proven to be beneficial , the current challenge to all health providers is to improve these results by further reducing mortality and morbidity , while improving cost - efficiency . to achieve this 
 , databases for cholecystectomies must be studied to determine which aspects of the preoperative evaluation , surgery and postoperative treatment could be modified to achieve these goals . this paper attempts to address these goals with studying the charges for open , laparoscopic and converted laparoscopic cholecystectomy by evaluating matched pairs of patients in a large data set . 
 matched - pair comparisons of open and converted laparoscopic cholecystectomy provide a simple method of determining the true charges for laparoscopic cholecystectomy ( including increased operative time , anesthesia time , cholangiograms , instruments , etc . ) . with similar length - of - stay ( los ) , and averaging the charges over a large number of matched pairs , 
 the main independent variable remaining is that of the charge for the laparoscopy . in this comparative series , 
 reduction of this charge by applying reusable instruments and having surgeons with advanced laparoscopic skills operate on patients that have a higher probability of conversion would make laparoscopic cholecystectomy more cost - effective . 
 consider that the average charge for laparoscopic cholecystectomy in the 633 patients equaled $ 16,011 and the average charge for open cholecystectomy was $ 13,042 . 
 a decision - tree analysis of these values shows that managed - care contractors would prefer open cholecystectomy for all patients with cholecystitis at mmc ( figure 1 ) . 
 however , most patients would not enroll in a health plan or go to a hospital which did not provide laparoscopic cholecystectomy , so an evaluation of what could be done to reduce the charges for laparoscopic cholecystectomy must be done to make laparoscopic cholecystectomy attractive to the buyer . the most important question is , "" how much of a reduction in charge ( cost ) would be necessary to make laparoscopic cholecystectomy cost - efficient ? "" this depends not only on a reduction in charges but on the percentage of conversions ( because of the high cost of conversions ) . 
 sensitivity analysis of these data shows that if the conversion rate equals 5% , a charge of < $ 12,679.90 would be needed to make laparoscopic cholecystectomy cost - effective ( figure 2 ) . 
 therefore , a $ 3,331 ( $ 16,011 - $ 12,680 ) reduction in the charge of laparoscopic cholecystectomy would be needed before all patients could be considered for laparoscopic cholecystectomy . 
 therefore , a $ 4,689 ( $ 16,011 - $ 11,322 ) reduction would be needed to consider all patients for laparoscopic cholecystectomy . 
 therefore , the greater the amount of reduction needed , the less likely efficiency can be achieved by reducing laparoscopic cholecystectomy charges . 
 vanek and borguet reviewed the cost of laparoscopic versus open cholecystectomy in a community hospital.5 they demonstrated that conversion from laparoscopic to open cholecystectomy resulted in a 57% increase in total charges over laparoscopic cholecystectomy . 
 they suggested that this is due to the increase in hospital los of 4.7 days . 
 they further demonstrated a charge for open cholecystectomy of $ 8,160 versus $ 10,210 ( p<0.001 ) for laparoscopic cholecystectomy . 
 furthermore , the mean total hospital charge for converted laparoscopic cholecystectomies was $ 16,070 . given the literature average of return to work of 8.4 days for laparoscopic cholecystectomy and 27.5 days for open cholecystectomy , a savings of $ 2,483 accrues with the selection of laparoscopic cholecystectomy . 
 comparative analysis is difficult because many of these studies include all cholecystectomy categories while other studies were limited to elective laparoscopic cholecystectomy patients . 
 furthermore , these studies reflect marked geographical and socioeconomic factors as well as severity of disease mixes . 
 it therefore becomes advisable that health care payers evaluate charge data for each of the regional hospitals before contracting with them for medical services . 
 since laparoscopic cholecystectomy is demanded by most patients , knowledge based on laparoscopic charges and conversion rates give the hospital or health care provider information to use in evaluating surgical costs . 
 obviously , the lower the charge , all else being equal , the more likely the contract . 
 health care providers , including hmos , are looking for the most cost - effective provider and institutions which wish to compete must evaluate their procedures to inform health care providers that they are cost - efficient . matched - pair analysis , decision - tree and sensitivity analyses , similar to those described above , facilitate appraisal by each institution as to what can be done to improve care , reduce charges and become cost - efficient in performing laparoscopic cholecystectomy . 
 since there is reduced los and short - term dis - ability , hospital charges should not be in excess of that for open cholecystectomy . only evaluation and modification of the charges which contribute to the excess will lead to validated cost efficiencies and subsequent recruitment by providers for hospital service .","background and objectives : maricopa medical center ( mmc ) was found to have higher charges and length - of - stays than 16 other regional hospitals in an analysis of drg categories for gallbladder disease . 
 these comparative figures identified mmc as being inefficient and demanded review to determine the reasons for the inefficiencies.methods:in an attempt to determine the reason for inefficiency of charges and length - of - stay for the laparoscopic portion of laparoscopic cholecystectomy , matched pairs of open cholecystectomy and converted laparoscopic cholecystectomy from a data base of 633 patients with cholecystectomies were reviewed . 
 thirty - five matches for age , sex and similar diagnosis were successful.results:matched pair evaluation disclosed a $ 6,880 difference in charges , which was attributed solely to the charge for laparoscopy . 
 subsequent chart analysis showed a high charge for instrumentation , prolonged anesthesia and operative times and longer preoperative delays before surgery . 
 moreover , no matter what the conversion rate is , open cholecystectomy was more cost effective . 
 however , if there is a conversion rate of 5% , total hospital charges for laparoscopic cholecystectomy would have to be reduced to $ 12,679 ( a reduction of $ 3,332 from $ 16,011 ) to make laparoscopic cholecystectomy cost-effective.conclusions:cost-effective decision tree analysis of matched pair comparisons and sensitivity analysis proves to be an effective technique in evaluating the cost - effectiveness of laparoscopic cholecystectomy in a hospital population .",pubmed
"type 1 diabetes is characterized by progressive autoimmune destruction of pancreatic  cells   that leads to symptoms of insulinopenia when -cell mass is deficient . 
 glucose homeostasis is restored with administration of exogenous insulin and some patients experience a reduction of their daily insulin requirements ( dir ) with maintenance of optimal glycemia and hba1c ( a1c ) levels . due to its impact on disease control , this phenomenon is called  partial remission  ( pr ) or 
 honeymoon period  and is variable both in intensity ( with sometimes interruption of insulin ) and duration . 
 definition of pr was recently revised based on dir and a1c levels ( i.e. , insulin - dose - adjusted a1c , idaa1c ) , after evidence of strong correlation between idaa1c and stimulated c - peptide levels 6 and 12 months after diagnosis . underlying mechanisms of pr remain largely unknown , but restoration of residual -cell mass activity   in an otherwise insulin - sensitive environment   is believed to account for the condition . 
 pr has driven much interest as a crucial target period for interventions aiming at preserving the residual -cell mass , but immunomodulation trials have not proven to be efficient so far except for secondary study endpoints . besides potential therapeutic implications , the significance of pr is unclear , in terms of both disease progression and complication risks . 
 reduction of diabetes - related complications ( retinopathy , nephropathy ) has been described in patients with residual c - peptide secretion , but association factors are unknown and regular c - peptide measurements are included neither in current pr definition nor in clinical routine guidelines . moreover , pr impacts preliminary experiences of patients with type 1 diabetes , which change abruptly ( with rise of both dir and a1c ) as pr ends . 
 the objectives of this retrospective study are to evaluate the characteristics and potential determinants of pr according to the new definition   and to evaluate the presence of correlations between pr and long - term type 1 diabetes evolution ( metabolic control and complications ) and occurrence of severe hypoglycemia . 
 in our study , we included a total of 242 children aged 0.9 to 16.4  years , followed up in our pediatric diabetes clinic from diagnosis ( established from 1994 to 2008 ) to adulthood ( 1820  years of age ) . 
 type 1 diabetes was diagnosed according to international society for pediatric and adolescent diabetes guidelines and based on symptoms of insulinopenia , elevated blood glucose ( bg ) and a1c , positive anti - islet antibodies ( gad65 , ia2 , and insulin ) , and lack of family history of genetic diabetes . biometrics ( age , height z - score , and bmi z - score ) and biological features ( bg , a1c ) were collected at diagnosis and at each consultation ( postdiagnosis consultations occurred at 15 days and 1 month and then every 3 months ; only fully adherent patients were recorded ) . at diagnosis , 
 measures included screening of dka ( defined as ph < 7.3 and/or bicarbonate < 16  mm ) and postprandial c - peptide levels ( autodelfia c - peptide , perkinelmer life and analytical sciences ) , which were assayed every year . 
 bmi was calculated by using the formula ( bmi = weight [ kg]/height [ m ] ) . 
 pr was defined as idaa1c  9 , according to definition by mortensen et al . : 
 a1c ( % ) + [ 4  insulin dose ( u / kg / day ) ] . 
 insulin doses were adjusted for pre- and postprandial glycemic targets according to ispad guidelines , when available , or to our institution 's guidelines . 
 screening for complications was performed yearly , starting from 2 years after diagnosis until adulthood , and included determination of microalbuminuria ( urine spot ) , ophthalmoscopy , peripheral and autonomic neuropathy by physical examination , and blood lipids . 
 a patient was considered having complications when a single anomaly or more anomalies were diagnosed . 
 severe hypoglycemia was defined as loss of consciousness , coma with or without convulsions , or alteration of consciousness impeding the capacity for oral sugar ingestion ( need of a tier for i m glucagon administration ) . 
 occurrence of severe hypoglycemia was monitored at each consultation ( as per our institution 's guidelines ) . for data analysis , 
 3 age subgroups were constituted ( 04  years , 59  years , and > 10  years ) in order to include most pubertal patients into one subgroup ( > 10  years ) . 
 categorical variables were analyzed using chi - square test and continuous variables were analyzed using chi - square trend test , unpaired t - test , or mann - whitney u test , according to the statistical distribution . 
 data were submitted to d'agostino and pearson omnibus normality test and levene 's test for equality of variances . 
 correlation analysis was used to evaluate relationship between variables . to assess the relative contribution of each variable ( 
 initial c - peptide , initial a1c , sex , and dka at diagnosis ) to chances of remission , logistic regression models were built . 
 in our clinical series , pr occurred in 56.2% of patients with type 1 diabetes ( table 1 ) , without any case of complete remission . 
 the proportion of girls ( 47.5% ) and boys ( 52.5% ) was comparable among remitters and nonremitters . 
 mean age at diagnosis was 8.8  3.8 years with no difference between subgroups ( pr versus no pr , girls , boys , and age subgroups ) . 
 height and bmi z - scores , measured at first follow - up consultation to avoid influence of insulinopenia on weight , were also similar between subgroups and to the distribution of healthy belgian children from the reference database . at diagnosis 
 , most patients had no dka ( 74.4% versus 25.6% with dka , p < 0.0001 ) and the absence of dka was significantly higher in pr ( 82.3% ) than in no pr ( 63.5% ) patients ( p = 0.0047 ) ( table 2 ) . 
 this effect was more pronounced in boys , who had a higher pr frequency ( 69.4% versus 30.6% no pr , p = 0.0023 ) when no dka occurred at diagnosis . 
 furthermore , multivariate logistic regression analysis showed that chances of pr were higher when there was no dka at diagnosis ( or = 0.43 , p = 0.018 ) ( cf . below and in table 3 ) . 
 mean pr duration was 279.6 days ( range 151722 ) or 9.2 months ( range 0.556.6 ) with no differences between gender and age subgroups ( figure 1(a ) ) . 
 in 71.6% of patients , pr duration was shorter than 1 year and pr mostly occurred during the first 6 months ( figure 1(b ) ) . 
 however , 7.5% of patients experienced pr during more than 2 years ( mean 992 days ; 95% ci [ 797 , 1187 ] ) . at diagnosis , patients had an overall a1c at 10.4  2.8% ( figure 1(c ) ) , which then dropped after 2 , 3 , and 5 years . 
 patients entering pr had a lower baseline a1c ( 10.1  2.7% ) than patients with no subsequent pr ( 10.8  2.8% ) ( p = 0.049 ) ( figure 1(d ) ) . 
 this effect was evidenced in girls entering pr who had a lower a1c at diagnosis ( 10.0  2.9% ) than girls without pr ( 11.2  3.1% ) ( p = 0.028 ) . 
 however , boys had similar a1c levels whether they entered pr or not . in multivariate logistic regression models , lower a1c at diagnosis 
 was associated with higher chances of pr ( or = 0.87 , p = 0.03 ) ( table 3 ) , but levels of a1c at diagnosis did not correlate with pr duration when analyzed for all patients ( cf . 
 yet when age subgroup 04  years was considered , a1c levels at diagnosis negatively correlated with longer pr durations ( p = 0.01 , r = 0.15 ) . 
 initial a1c did not impact long - term a1c levels since no correlation could be observed between a1c at diagnosis and a1c at 2 years after diagnosis ( a1c+2y ) or at 3 years after diagnosis ( a1c+3y ) , either in pr or in no pr subgroups ( see figures s1(a)-s1(b ) in supplementary material available online at http://dx.doi.org/10.1155/2014/851378 ) . 
 a1c+2y was significantly lower in the pr ( 7.5% ; 95% ci [ 7.3 , 7.7 ] ) than in the no pr population ( 8.2% ; 95% ci [ 7.9 , 8.5 ] ) ( p < 0.0001 ) ( figure 1(e ) ) , which is partly explained by the fact that pr duration is longer than 2 years in 7.5% of the pr patients . 
 this observation was corroborated by the fact that a1c at 3 and 5 years after diagnosis ( resp . 
 , a1c+3y and a1c+5y ) did not differ between pr and non - pr groups . however , a1c+3y was lower ( 6.7% ; 95% ci [ 4.7 , 8.8 ] ) in patients having a remission lasting between 510 and 570 days as compared to nonremitters ( 8.2% ; 95% ci 
 [ 7.8 , 8.5 ] ) ( p = 0.045 ) , suggesting a prolonged effect on a1c after the pr period in this subgroup . furthermore , a1c levels at 3 , 6 , 9 , and 12 months after diagnosis were strongly associated with pr duration when subgrouped ( figure 2(a ) ) . 
 indeed , low ( 6% ) a1cs at 3 to 12 months were associated with longer pr ( mean 320 to 515 days ) , whereas pr durations dropped to mean 169 to 222 days for a1cs > 6 to 7% ( p < 0.01 ) and to mean 62 to 82 days for a1cs > 7 to 8% ( p < 0.01 ) . with high 
 a1cs ( > 8 to 9% ) , pr durations were only negligible ( 1446 days , p < 0.05 ) and not different from patients with poorly controlled diabetes ( a1c > 9% ) . 
 hence , a1c levels at 3 months allowed us to estimate the proportion of patients that will experience pr for more than 1 year at 34% ( n = 15/44 ) when a1c levels were 6% , 16% ( n = 16/102 ) for a1cs > 6 - 7% , and 5% ( n = 2/36 ) for a1cs > 7 - 8% . 
 similarly , at 6-month follow - up , these proportions changed to 46% ( n = 23/50 ) for a1cs  6% , 8% 
 ( n = 6/74 ) for a1cs > 6 - 7% , and 4% ( n = 2/50 ) for a1cs > 7 - 8% . 
 no differences could be observed in the titers of anti - gad65 and anti - ia2 antibodies at diagnosis among the different analyzed populations ( girls , boys , and age subgroups ) ( table s1 ) . 
 however , pr duration was significantly shorter ( 85.8 days ; 95% ci [ 33.9 , 137.6 ] ) in patients having 2 positive anti - gad65 and anti - ia2 antibodies at diagnosis compared with patients with only 1 positive antibody ( 198.0 days ; 95% ci [ 70.9 , 325.0 ] ) ( p = 0.04 ) . 
 additionally , anti - ia2 antibody titers positively correlated with a1c+2y levels ( p = 0.006 , r = 0.16 ) , but this correlation was not found with a1c+3y and a1c+5y levels or with the other antibodies , partly due to smaller sample sizes . 
 c - peptide secretion was significantly different in pr versus no pr patients , from diagnosis until 3 years after diagnosis ( figure 2(b ) ) . 
 however , this difference could not be observed after 4 and 5 years after diagnosis , perhaps in part because of a bias of patient selection for c - peptide screening . 
 higher baseline c - peptide concentrations were observed both in girls and in boys entering pr compared to patients without pr and this effect was more pronounced in boys ( figure 2(c ) ) . an age effect on c - peptide levels 
 was observed not only at diagnosis where higher c - peptide levels were found in patients aged > 10  years , as compared with other age groups ( 04  years , 59  years ) ( p < 0.0001 ) ( figure 2(c ) ) , but also later on since age at diagnosis positively correlated with c - peptide levels at 2 years after diagnosis ( p < 0.0001 , r = 0.21 ) . in multivariate logistic regression , other variables than a1c levels and dka at diagnosis ( i.e. , age , sex , height and bmi z - scores , c - peptide levels , and anti - gad65 and ia2 titers ) were not significantly associated with a higher chance of pr ( table 3 ) . when pr was considered on a continuous scale ( pr duration ) , none of the estimates was significant in linear regression , even if consistent with logistic regression estimates . 
 pr duration or a1c+2y levels did not correlate with occurrence of severe hypoglycemia as these parameters were not different in patients without documented episodes of severe hypoglycemia during the follow - up period as compared to patients having at least one episode ( n = 97 ; 39.7% ) or more than 2 ( n = 32 ; 13.2% ) episodes of severe hypoglycemia . 
 furthermore , only few patients ( n = 11 ) developed diabetes - related complications during follow - up until adulthood ; thus , no differences with complication - free patients could be observed in terms of pr duration and  a1c+2y . 
 in our study , overall occurrence of pr was 56.2% and mean pr duration was 9.2 months , which was comparable to other studies using idaa1c or dir and a1c levels as pr definition ( table s2 ) . 
 table s2 shows studies that included a1c and dir in pr definition since pr defined with dir only   or a1c only   overestimates pr rates . while some data   showed that young children ( < 5  years ) have lower rates of pr , in our study , young children ( 04  years ) had similar pr rates ( 47.7% ) than older children ( 60.2% and 56.3% , resp . 
 similar to other studies [ 10 , 11 ] , even with different pr definitions [ 7 , 12 ] , we did not find gender differences . however , dost et al . 
 reported longer pr in boys below 10  years of age , but in the same study pr durations were shorter for patients < 10  years when the global cohort was analyzed . 
 furthermore , we did not observe any complete remission as per idaa1c definition or defined as a complete interruption of insulin administration , contrary to data from other groups [ 11 , 14 ] showing complete remission in up to 4% of patients for a mean period of 3 months . 
 our finding that dka is associated with a lower frequency of pr is in concordance with the literature . 
 however , this effect was more pronounced in boys than in girls in our series . 
 furthermore , we did not find a higher frequency of dka in younger children ( 04  years ) as described elsewhere [ 9 , 10 , 15 , 16 ] . whether dka is 
 a variable independent of pr is unclear , since dka might reflect rapid -cell destruction   and thus preclude ongoing pr development , by contrast to patients without dka whose -cell mass is partially preserved . in our study , 
 however , only girls had lower a1c levels at diagnosis when entering pr , and only young patients ( 04  years ) had their a1c levels at diagnosis correlating negatively with pr duration . 
 we did not find similar gender- or age - related correlations in the literature , whether as it was not measured or not found . 
 interestingly , our findings show that early a1c levels might be helpful to predict pr durations since , as soon as 3 months after diagnosis , lower a1cs were significantly associated with a higher incidence of longer prs , this association being present at 6- , 9- , and 12-month follow - up . likewise to a1c , patients entering pr had higher c - peptide levels at diagnosis , and this effect was more pronounced in older ( > 10  years ) patients . also , older children at diagnosis had more chance to have higher c - peptide levels after 2 years . however , initial c - peptide levels were not significantly associated with higher chances of pr . in our study , we randomly assayed postprandial c - peptide levels , making definitive analysis of -cell reserve difficult . 
 proper analysis of stimulated c - peptide with mixed meal tolerance testing was not available in our retrospective study . 
 however , since c - peptide secretion is not affected by exogenous insulin and higher c - peptide levels may be reached by random rather than stimulated sampling , we believe that our data provide a valuable basis for correlation analyses . 
 furthermore , the age effect in c - peptide levels observed in our series might in part be explained by differences in treatment modalities . 
 indeed , in our center during the study period , adolescent patients were systematically proposed multiple daily injections ( mdi ) whereas younger children were offered bis in die insulin regimens . 
 whereas similar glycemic and a1c targets were applied in both groups , mdi - treated patients might have benefited from intensive insulin therapy which was shown to prolong c - peptide levels in the dcct trial . nevertheless , as for dka , a1c and c - peptide levels at diagnosis may perhaps not be considered independent of pr , since they reflect , directly or not , residual -cell mass . 
 while young children were shown to have either higher   or lower   rates of anti - islet antibodies at diagnosis of type 1 diabetes , we found no difference in anti - gad65 or anti - ia2 titers in any of the analyzed subgroups . only in patients without pr and aged 04 
 years a trend towards higher anti - gad65 titers was observed , while this was not significant ( p = 0.06 ) . 
 our finding that pr duration was shorter in patients with 2 positive autoantibodies , as compared to patients with a single autoantibody , corroborated a recent study   showing higher probability for developing type 1 diabetes in children having 2 anti - islet antibodies ( 69.7% ) versus one autoantibody ( 14.5% ) . in this prospective cohort , progression towards type 1 diabetes after seroconversion was also accelerated in the group having 2 autoantibodies whereas no data were provided regarding evolution of type 1 diabetes after diagnosis . 
 finally , we did not observe long - term effects of pr on diabetes complications or incidence of severe hypoglycemia , partly due to low numbers of events . yet a short - term effect of pr on metabolic control was suggested in patients having pr durations between 510 and 570 days , since they had lower a1c levels after 3 years . 
 however , no difference in a1c levels was detected after 5 years , showing the rapid loss of this effect . in conclusion 
 , our comprehensive analysis of clinical and biological features of newly diagnosed belgian type 1 diabetes patients shows that , at diagnosis , parameters directly or indirectly associated with -cell mass reserve ( a1c and dka ) correlate with pr and that longer pr duration is associated with a lower a1c even 18 months after pr ends . 
 further long - term research is needed to determine if this effect is associated with a reduced rate of complications and severe hypoglycemia .","to evaluate the characteristics and determinants of partial remission ( pr ) in belgian children with type 1 diabetes ( t1d ) , we analyzed records of 242 children from our center . 
 clinical and biological features were collected at diagnosis and during follow - up . 
 pr was defined using the insulin - dose - adjusted a1c definition . 
 pr occurred in 56.2% of patients and lasted 9.2 months ( 0.5 to 56.6 ) . 
 25.6% of patients entered t1d with dka , which correlated with lower pr incidence ( 17.6% versus 82.3% when no dka ) . in our population , lower a1c levels at diagnosis were associated with higher pr incidence and in young children ( 04 years ) initial a1c levels negatively correlated with longer pr . 
 early a1c levels were predictive of pr duration since 34% of patients had long prs ( > 1 year ) when a1c levels were 6% after 3 months whereas incidence of long pr decreased with higher a1cs . 
 c - peptide levels were higher in patients entering pr and remained higher until 3 years after diagnosis . 
 initial antibody titers did not influence pr except for anti - ia2 titers that correlated with a1c levels after 2 years . 
 presence of 2 versus 1 anti - islet antibodies correlated with shorter pr . 
 pr duration did not influence occurrence of severe hypoglycemia or diabetes - related complications but was associated with lower a1c levels after 18 months . 
 we show that , at diagnosis of t1d , parameters associated with -cell mass reserve ( a1c , c - peptide , and dka ) correlate with the occurrence of pr , which affects post - pr a1c levels . further research is needed to determine the long - term significance of pr .",pubmed
"oral health is important to the quality of life of individuals of all age groups . 
 oral lesions can lead to interference of daily activities due to discomfort or pain that interferes with mastication , swallowing , and speech , producing additional symptoms such as halitosis , xerostomia , or oral dysesthesia , which hampers an individual s daily social activities . 
 as has been established by the who ( 1984 ) , a population aging more than 60 years old should be considered , to be an elderly population . 
 the last national census of population conducted in india in 2011 showed that 8.6% of the population was 60 years or more . 
 these figures indicate that geriatric health care should be given due importance , since there has been little research done in the field of gerodontology . 
 the prevalence of oral lesions in the major adult has been reported by few researchers in colombia , mexico , chile , spain , brazil , israel , argentina , usa , and a few asian countries like cambodia and china . 
 these studies were mainly based on clinical evaluation of the lesions . the prevalence based on the biopsies of the observed lesions 
 the oral lesions and treatment needs of the elderly patients differ according to every nation , geographic region within a country , and even in different communities . therefore , 
 identification of the prevalence of oral lesions in this section of the population is due to socioeconomic differences , medical and dental history , treatment expenditure , and any handicap . 
 no previous studies have been done till date in the indian subcontinent estimating the prevalence of oral mucosal lesions in the elderly population . 
 epidemiologic studies provide information that is important to understand the prevalence , incidence , and severity of oral disease in a specific population , but the results of such studies have rarely been published worldwide . 
 earlier epidemiologic studies of the oral health status of the general population in india provided very little information about oral mucosal lesions particularly as seen in the elderly . 
 the present study was carried out to find the prevalence of oral mucosal lesions in 5,100 geriatric patients in india . 
 this study consisted of 5,100 patients , 3,100 ( 60.8% ) men and 2,000 ( 39.2% ) women with age range , 60 to 98 years , attending the department of oral medicine and radiology , jodhpur dental college general hospital , from september 2008 to october 2012 . 
 a written informed consent was obtained from the patients prior to the inclusion for the study . 
 all elderly patients who were not willing or could not be allowed the clinical oral examination were excluded . 
 all the examinations were carried out by one researcher . before recording the clinical parameters , the examiner and another researcher calibrated the clinical examination . 
 both a dental and a general medical history of the patients were obtained , and a questionnaire was completed by each patient , after which a clinical examination was performed . 
 some of the mucosal changes were diagnosed solely by clinical examination ( e.g. , linea alba , fissured tongue ) . when clinical features were not diagnostic , a biopsy was undertaken . 
 the clinical diagnosis was established according to the correlation in the etiological factor associated to the lesion by systematic examination of oral mucosa and classifying those alterations according to the epidemiology guide for the diagnosis of oral mucosal diseases ( who ) . 
 the elements to evaluate during the questionnaire included : general status of the patient , systemic diseases , medications used , age , gender , alcohol and tobacco consumption , habits ( trauma ) , and prosthetic or other appliances use . during the clinical examination 
 the following elements were analyzed : features of the lesion , anatomical location , extension , etiological factors or related factors , dental status , alcohol , tobacco , trauma , use of prosthesis and whether or not these were well adapted . 
 all non - pathological conditions or developmental such as leukoedema , lingual varices , fordyce granules , benign migratory glossitis , and fissured tongue were excluded from the present study . 
 there were 3,100 ( 60.8% ) men and 2,000 ( 39.2% ) women with the age range 60 to 98 years . 
 of these 5,100 patients , 3,264 patients ( 64% ) presented with various oral lesions . 
 males were more affected ( 66% ) than females ( 34% ) , and this difference was not statistically significant ( p > .05 ) . in the majority of the cases , only one lesion was found ( 42% ) ; however , some patients exhibited more than one oral lesion simultaneously . 
 the majority of the oral lesions were observed in patients between 65 to 70 years of age . 
 the hard palate had most lesions ( 23.1% ) , followed by the tongue ( 16.4% ) and gingiva ( 14.7% ) , whereas soft palate ( 3.6% ) was the least affected site . 
 the lesions identified during the study were classified according to clinical , histopathological , and microbiological diagnosis and were distributed in 27 different clinical entities ( table 2 ) . 
 nicotinic stomatitis ( 43% ) due to tobacco use and denture stomatitis ( 34% ) due to long use of prostheses were the most commonly diagnosed lesions . 
 osmf ( 30% ) was also a commonly seen premalignant lesion due to high percentage of people with the habit of betel nut chewing . 
 according to different studies , it is a common finding to observe oral pluripathology in the elderly , due to the aging process , systemic complexity involvement in these patients , metabolic changes , nutritional factors , prosthetic use , medications , psychobiological habits , and alcohol or tobacco habits . therefore , several conditions are to be encountered in this particular age group which include neoplasms , infections , immunological , hematological , and systemic disorders , leading to oral pain and discomfort in the elderly patient . among the various causes leading to changes in the oral mucosa due to aging 
 are bacterial , fungal , viral , parasitic infections and infections caused by other agents , physical and thermal alterations , immune system changes , systemic diseases , tumors , trauma , and other factors . 
 the prevalence of oral mucosal lesions has been found to be higher in older patients than in the younger population . 
 the prevalence of mucosal changes or lesions was 64% in the present study , which was higher than that of the findings of mujica et al . and espinoza et al . who noted the prevalence of mucosal changes to be 57% and 53% , respectively 
 however , cebeci et al . observed the prevalence of oral changes to be 15.5% in the elderly patients . 
 most population - based studies correlate oral mucosal disease with oral cancer and precancerous conditions , but few authors have recorded overall oral mucosal lesions or mucosal changes . 
 the most common mucosal lesion found in the present study was nicotinic stomatitis ( 43% ) , whereas mujica et al . and cebeci et al . 
 showed that the most common lesion was denture stomatitis ( 18% ) and anatomical changes ( 7% ) , respectively . in the present study , 
 denture stomatitis was the second common lesion and was observed in 34% of patients , which was more when compared to the other studies . in the present study , 
 osmf was recorded in 30% of population , but this condition was not at all seen in the studies by mujica et al . who reported leukoplakia to be the most common premalignant lesion ( 13% ) . 
 leukoplakia was seen in 22% of the patients , in the present study which , however , was more when compared to results of the above study and the findings of cebeci et al . who noted leukoplakia in 0.4% . in the current study , 
 lichen planus was detected in 18% of patients , whereas in the mujica and cebeci   studies , lichen planus was noted in 3% and 0.8% . respectively . in the present investigation , 
 110 patients ( 2% ) were diagnosed as having malignancies , all of whom were diagnosed as having squamous cell carcinoma . 
 the oral cavity is one of the most suitable locations for the development of cancerous diseases . 
 most oral cancers , especially squamous cell carcinomas involving the mucosal tissue , are usually evident . 
 however , all such potentially malignant lesions should be confirmed by microscopic analysis . in the present study , tobacco and its related products 
 the odds of having a lesion increased with age , even after adjusting the tobacco use , the presence of faulty dentures , race / ethnicity and sex . 
 the use of prostheses or their condition in the elderly helps to determine the number of oral lesions present in these individuals . in the present study 
 , 42% of patients exhibited one lesion , similar to the findings of mujica et al . 
 espinoza et al . stated that the use of poorly adapted prostheses should be considered as a relevant factor , explaining the fact that in some aged populations , where the use of prosthesis is low or limited , the prevalence of oral pathologies is relatively low . 
 this fact could be evidenced in reports from hoad - reddick and corbet et al . who noted a prevalence of 41% and 31% , respectively , due to poorly adapted prosthesis . 
 in the present study , however , of the lesions related to prosthesis use , denture stomatitis , was the most common one , representing 1,734 cases ( 34% ) as reported in the literature . 
 the length of denture use and diabetes mellitus can be considered as significant risk factors for denture stomatitis and denture hyperplasia . some important associations between denture - related lesions , systemic diseases , and medication use which require further investigation has also been observed . 
 certain lesions are more commonly seen on lower lip , while few are seen most commonly on upper lip . 
 different interactions between genetic and environmental factors in the oral mucosa lead to the formation of different lesions . 
 hard palate ( 23.1% ) was the most commonly affected site in the present study , whereas the soft palate was the least involved ( 3.6% ) . 
 knowledge of site predilection for different diseases will be useful in acknowledging the factors responsible for the same . according to gender distribution 
 , the majority of the studies have not demonstrated differences in the prevalence of the lesions . 
 in contrast to these findings , a slight male preponderance is noted in the present study . 
 in conclusion , the present study has provided information about the epidemiologic aspects of oral mucosal lesions in the geriatric population that may prove valuable in the planning of future oral health studies of this group of the population in india . 
 the increased number of aged individuals implies an important demographic change all over the globe . 
 therefore , it is important to plan accordingly in the social- , economic- , and health - related areas . 
 further , community programs should be enhanced to educate the elderly population along with the younger generation to get the elderly screened for any oral - mucosal lesions by availing adequate clinical and pathological laboratory facilities .","backgroundoral health is important to individuals of all age groups . 
 previous epidemiologic studies of the oral health status of the general population in india provided very little information about oral mucosal lesions in the elderly . hence , the purpose of the present study was to determine the prevalence of the oral lesions in a geriatric indian population.methods5,100 patients were clinically evaluated , with age ranging from 60 to 98 years . 
 there were 3,100 males and 2,000 females , with a mean age of 69  6.3 yrs . 
 the statistical analysis was done using the spss software , where p < .05 was considered to be significant.results64% of the patients presented with one or more oral lesions , associated to tobacco , betel nut consumption , and lesions secondary to trauma and prosthesis . 
 males were more affected than females and this difference was clinically not significant ( p > .05 ) . 
 the lesions were more frequently observed between 65 to 70 yrs . 
 the most common alterations observed were smoker s palate ( 43% ) , denture stomatitis ( 34% ) , oral submucous fibrosis ( 30% ) , frictional keratosis ( 23% ) , leukoplakia ( 22% ) , and pyogenic granuloma ( 22% ) . 
 hard palate was the most commonly affected site ( 23.1%).conclusionsthe findings of the present study provide important information when clinically evaluating oral cavity in elderly . 
 close follow - up and systematic evaluation is required in the elderly population to plan future treatment needs .",pubmed
"chondroblastoma is a rare osseous tumor which accounts for less than 1% of all primary bone tumors , and affects the epiphyseal regions of the tubular bones , especially the ends of tibia , femur , and humerus . 
 chondroblastoma localized in the head and neck region is uncommon but characteristically occurs in the temporal bone or the temporomandibular joint ( tmj ) and results in various symptoms such as occlusal abnormalities , trismus , clicking of the tmj , hearing impairments , dizziness , and otorrhea . 
 malignant changes in chondroblastoma , although rare , have been observed after irradiation [ 3 , 4 ] . in order to avoid the inherent risk of malignant progression , radiation therapy should not be considered . 
 however , chondroblastoma in the temporal bone has unusual histological features that make diagnosis difficult , and incomplete resection carries a high risk of recurrence [ 5 , 6 ] . 
 thus , it would seem advisable , provided the diagnosis is accurate , to attempt a complete surgical removal for the curative treatment of chondroblastoma . following 5 years of unsuccessful treatment , 
 the patient was referred to our department presenting with chronic hearing impairments and subsequently progressive trismus . 
 a tumor mass was present in the middle cranial fossa , including the tmj and cochlea , in spite of earlier treatment regimens . by reporting this initially misdiagnosed case 
 an accurate diagnosis was delayed because the original diagnosis relied on imaging analysis with neither careful magnetic resonance imaging ( mri)/computed tomography ( ct ) scanning analysis nor histopathologic examination . 
 this less than thorough approach led not only to a delay of an accurate diagnosis and to unnecessary treatments , but also to a delay of suitable treatments for this rare chondroblastoma with malignant potential . 
 this study presents methods which are helpful in diagnosing chondroblastoma , as well as suitable surgical procedures for maintaining postoperative functionality . 
 a 41-year - old male with tmj disorder , initially diagnosed with otitis media , presented with a history of chronic ( lasting more than 1 year ) hearing loss on the right side . 
 the patient underwent several attempts at antibiotic therapy and treatment for eustachian tube blockage , but his symptoms were unresponsive to both systemic antibiotic agents and tubing therapy . 
 his initial diagnosis of tmj disorder was based on physical assessment of malocclusion and complaints of subsequently developed trismus . 
 after these 2 years , endoscopic surgery , followed by several attempts to correct the malocclusion for the tmj disorder , was implemented but resulted in no significant improvement in clinical manifestations . 
 the patient was further assessed and a ct scan and pathologic examination showed the presence of a giant cell tumor in the temporal bone . 
 the patient harbored the primary disease and consulted us for persistent restriction of the mouth opening as well as hearing loss and dizziness , which he had noticed 5 years earlier . 
 re - examination of the ct scan revealed that the tumor mass had progressively enlarged and had widely destroyed the middle cranial fossa in the temporal lobe ( fig . 
 1 ) . mri displayed the presence of a poorly defined mass , with predominantly low signal and relatively high signal intensities , on t1- and t2-weighted sequences , respectively , involving the right middle cranial fossa and infratemporal fossa floor . 
 the tumor mass invaded the right temporal lobe and destroyed the subzygomatic fossa ( fig . 
 hearing loss in the right ear , trismus , and paralysis in v2 had not been reported by the patient before visiting our department . 
 no paralysis was observed in v1 and v3 , nor in the facial , glossopharyngeal , or hypoglossal nerves . 
 re - examination of combined mri / ct scanning indicated rapid enlargement and widespread invasion of the mass into the cranial bone , with enlarged lymph nodes in the cervical region , providing evidence for chondrosarcoma or chondroblastoma in the temporal bone ( data not shown ) . 
 preoperative histopathologic studies of the biopsy specimens for s-100 protein ( rabbit polyclonal antibody ; dako , denmark ) were performed on formalin - fixed , paraffin - embedded tissues , using an avidin - biotin - complex method as instructed by the manufacturer . 
 histopathologic findings showed extensive proliferation of multinucleated giant cells with circular nuclei showing a sheet - like structure , formation of a small quantity of acidophilic substrate in the tumor mass , large cystic areas , and the presence of hemorrhage and aneurismal bone cysts indicating secondary aneurism - like bone cyst formation ( fig . 
 preoperative re - examinations of combined mri / ct scanning and pathologic findings revealed the features of chondroblastoma . 
 a generous c - shaped postauricular incision was made through the skin and subcutaneous tissue , which was elevated to expose the underlying fascia , periosteum , and muscle . 
 the incision was extended down to the cervical region to allow exposure of the sternocleidomastoid ( scm ) muscle , the carotid artery , and the internal jugular vein . 
 neck dissection was performed ranging in scope from removal of level ii and iii nodes to possible radical neck dissection , and total parotidectomy was performed while leaving facial nerves intact ( fig . 
 a radical mastoidectomy was performed to achieve complete exposure of the mastoid and tympanic segments of the facial nerve . 
 subtotal resection of the temporal bone and skull base dissection with resection of tmj using a zygomatic - extended middle fossa approach was carried out . 
 the temporal lobe dura was resected about 30 cm together with part of the temporal lobe . 
 the dura was reconstructed using fascia lata and covered with the pericranial flap and the temporalis muscle , and reconstruction using the scm and the platysma muscle was performed to prevent infection and dysfunction ( fig . 
 reconstruction of the facial tissue allowed the patient to open his mouth to the extent of 40 mm with no pain ( fig . 
 this study underlines an attempt to use conventional surgical procedures , with some modifications , for the treatment of chondroblastoma in order to prevent postoperative tmj dysfunction , including malocclusion , trismus , and dysphagia , while preserving the masseter , the coronoid process , and the anterior part of the temporal muscle . 
 reconstruction for facial tissues was carried out by inserting the scm muscle flap into the infratemporal fossa . 
 the scm flap was then fixed to the resected end of the zygoma inside the mandible . 
 four and a half years after surgery , enhanced ct imaging showed no residual tumor . 
 the patient had no clinical evidence of tumor recurrence during this period , nor of dizziness , with improvement of the occlusal abnormalities and trismus , indicating no postoperative dysfunction . 
 the patient did not undergo postoperative irradiation since this is contraindicated with a diagnosis of chondroblastoma . 
 the present study underlines the importance of an accurate diagnosis especially for a mass in the temporal bone . 
 tumors such as chondroblastoma , which may originate as benign , may become malignant under inappropriate treatment . 
 in fact , a combined radiotherapeutic - surgical approach may provoke neoplastic transformation in the residual tumor tissues of chondroblastoma [ 4 , 5 , 7 ] . following an inaccurate diagnosis , 
 the misdiagnosis was attributable to the difficulty of a differential diagnosis since the clinical manifestations can be indistinguishable from giant cell tumor , chondrosarcoma , aneurismal bone cyst , enchondroma , or chondromyxoid fibroma . immediately 
 before being referred to our department , the patient was misdiagnosed with giant cell tumor in the temporal region on the basis of ct scan / mri plus pathology . 
 re - examination of ct / mri indicated rapid enlargement and widely - spread invasion of a mass into the cranial bone , along with enlarged lymph nodes in the cervical region , showing a possibility of chondrosarcoma in the temporal bone . 
 however , the widely- and rapidly - enlarged mass was found to have infiltrated the temporal lobes , and to have destroyed the middle cranial fossa . 
 these findings provided evidence of malignant chondroblastoma in the temporal bone . the subsequent immunohistochemical examination of the lesion showed reactivity of the neoplastic protein mass for s-100 protein . 
 the presence of s-100 protein in tumor cells differentiates chondroblastoma from giant cell tumor , chondrosarcoma , and aneurismal bone cysts , favoring the diagnosis of chondroblastoma [ 8 , 9 , 10 ] . 
 the presence of associated aneurismal bone cysts is , while not absolute , a supplemental criteria for the diagnosis of chondroblastoma . these findings strongly suggest potentially malignant chondroblastoma . 
 treatment of a chondroblastoma in the temporal bone requires total en bloc excision , curettage , and irradiation . 
 postoperative irradiation has been performed with the intention to reduce the risk of postoperative recurrence . 
 however , it is not universally recommended due to malignant changes in chondroblastoma observed after irradiation . 
 , a total en bloc excision should be the first choice as a therapeutic approach for chondroblastoma in the temporal bone . in the case presented here , the tumor was located in a site that included important neurovascular components , which generally makes surgical resection impractical . 
 thus , accurate , early - stage diagnosis is a vital component to successful treatment . 
 for patients with later - stage chondroblastoma , our modified surgical procedure is a valuable alternative . 
 ","we present a case involving a late diagnosis of chondroblastoma of the temporal skull base involving the temporomandibular joint ( tmj ) . following an initial misdiagnosis and unsuccessful treatment over a period of 5 years 
 , the patient was referred to our department for further evaluation and possible surgical intervention for occlusal abnormalities , trismus , clicking of the tmj , and hearing impairment . based on preoperative immunochemical studies showing positive reaction of multinucleated giant cells for s-100 protein , 
 the final diagnosis was chondroblastoma . the surgical approach 
 postauricular incision and total parotidectomy , with complete removal of the temporal bone , including the tmj via the extended middle fossa  was successful in preserving facial nerves and diminishing clinical manifestations . 
 this study highlights a misdiagnosed case in an effort to underline the importance of medical examinations and accurate differential diagnosis in cases involving any tumor mass in the temporal bone .",pubmed
"the benefits of regular structured exercise are well documented and include improvements in cardiovascular and metabolic function , which reduce the risk of chronic health conditions such as type 2 diabetes and cardiovascular disease . 
 recent evidence also demonstrates that exercise alone ( in the absence of concomitant dietary restriction ) reduces visceral adipose tissue ( vat )   and intrahepatic lipid ( ihl ) [ 35 ] , both of which are independently associated with cardiovascular and metabolic risk . while there is strong evidence for the benefits of regular structured exercise ( i.e. , exercise which is planned and involves repetitive movement ) , it is currently unclear whether increased levels of habitual activity ( activity that is unstructured and accumulated during daily activities outside of structured exercise ) and reduced sedentary behavior ( activity with low metabolic rate ( < 1.6 mets ) and in a reclining or seated position ) have similar health benefits in the reduction of these adipose tissue depots . understanding such associations has implications for policy as , because of low participation rates in structured exercise , there is an increasing emphasis in public health messages on the importance of habitual physical activity levels and reduced sedentary time . 
 cross - sectional analyses indicate an inverse relationship between central adiposity as inferred by waist circumference , dual energy x - ray absorptiometry , or computed tomography ( ct )   and self - reported physical activity levels . 
 a similar inverse relationship has been observed for nonalcoholic fatty liver disease ( nafld ) [ 1214 ] . 
 prospective studies have further supported this association [ 1517 ] . in the finnish twin cohort ( twinactive ) 
 study , physically inactive cotwins were found to have liver fat scores ( assessed via difference in mean signal intensity from magnetic resonance imaging ) 170% greater than that of their active cotwin , and habitual physical activity , performed outside of work hours , was the strongest correlate of central abdominal fat after controlling for both environmental and genetic factors . 
 however , self - reported behavior is subject to reporter bias and tends to overestimate activity levels , especially in overweight and obese populations . 
 emerging evidence using objectively measured physical activity via accelerometry supports these observations [ 2124 ] ; however research using accurate assessments of vat and ihl is limited . 
 if the above epidemiological data [ 1114 ] can be confirmed by high quality objective assessments , encouraging habitual physical activity and reducing sedentary behavior will have important clinical ramifications . 
 hence , in this study , we aimed to evaluate whether levels of accurately quantified sedentary behavior and habitual physical activity could predict levels of vat and ihl in adults with overweight / obesity . based on current guidelines for the management of obesity which recommend high volumes of moderate intensity activity , we hypothesised that there would be a negative relationship between time spent in moderate intensity physical activity and levels of vat and ihl and a positive relationship between time spent in sedentary behavior and vat and ihl content . 
 furthermore , we hypothesised that levels of sedentary behavior and moderate physical activity would explain a significant proportion of variance in vat and ihl when modelled with known obesity - related health risk factors ( including age , sex , body mass index ( bmi ) , waist circumference , fasting glucose , fasting insulin , high density lipoprotein cholesterol , triglycerides , and resting blood pressure ) . 
 participants were recruited via noticeboards , electronic bulletins , and clinical databases between june 2011 and january 2015 from a community - based sample of adult men and women ( aged 1860 years ) with overweight or obesity ( bmi  25.0  kgm ) . volunteers were excluded if they reported being physically active ( exercising > 3 days per week or meeting current guidelines for physical activity ) or were cigarette smokers . 
 participants provided clearance for participation from a medical practitioner prior to involvement and were excluded if there was evidence of an unstable cardiac condition , type 2 diabetes , hypertension , or liver disease other than nonalcoholic fatty liver disease ( nafld ) or secondary causes of steatohepatitis . 
 other exclusion criteria included taking lipid - lowering or insulin sensitizing medication , reporting taking n-3 polyunsaturated fatty acids within the last 6 months , or a change in medication within the last 12 months , regular alcohol consumption > 20  g / day , or a significant change in body weight ( > 5% weight change ) in the previous three months . 
 the analysis included 82 volunteers from larger intervention trials ( anzctrn12610000351011 ; actrn12614000723684 ) , the results of which have been published elsewhere . 
 the study conformed to the ethical guidelines of the 1975 declaration of helsinki and was approved by the university of sydney human research ethics committee and the sydney local health district ethics review committee . 
 eligible participants were screened via telephone interview and those meeting criteria provided informed consent and were invited to attend baseline assessments which included measures of anthropometry , a fasting blood test , magnetic resonance imaging ( mri ) and spectroscopy ( h - mrs ) , and accelerometry . 
 stature was recorded by stadiometer ( seca model 220 telescopic height rod , hamburg , germany ) . 
 weight was measured ( tanita bc-418 body composition analyzer ; tanita corporation , tokyo , japan ) to the nearest 0.1  kg and bmi ( kgm ) was calculated . 
 waist circumference was measured in triplicate at the horizontal plane , midway between the inferior margin of the ribs and the superior boarder of the iliac crest at end - expiration . 
 systolic ( sbp ) and diastolic ( dbp ) blood pressure were measured manually on each arm , after 1015  min of quiet sitting . 
 venous blood was collected from the antecubital vein after an overnight fast ( > 10 hours ) . on the same day 
 serum glucose , insulin , total cholesterol ( tc ) , triglycerides ( tg ) , high density lipoprotein cholesterol ( hdl ) , low density lipoprotein cholesterol ( ldl ) , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , and high sensitivity c - reactive protein ( hs - crp ) were analysed by a commercially accredited laboratory . 
 visceral adipose tissue was assessed via mri and intrahepatic lipid via h - mrs using a 1.5 tesla achieva whole - body system ( philips medical systems ; best , netherlands ) as previously outlined [ 5 , 26 ] . 
 the cross - sectional areas for abdominal vat were analysed using automated software ( hippo fat )   with manual editing as necessary . 
 total vat volumes were calculated by summation of slice areas with adjustment for slice thickness . 
 liver spectra data were analysed by magnetic resonance user interface software ( jmrui version 4.0 , eu project ) . 
 current mean time spent in physical activity , sedentary behavior , daily steps , and energy expenditure were assessed by triaxial accelerometer ( sensewear , bodymedia inc . 
 data were collected from all participants for four days ( 3 weekdays and 1 weekend , which was the minimum inclusion criteria ) for 24 hours per day , except during water - based activities including showering , bathing , or swimming . 
 physical activity levels and sedentary behavior were quantified ( bodymedia software , v8.0 ) with sedentary behavior defined as < 1.6 mets ,  light 
 activity as 3 < 6 mets ,  vigorous  activity as 6 < 9 mets , and  high  activity as 9 mets . 
 data were included if the monitor was worn for > 85% of a 24-hour day . 
 all descriptive data are reported as means  standard error from the mean ( se ) . 
 differences between groups were compared by student 's t - test with homogeneity of variances assessed via levene 's test for equality of variance or  for categorical data . 
 simple anthropometric and demographic variables were entered at block 1 for both vat and ihl . 
 known cardiometabolic risk variables were entered into the regression at block 2 , based on the variables that were significantly associated with vat and ihl , respectively , in our sample ( table 2 ) . 
 these included tg , glucose , insulin , sbp , dbp , alt and bmi for vat and glucose , insulin , alt , hdl , and bmi for ihl . 
 while tg was not significantly associated with vat in our analysis , it was included given its previously established association with excess vat . 
 finally , mean time spent in sedentary behavior , moderate intensity activity , and vigorous intensity activity were entered into block 3 of the regression analysis . 
 data were analysed using statistical package for the social sciences ( spss version 22.0 ; imb corp . , armonk , ny , usa ) ; p values were based on two - sided tests and considered statistically significant at p < 0.05 . 
 mean age was 40.1  1.9 years , bmi 30.9  0.5  kgm , waist circumference 100.0  1.1  cm , and liver fat 5.3  0.5% . 
 thirty - five percent of the participants were classified as having nafld based on ihl > 5.5% . 
 participants with nafld had a significantly higher bmi , waist circumference , sbp , vat , fasting glucose , and fasting insulin ( p < 0.05 ) . 
 there were no differences in objectively measured levels of physical activity , sedentary behavior , total daily energy expenditure , or the number of steps taken per day between participants with and without nafld ( table 1 ) . 
 time spent in high intensity activity ( > 9 mets ) was negligible ( mean 37  27 seconds ) with only 3/82 participants ( 4% ) engaging in high intensity activity for more than an average of one minute in total . when high intensity activity was included in the analysis , the results were not altered ( data not shown ) . while there are no established critical thresholds for vat volume with respect to morbidity and mortality risk , based on gender specific waist circumferences for caucasian populations , 51% of men and 
 bivariate correlations between vat and ihl and biochemical , anthropometric , and accelerometry assessed activity variables are summarised in table 2 
 . a higher volume of vat was associated with higher ihl , bmi , waist circumference , sbp , dbp , fasting glucose , fasting insulin , and alt ( table 2 ) . 
 there was a weak association between higher levels of vat and higher amounts of moderate intensity physical activity ( r = 0.294 , p = 0.007 ) . 
 higher levels of ihl were associated with higher bmi , waist circumference , vat , fasting glucose , and fasting insulin and lower levels of hdl , but there was no association between ihl and total energy expended , time spent in sedentary behavior , and time spent in any level of activity ( table 2 ) . 
 demographic and anthropometric variables ( waist circumference , sex , and age ) accounted for 54% and 30% of the variance in vat and ihl , respectively ( p < 0.001 ; tables 3 and 4 ) . 
 the addition of tg , glucose , insulin , sbp , dbp , and bmi in block 2 accounted for a further 9% of variance in vat ( p = 0.032 ) . 
 the addition of glucose , hdl , alt , insulin , and bmi increased the prediction of ihl to 40% ; however this was not significant ( p = 0.06 ) . 
 the addition of time spent in sedentary behavior , moderate and vigorous intensity activity ( block 3 ) , increased the prediction of vat to 65% ( p = 0.14 ) and did not change the prediction of ihl ( p = 0.96 ) . 
 waist circumference , triglyceride , and fasting glucose contributed significantly to the prediction of high levels of vat while waist circumference and fasting insulin contributed significantly to the prediction of high levels of ihl . 
 we aimed to evaluate whether levels of accurately quantified sedentary behavior and habitual physical activity could predict levels of visceral adiposity and liver fat . surprisingly , while traditional risk factors for cardiovascular disease ( e.g. , sex , age , and waist circumference ) were associated with vat and ihl , no relationship was observed between objectively measured physical inactivity and raised liver fat . 
 further , we unexpectedly observed a weak positive association between vat volume and time spent in moderate activity ( i.e. , increased vat was associated with higher time spent in moderate intensity physical activity ) and also total energy expenditure . 
 the inclusion of objectively measured levels of moderate activity and sedentary behavior did not improve the potency of traditional risk factors ( waist circumference , triglycerides , fasting glucose , or fasting insulin ) for predicting the liver fat concentration or level of visceral adiposity . as a cross - sectional analysis 
 , this study demonstrates that current habitual activity levels are not related to vat or liver fat ; however it does not account for lifetime levels of habitual activity . 
 further , while objective assessment of activity via accelerometry is considered more accurate than self - report methods , participants may still have altered their usual behavior during monitoring . while a significant reduction in liver fat has been observed with just 4 weeks of structured aerobic exercise , it is uncertain how long current habitual activity patterns may take to affect vat and liver fat . on the basis of epidemiological data at the population level , physical inactivity 
 is associated with the development of nafld [ 1214 ] and visceral adiposity , and reducing sedentary behavior ( including sitting time ) should be generally advocated in all populations and is now recognized in public health messages . however , our findings suggest that objectively measured physical activity is not greatly predictive of risk at an individual level and the associations between habitual physical activity levels , sedentary behavior , and metabolically detrimental fat depots such as vat and ihl remain uncertain . while our findings appear counterintuitive to epidemiological data , unlike these studies , we accurately quantified both liver fat and vat , as well as 24-hour habitual physical activity behavior , suggesting that the relationship may not be as straightforward . there is now strong and consistent evidence that regular structured physical activity ( exercise training ) reduces vat   and ihl [ 35 ] . 
 this reflects current physical activity guidelines which advocate structured exercise for health benefits . with the less potent stimulus of general habitual activity 
 , the volume of activity required for benefit is much greater ( e.g. , the accumulation of > 470  min of physical activity for weight loss ) . 
 while increasing incidental activity is important for increasing total daily energy expenditure , there is an established dose potency of structured exercise for a range of cardiovascular and metabolic health risk variables . 
 additionally , there are well - established inverse relationships between cardiorespiratory fitness and nafld , histological severity of liver disease , and vat . indeed high levels of cardiorespiratory fitness is a characteristic of the phenomenon termed  metabolically healthy obesity    and low cardiorespiratory fitness is an independent predictor of all - cause and cardiovascular disease mortality . in consideration of this , 
 the emphasis for behavior change in this population should arguably be on adopting and adhering to structured exercise programs , cognizant of individual barriers to participation in exercise and exercise preferences , which may require facilitation with behavioral management strategies . 
 obesity in developed nations is linked with an obesogenic environment which encompasses a calorie - dense diet ( overnutrition ) and a physical activity hostile ( or physical inactivity - promoting ) environment . however , while both physical inactivity and excess adiposity are independently associated with a greater risk of mortality , it is contentious whether physical inactivity is a cause or an effect of obesity [ 39 , 40 ] . 
 therefore , while it is established that low amounts of physical activity and high amounts of sedentary behavior predict mortality , the role of these behaviors in the development of abdominal obesity and liver fat is unclear and perhaps minimal . 
 recent research has suggested that prolonged sitting ( 8  hours / day ) is associated with an increased risk of all - cause mortality [ 41 , 42 ] and type 2 diabetes . 
 however , in support of our findings , the association between sedentary behavior and markers of cardiometabolic risk disappear when adjusted   or modified   for total physical activity levels . 
 hence only individuals with low activity and high levels of sedentary behavior such as sitting may be at increased health risk . 
 while there is a heightened understanding of the detrimental effects of increased sedentary behavior and sitting time [ 42 , 45 ] and interrupting sitting time may confer some health benefit , this study suggests that a direct link between unstructured , objectively measured levels of physical activity and sedentariness and levels of metabolically risky fat depots is insignificant . in concordance with these findings , despite significant associations with moderate - to - vigorous physical activity , no associations were observed between objectively quantified sedentary time and hepatic steatosis ( estimated via ct ) in adults with nafld   or with vat volume in men or women . 
 a large analysis in 3056 volunteers observed that individuals with nafld ( defined by the fatty liver index ) spent less time in light , moderate , or vigorous activity than non - nafld counterparts and that the lowest quartile of physical activity was found in individuals with nafld and comorbid type 2 diabetes . 
 it has also been observed that individuals meeting 150  minutes / week of moderate - to - vigorous physical activity as assessed via accelerometry had the lowest odds of hepatic steatosis ( assessed via ct ) independent of bmi ( or 0.77 ) . 
 however , in these investigations , it is unclear whether the low levels of physical activity caused the elevated liver fat content or whether nafld itself caused patients to have low participation in physical activity . 
 a limitation of this cross - sectional analysis is that the non - nafld cohort had low mean liver fat levels which reduced the range in the data . 
 an important consideration is that we recruited adults with overweight / obesity who had no known comorbid cardiovascular or metabolic disease , which probably contributed to this limited spread . 
 further research examining the interaction between sedentary behavior , physical activity levels , and metabolically detrimental fat depots , employing gold - standard methods , across a more diverse population , is therefore warranted . 
 another limitation is that the aforementioned associations between physical activity , fitness , and nafld may not translate to disease severity . a retrospective analysis of 813 adults with biopsy - proven nafld demonstrating that only those who reported meeting the minimal guidelines for vigorous intensity activity ( 75  min / week ) and not moderate physical activity guidelines ( 150 to 300  min / week ) reduced the odds of developing nash ( or 0.65 ) with further reduced odds ( or 0.56 ) when the extensive guidelines for vigorous intensity activity ( 150  min / week ) were met . 
 furthermore , the odds of advanced fibrosis were only reduced in patients meeting the extensive guidelines for vigorous activity . 
 these data suggest that vigorous activity per se may be necessary for improvements in the progressive forms of nafld although longitudinal data from clinical trials are lacking and limited by the invasive nature of liver biopsy to inform disease severity . in conclusion 
 , there were no associations between sedentary behavior and levels of ihl or vat and low levels of objectively measured physical activity did not predict high levels of vat or ihl . 
 these findings suggest that while traditional risk factors such as waist circumference , triglycerides , fasting glucose , or fasting insulin may predict the incidence of nafld or high levels of vat , objectively quantified physical activity levels and sedentary behavior did not predict those at risk .","objective . 
 epidemiologic studies suggest an inverse relationship between nonalcoholic fatty liver disease ( nafld ) , visceral adipose tissue ( vat ) , and self - reported physical activity levels 
 . however , subjective measurements can be inaccurate and prone to reporter bias . 
 we investigated whether objectively quantified physical activity levels predicted liver fat and vat in overweight / obese adults . methods . 
 habitual physical activity was measured by triaxial accelerometry for four days ( n = 82 ) . 
 time spent in sedentary behavior ( met < 1.6 ) and light ( met 1.6 < 3 ) , moderate ( met 3 < 6 ) , and vigorous ( met 6 < 9 ) physical activity was quantified . 
 magnetic resonance imaging and spectroscopy were used to quantify visceral and liver fat . 
 bivariate correlations and hierarchical multiple regression analyses were performed . 
 results . 
 there were no associations between physical activity or sedentary behavior and liver lipid . 
 sedentary behavior and moderate and vigorous physical activity accounted for just 3% of variance for vat ( p = 0.14 ) and 0.003% for liver fat ( p = 0.96 ) . 
 higher levels of vat were associated with time spent in moderate activity ( r = 0.294 , p = 0.007 ) , but there was no association with sedentary behavior . 
 known risk factors for obesity - related nafld accounted for 62% and 40% of variance in vat and liver fat , respectively ( p < 0.01 ) . conclusion . 
 objectively measured levels of habitual physical activity and sedentary behavior did not influence vat or liver fat .",pubmed
"this is a retrospective , observational , consecutive case series conducted in a tertiary eye care hospital in north india . 
 all patients with history of fireworks - related injuries , who attended the emergency eye care services and outpatient services during the 5 days of the diwali festival  2 days before diwali , day of diwali , and 2 days after diwali  for 5 consecutive years from 2011 to 2015 , were included in this study . 
 all relevant data related to age , sex , laterality , type of firework , whether user or bystander , baseline visual acuity ( va ) , details of injuries , diagnosis , whether requiring surgical or conservative management , and final best - corrected va ( bcva ) at the last follow - up were recorded . 
 patients with lid tears , corneal tears , scleral tears , and intraocular foreign body ( iofb ) were admitted and were given immediate medical and surgical attention , while those with nonpenetrating injuries were treated on an outpatient basis . 
 the immediate management of ocular burns comprised thorough eye wash with normal saline , removal of dust , soot particles and debris , and finally ruling out open - globe injury . 
 depending on the severity of ocular injury , patients were started on copious lubricants , topical antibiotics , and steroid combination eye drops and cycloplegics . 
 a total of 53 eyes of 45 patients were seen during the 5 consecutive days of diwali festival from 2011 to 2015 . 
 the right eye was affected in 30 eyes , while in 23 , the left eye was affected . 
 the characteristics of injury at presentation are summarized in table 1 . among the patients who could recall the type of the firecracker causing injury , 
 the severity of ocular damage was more among the patients who ignited the fireworks themselves than the bystanders [ table 1 ] . 
 five eyes ( 9.43% ) had an open - globe injury while 48 ( 90.56% ) had closed - globe injury . 
 six patients had undergone primary treatment elsewhere and were referred to our hospital for further management . 
 one patient with corneal epithelial defect required amniotic membrane transplant and glue with bandage contact lens . 
 eighteen ( 33.96% ) eyes underwent surgical management at our institute , 34 ( 64.15% ) eyes were managed conservatively , and only 1 ( 1.88% ) eye was advised primary enucleation . 
 age distribution of patients presenting with fireworks - related ocular injuries characteristics of ocular injury out of the 18 surgically managed eyes , three eyes underwent corneal tear repair , one eye underwent scleral tear repair , cataract extraction with posterior chamber intraocular lens implantation , and lensectomy in one eye each . 
 pars plana vitrectomy for the management of vitreous hemorrhage , retinal detachment , and iofb removal was done in 5 , 2 , and 1 eyes , respectively . 
 twenty - two eyes had bcva of 6/6 at final follow - up , 11 eyes had between 6/60 and 6/9 , < 6/60 to perception of light ( pl+ ) in 7 eyes , and no pl in 1 eye [ table 2 ] . 
 five eyes eventually developed phthisis bulbi , of which three eyes underwent secondary enucleation with ball implant and ocular prosthesis . 
 va could not be assessed in 12 injured eyes , of which three patients ( 6 eyes ) were of the pediatric age group . 
 three patients ( 6 eyes ) were lost to follow - up . visual outcome following ocular firecracker injury 
 diwali or deepawali  is described as a festival to celebrate lord rama 's return from exile . 
 this 5-day festival is also called the  festival of lights ,  where people light their homes with 
 this gesture brings out a feeling of joy and happiness and an expression of gratitude to the lord . 
 the firecracker industry gives livelihood to over 0.3 million people directly and indirect employment to about 1 million people . 
 sound and dust pollution and firework - related burn injuries are the most common problems related to the use of firecrackers . horrifying industrial accidents arising from the manufacture of fireworks have also been reported . 
 our study was a hospital - based , single - center , retrospective case series of firecracker injuries documented over a 5-day long period during diwali over the past 5 years . 
 our hospital is a tertiary eye care hospital located in the central district of the state of new delhi . 
 we cater our services to a large population of northern and central india including the adjoining states of haryana , rajasthan , uttar pradesh , and punjab . in our study , the age range was 353 years with mean age of 20.55 years . among the 45 patients , the majority ( 39 ) were males , consistent with findings from previous studies , probably reflecting the males to be more adventurous and aggressive . 
 a similar study was conducted in a multispecialty hospital located in the vellore district , tamil nadu , by john et al . , which included patients < 18 years during the 3 days of diwali over the past 5 years . 
 eighty - four children presented with firework - related ocular injuries during the study period , of which 44% required hospitalization . the prevalence of unilateral blindness in children due to fireworks was 8% . in our study , 53 eyes of 45 patients presented with firework - related eye injuries . 
 we included patients of all age groups and all cases who presented to our hospital during the 5 days of diwali in the past 5 years . 
 the possible reasons for the lesser incidence of firework - related injuries in our study were as follows : ( 1 ) our hospital is a tertiary eye care center where only cases related to eye pathologies are referred . 
 ( 2 ) our hospital is surrounded by muslim - dominated population who do not celebrate the hindu festival of diwali . 
 ( 3 ) the urban location of our hospital may be one of the reasons for the fewer cases being reported . 
 there are multiple tertiary referral centers in the city that are capable of managing ocular trauma - related cases . 
 ( 4 ) more firecracker manufacturing industries are located in south india ; increased risk of fireworks - related trauma . 
 ( 5 ) better awareness regarding safety and legislation related to fireworks among the urban population . in our study , only one eye ( 1.88% ) with ocular injuries did not have even pl while seven eyes ( 13.2% ) had vision worse than 3/60 . 
 five eyes had phthisis bulbi  three eyes following irreparable retinal detachment , one eye following severe ocular hypotony , and one eye following a failed retinal detachment surgery . 
 , three children ( 5.1% ) with ocular injuries did not have even pl while 10 ( 11.9% ) children had vision worse than 3/60 in the injured eye . 
 this clearly demonstrates the extent of ocular morbidity and visual loss due to firecracker injuries . in our study , 
 serious ocular injuries occur due to the sudden appearance of unpredictable firecrackers before a person can deploy any defense mechanism . 
 the most common types of firecracker causing injury were bombs as reported by other indian studies . 
 high - velocity fireworks such as rockets and bombs are more frequently associated with facial lacerations , iofb , endophthalmitis , and subsequently , poorer visual outcome . 
 a study from china documenting fireworks - related injuries during the spring festival have reported a higher preponderance of open - globe injuries . in our study , 
 corneal abrasions , corneal foreign body , and hyphema were the most common findings involving the anterior segment , while the most common posterior segment finding was vitreous hemorrhage . 
 unfortunately , fireworks - related ocular trauma can be vision - threatening and can cause permanent facial disfigurement . 
 the central pollution control board estimates that 95% of firecrackers violate noise and pollution norms . in india , the manufacture , sale , and use of firecrackers generating noise levels exceeding 125 db or 
 the supreme court has banned setting off firecrackers between 10 p.m. and 6 a.m. during festival seasons . however , in india , laws regarding displays and celebrations involving firecrackers and those regulating bursting of firecrackers by individual members of the public are virtually absent . 
 deregulating the sale of fireworks can cause increased burden of firework related morbidity as seen in studies from northern ireland and south africa . 
 chan et al . reported tripling of ocular injuries after the removal of a ban on fireworks in 1996 in ireland . 
 kuhn et al . showed a reduced incidence of firework related injuries , following the strict enforcement of fireworks ban at private places . 
 the limitation of our study was the retrospective method of capturing data and inclusion of cases that got injured only during the 5 days around diwali . 
 this would have resulted in missing out additional cases of firework - related eye trauma and underestimating the actual magnitude of this vision - threatening problem . 
 a significant percentage of patients end up losing their vision due to firework - related eye injuries . 
 immediate measures such as prohibiting the manufacture and sale of dangerous fireworks such as rockets and those which produce loud noise are required in the reducing significant ocular morbidity and noise pollution . 
 public awareness about the possible dangers , prophylactic measures , and expert primary ophthalmic care of the injured are the most realistic options available at present . 
 firework displays at public places by professionals should be encouraged during diwali and other major celebrations . 
 firework - related eye injuries are an important cause of preventable blindness , especially in the younger economically productive population . 
 ocular trauma from firecrackers when serious can lead to blindness , facial burns , and permanent disfigurement . in a country like india , 
 festivals have their own social , cultural , and economic significance . with the concept of cracker - free festival not working , 
 a combined approach of creating public awareness and strong legislation is required to regulate the manufacture , sale , and use of consumer fireworks to reduce this preventable cause of ocular morbidity . 
  
 ","purpose : to evaluate the epidemiological characteristics and outcomes of ocular injuries resulting from the use of firecrackers during the diwali festival in all age groups.materials and methods : a single - center , retrospective , hospital - based case series presenting with ocular trauma consequent to fireworks usage in a tertiary eye care center in north india during the 5 days of diwali festival from 2011 to 2015 was conducted.results:a total of 53 eyes of 45 patients were included in the study , out of which the vast majority ( 39/87% ) were males . 
 the mean age was 20.55 years . 
 almost an equal number of bystanders ( 25/55.5% ) were affected as compared to people handling the fireworks ( 20/44.44% ) . 
 five ( 9.43% ) eyes had open - globe injury , whereas 48 ( 90.56% ) eyes had closed - globe injury . 
 eighteen ( 33.96% ) eyes underwent surgical intervention . 
 thirty - three ( 62.26% ) eyes had final vision > 20/200 with eight ( 15.09% ) eyes being vision < 3/60 in the affected eye.conclusion:firework-related ocular trauma can lead to serious visual impairment . 
 mandatory legislative laws pertaining to the manufacture , sale , and use of fireworks and creating public awareness can reduce the incidence of this preventable cause of blindness in the society . initiating new policies for retailers involved in sale of these firecrackers can also bring in decrease of such morbidities .",pubmed
"subclinical hypothyroidism ( sch ) , which is defined as an isolated elevation of thyroid - stimulating hormone ( tsh ) levels with a normal free thyroxine ( ft4 ) level , is a relatively common disorder . 
 most experts agree that sch represents early , mild thyroid failure   and this issue is becoming increasingly recognized because the performance of thyroid function tests have substantially increased in primary care settings . despite the intuitive appeal of the relationship between elevated tsh levels or sch and adverse cardiovascular ( cv ) outcomes , 
 it remains unclear whether variations in thyroid hormone levels within the normal range are also associated with mortality endpoints . 
 the precise definition and clinical significance of sch are also confounded by controversies regarding the correct upper limit of the reference range for serum tsh . 
 in fact , the optimal tsh cutoff values and clinical significance of these subclinical abnormalities are even more controversial . 
 typically , the treatment of sch is considered only for patients who are pregnant , infertile , exhibit associated symptoms , or have a high risk of progression to overt hypothyroidism . 
 although there is a high prevalence of sch , the evidence supporting screening for this disorder and the benefits and risks of its treatment remain controversial . therefore , the primary goals of the present narrative review were to assess current evidence regarding the clinical aspects of sch and cardiovascular disease ( cvd ) and to evaluate the mechanisms that are likely to underlie this relationship . to accomplish these goals , an updated search of pubmed was conducted for the most recent articles investigating the risks and treatment of sch that were published prior to 2015 . 
 sch has various causes of which the most common ( 60% to 80% ) is chronic autoimmune thyroiditis associated with the presence of anti - thyroid peroxidase antibodies , which is a marker of chronic lymphocytic ( hashimoto 's ) thyroiditis . 
 hashimoto 's thyroiditis is more common in girls and women , but the overall incidence increases with age in both sexes . 
 patients who have been treated for overt thyroid failure often exhibit sch due to inadequate thyroid hormone supplementation , poor adherence to the treatment regimen , drug interactions , or the inadequate monitoring of treatment . 
 the prevalence of sch varies according to population , age , sex , race , region , and method of tsh measurement . 
 thus , it is necessary that the normal reference range for tsh levels be standardized and that appropriate quality control procedures be established by each laboratory investigating this phenomenon . in general , 
 sch is defined as a tsh level greater than 4.0 to 6.0 miu / l . 
 the prevalence of sch is relatively high and ranges from 4% to 20% in the adult population with women , elderly people , and iodine - sufficient populations being affected more often . 
 for example , in a recent study using korean population - based cohorts , the prevalence of sch was reported to be 11.3% . 
 the adverse clinical effects associated with overt thyroid disorders are well known and , given the multiple actions of thyroid hormones on the heart , vessels , bones , and brain , long - term adverse outcomes may manifest as subclinical levels of dysfunction . 
 table 1 provides an overview of the findings from studies investigating the associations of sch with various clinical conditions . 
 for example , the relationship between pathological thyroid dysfunction and body weight is well established . 
 cross - sectional studies using population cohorts have demonstrated that variations in thyroid function within the population reference range have a substantial impact on weight and body mass index ( bmi ) . 
 higher levels of tsh are associated with an increased bmi   and rising tsh levels which , in turn , are associated with enhanced weight gain . 
 additionally , an increasing amount of evidence indicates that higher levels of tsh are associated with worsening blood pressure ( bp ) and altered lipid levels . 
 a cross - sectional study demonstrated that subjects with sch exhibit increased bp   and a population - based study observed positive linear associations between tsh levels and systolic and diastolic bp . 
 similarly , a recent meta - analysis found that sch is associated with higher systolic bp . 
 thyroid hormones exert multiple effects on the regulation , absorption , and metabolism of lipid synthesis as well . 
 previous studies have consistently demonstrated that elevated levels of serum total cholesterol ( tc ) , low density lipoprotein cholesterol ( ldl - c ) , apolipoprotein b , lipoprotein(a ) , and possibly triglycerides ( tg ) are reversed in individuals with overt hypothyroidism following levothyroxine ( t4 ) therapy . 
 a number of studies have reported significant increases in serum tc , ldl - c , and tg in patients with sch   and the findings of a meta - analysis found that the tc , ldl - c , and tg levels of sch subjects are significantly increased relative to euthyroid individuals . 
 similarly , a korean study demonstrated that sch subjects with an intermediate - to - high risk of cvd had higher coronary calcium scores than euthyroid subjects   and marfella et al . 
 identified enhanced inflammatory activity in the asymptomatic carotid plaques of sch subjects compared to euthyroid subjects . 
 sch can also lead to vascular problems characterized by increased vascular stiffness and endothelial dysfunction . 
 studies of euthyroid individuals have shown that ft4 and triiodothyronine ( t3 ) levels , even within the normal range , are inversely associated with the presence and severity of coronary and carotid atherosclerosis as well as carotid artery intima media thickness . 
 other possible explanations for the increased cv risk in persons with sch include hypercoagulability , insulin resistance , oxidative stress , and endothelial dysfunction . 
 metabolic syndrome ( mets ) is a condition characterized by a cluster of metabolic disorders including abdominal obesity , insulin resistance / glucose intolerance , dyslipidemia , and hypertension . 
 it has been well documented that mets is associated with increased risks of all - cause mortality and cvd . a systemic review by iwen et al 
 .   found convincing evidence supporting the major impact of sch on all components of mets and a cross - sectional analysis of cohort studies highlighted the fact that the odds of having mets are positively associated with tsh levels within the reference range . additionally , cross - sectional and longitudinal follow - up studies from japan observed strong associations between sch and mets . 
 in korea , high normal tsh levels were significantly associated with the increased prevalence of mets in 7,270 euthyroid men and women   and another study of 2,760 young korean female volunteers with normal tsh levels found that the high tsh group had a 2-fold greater risk of mets than subjects in the low tsh group . 
 it is well known that thyroid hormones act on the heart and vasculature and , as a result , the impact of subclinical thyroid dysfunction on the cv system has recently become an important topic of research . 
 sch can lead to impaired systolic and diastolic cardiac function   and symptomatic sch patients often complain of reduced exercise tolerance during effort . 
 this same study found that a slowed rate of left ventricular relaxation might critically impair ventricular filling during exercise and result in left ventricular systolic dysfunction . 
 older people with sch and a high cv risk appear to be at an increased risk of incident heart failure   and sch was found to affect left ventricular dysfunction , although this abnormality may be reversed by t4 substitutive therapy . 
 likewise , a recent cardiac magnetic resonance spectroscopic study demonstrated that early cardiac bio - energetic impairments in sch patients are reversible with t4 therapy . 
 a pooled analysis from six prospective cohorts also found that the risk of heart failure events increases in conjunction with higher tsh levels . 
 studies of euthyroid individuals observed a positive association between tsh levels and mortality due to coronary heart disease ( chd ) . 
 tsh levels within the normal range are inversely associated with all - cause mortality   and adult taiwanese subjects with sch were shown to have an increased risk for all - cause mortality and cvd death over a 10-year period . however 
 , other studies did not find any associations with all - cause   or cv   mortality rates . 
 these discrepant findings may be due to differences in sample size and power , the inconsistent age and sex ratios of the study populations , or the varied iodine intakes of different regions . 
 two meta - analyses   investigating this matter demonstrated that sch may be associated with a modest increase in the risks of chd and mortality , particularly in subjects with higher tsh levels . a more recent review of the clinical consequences of variations in thyroid function within the normal reference range found that even modest elevations of tsh may have substantial health outcomes , including cv mortality . in a korean cohort that was followed for 10 years , elevated serum tsh levels significantly increased the risk of cvd by approximately 20% per one standard deviation in males . 
 more recently , ft4 and ft3 levels within the normal range were inversely associated with the risks of all - cause and cancer mortality in a korean cohort of euthyroid men and women ( n=212,456 ) . 
 a number of placebo - controlled studies have assessed the effects of t4 replacement therapy on the symptoms of patients with sch . however , the reported benefits of various sch treatments are limited and evidence regarding the association of sch with cvd remains controversial . 
 an intervention study of 56 women with sch identified elevated systolic and diastolic bp and increased serum cholesterol and homocysteine levels compared to healthy controls ; these factors were normalized after 18 months of t4 therapy . 
 a systematic review of 13 heterogeneous studies concluded that t4 treatment leads to a reduction in serum tc and ldl - c levels in patients with sch ; this finding has been confirmed in subsequent randomized controlled trials ( rcts ) . 
 however , to date , clinical trials have not consistently shown the beneficial effects of t4 treatment on serum lipid levels in sch patients . 
 nonetheless , reductions in carotid intima - media thickness   and improvements in brachial artery endothelial function have been observed in individuals with sch following t4 replacement . 
 thus , the negative effects of sch on cv function might be improved or reversed by replacement doses of t4 . 
 although only a few studies investigating the cv effects of t4 have been double - blinded and placebo - controlled , these trials concur that t4 replacement therapy improves systolic and diastolic heart function , endothelial function , and carotid intima - media thickness . 
 similarly , in a small rct , the normalization of tsh levels following t4 replacement therapy led to an improvement in cardiac function . moreover , there are new data supporting improved outcomes in younger ( < 70 years ) chd patients treated with t4 . on the other hand , a cochrane review from 2007 assessed 12 rcts of t4 replacement in patients with sch and concluded that replacement therapy for sch did not result in improved survival or decreased cv morbidity . 
 the indications for the screening of sch and the determination of tsh threshold levels for the treatment of sch have remained a clinical controversy for over many years . 
 additional observational data are unlikely to adequately address the uncertainties associated with the risks of sch or the potential benefits and risks of replacement therapy 
 . these concerns will be best addressed in an appropriately powered rct that assesses clinical outcomes . 
 the european commission has now funded such a trial ; the thyroid hormone replacement for untreated older adults with subclinical hypothyroidism ( trust ) study . 
 the trust study is a multicenter , double - blinded , placebo - controlled randomized trial designed to assess the multimodal effects of thyroid hormone replacement in 3,000 adults 65 years of age and older with persistent sch ( clinicaltrials.gov : nct01660126 ) . 
 in addition to cv events such as chd , stroke , and heart failure , this trial will evaluate disease - specific and health - related quality of life , muscle function , and cognitive function . 
 this study should definitively clarify the populations that may benefit from t4 replacement by assessing multiple clinical outcomes as well as whether these benefits may be offset by the potential adverse effects of replacement therapy , such as atrial fibrillation and osteoporosis . 
 the significance of sch is now widely studied and the data continue to accumulate . in summary , 
 the present review has highlighted the fact that sch is associated with an increased risk of developing a wide range of adverse health outcomes and that sch might represent a potentially modifiable risk factor of cvd and mortality . 
 therefore , understanding the prevalence and risk factors of sch could aid in the prevention of cvd in this population . 
 however , until the results of the trust study are published and current clinical recommendations   are updated , the decision to treat or not treat an sch patient will be based on clinical judgment , clinical practice guidelines , and expert opinion .","subclinical hypothyroidism ( sch ) is a common disorder that is characterized by elevated thyroid - stimulating hormone levels in conjunction with free thyroxine concentrations within the normal reference range . 
 thyroid hormones are known to affect the heart and vasculature and , as a result , the impact of sch on the cardiovascular ( cv ) system has recently become an important topic of research . 
 strong evidence points to a link between sch and cv risk factors such as alterations in blood pressure , lipid levels , and atherosclerosis . 
 additionally , accumulating evidence indicates that sch is associated with metabolic syndrome and heart failure . 
 the present review proposes that sch may be a potentially modifiable risk factor of cv disease and mortality . 
 however , large - scale clinical trials with appropriate power investigating the risks and benefits of sch treatment are required to determine whether these benefits can be achieved with levothyroxine therapy .",pubmed
"the anaerobic gram - positive bacillus clostridium difficile is a leading cause of antibiotic - associated diarrhea . 
 antimicrobial drugs disrupt the normal intestinal flora , allowing c. difficile to flourish and release c. difficile toxin a and toxin b that induce c. difficile - associated diarrhea ( cdad).1,2 although cdad is the common health - care - associated infectious disease , treatment or infection countermeasures are not applied to patients who do not present with diarrhea , including the tens of percents of asymptomatic adults who carry c. difficile in their intestines.35 cdad diagnosis widely uses an enzyme immunoassay ( eia ) kit to detect glutamate dehydrogenase ( gdh ) and toxins a and b ( cd toxins ) released by the c. difficile cells.6 however , the low detection sensitivity of eia for the cd toxins has been reported.7,8 compared to the 90% detection sensitivity of polymerase chain reaction ( pcr ) and cytotoxigenic culture , eia detection sensitivity is usually thought to be ~70% and has been reported to be as low as 50% or below.7,8 cdad diagnosis is based on eia results in japan , because pcr has not been clinically available . if eia testing is performed on c. difficile isolates cultured from the same specimen , an eia gdh - positive and toxin - negative stool specimen may give a toxin - positive result . 
 bacterial content is higher in specimens containing colonies in solution compared to stool ; therefore , toxin concentration may also be markedly higher . 
 if a specimen is gdh positive , follow - up testing with a nucleic acid amplification test , such as pcr , is recommended to compensate for the low sensitivity of eia.911 pcr detection of tcda or tcdb genes is the evidence for the presence of toxigenic c. difficile . while pcr findings have been reported to correlate well with clinical symptoms,12 bacterial toxin proteins are not detected ; therefore , positive results may also include patients carrying c. difficile without cdad.13 there is currently no definite method of determining whether diarrhea is caused by cd toxins present at concentrations below the detection sensitivity of eia in patients with eia toxin - negative stool or whether diarrhea is due to a cause other than cd toxins , despite the presence of c. difficile in the stool . 
 patients with toxin - negative stool may be dismissed as non - cdad and thus overlooked in terms of treatment and infection countermeasures . 
 the present study investigated patients who had submitted stool specimens for cdad diagnosis using eia . 
 differences in patient characteristics , diarrhea duration , antibiotics usage , and severity were compared among patients with toxin - positive stool , toxin - negative stool but toxin - positive isolates , and toxin - negative stool and isolates . 
 the aim of the study is to investigate clinical history and need for the treatment of patients with gdh - positive and toxin - negative stool specimens . 
 the present study retrospectively investigated patients ( age 2 years ) whose stool specimens were submitted to the microbiology laboratory at our institution for cd toxin testing between march 2013 and june 2014 . 
 patients with c. difficile growth on stool culture and stool specimens confirmed to be gdh positive on eia were included . 
 patients were divided into three groups based on the eia cd toxin results in stool specimens and isolates obtained from bacterial culture : a toxin - positive stool group ( positive stool group ) , a toxin - negative stool / toxin - positive isolate group ( discrepant negative / positive group ) , and a dual toxin - negative stool and isolate group ( dual negative group ) . c. diff 
 , blacksburg , va , usa ) eia was performed according to the manufacturer s instructions to determine stool specimen positivity or negativity for gdh and cd toxins . 
 samples of toxin - negative stool specimens were inoculated onto cycloserine - cefoxitin mannitol agar ( ccma ; nissui pharmaceutical co. , ltd . , 
 eia was performed on isolates from colonies using the same method as for the stool specimens to confirm the presence or absence of gdh and cd toxins . 
 bowel movement status was investigated using the electronic medical records ( emrs ) of bowel movement frequency and characteristics . 
 patients with diarrhea or muddy stool passed once per day or loose stool passed four times per day were identified as symptomatic . 
 clinical data relating to white blood cell ( wbc ) count , c - reactive protein levels , serum albumin ( alb ) levels , and body temperature on the day the specimens were submitted to the microbiology laboratory or , if measurements were unavailable for that day , on the day before or after , were also obtained from the emr . 
 one - way analysis of variance was performed to compare the continuous variables among the three groups , and the tukey 
 the ethics committee of the university of tokyo graduate school of medicine approved this study and waived the need for obtaining written informed consent from each patient . 
 the present study retrospectively investigated patients ( age 2 years ) whose stool specimens were submitted to the microbiology laboratory at our institution for cd toxin testing between march 2013 and june 2014 . 
 patients with c. difficile growth on stool culture and stool specimens confirmed to be gdh positive on eia were included . 
 patients were divided into three groups based on the eia cd toxin results in stool specimens and isolates obtained from bacterial culture : a toxin - positive stool group ( positive stool group ) , a toxin - negative stool / toxin - positive isolate group ( discrepant negative / positive group ) , and a dual toxin - negative stool and isolate group ( dual negative group ) . 
 c. diff quik chek complete ( techlab , inc . , blacksburg , va , usa ) eia was performed according to the manufacturer s instructions to determine stool specimen positivity or negativity for gdh and cd toxins . 
 samples of toxin - negative stool specimens were inoculated onto cycloserine - cefoxitin mannitol agar ( ccma ; nissui pharmaceutical co. , ltd . , 
 eia was performed on isolates from colonies using the same method as for the stool specimens to confirm the presence or absence of gdh and cd toxins . 
 bowel movement status was investigated using the electronic medical records ( emrs ) of bowel movement frequency and characteristics . 
 patients with diarrhea or muddy stool passed once per day or loose stool passed four times per day were identified as symptomatic . 
 clinical data relating to white blood cell ( wbc ) count , c - reactive protein levels , serum albumin ( alb ) levels , and body temperature on the day the specimens were submitted to the microbiology laboratory or , if measurements were unavailable for that day , on the day before or after , were also obtained from the emr . 
 cary , nc , usa ) . one - way analysis of variance was performed to compare the continuous variables among the three groups , and the tukey 
 the ethics committee of the university of tokyo graduate school of medicine approved this study and waived the need for obtaining written informed consent from each patient . 
 a flow chart of the study design and patient cohort is shown in figure 1 . during the study period 
 , 127 specimens were excluded for the following reasons : second of multiple specimens submitted during the same diarrhea episode ( n=54 ) , no record of eia results for isolates obtained from stool culture ( n=47 ) , patient age < 2 years ( n=4 ) , and bowel movement status unclear from emr or fever chart ( n=22 ) . 
 all specimens underwent eia , identifying 39 toxin - positive and 29 toxin - negative stool specimens . 
 eia performed on agar - cultured isolates from toxin - negative stool specimens identified 14 toxin - positive and 15 toxin - negative isolates . 
 no significant differences were observed in clinical characteristics such as the number of comorbidities , presence of malignant tumors , recent history of transplant , and use of tube feeding . 
 no significant differences were observed among the three groups with regard to number and respective administration frequencies of antimicrobial drugs received by patients within the 30 days prior to diarrhea onset ( table 2 ) . 
 a significant difference was observed in mean diarrhea duration between the 39 patients in the positive stool group and the 29 patients with toxin - negative stool specimens ( 10.6 days vs 5.6 days ; p=0.0179 ) . among the 29 patients with toxin - negative stool specimens , mean diarrhea durations were 7.9 days and 3.4 days for the discrepant negative / positive group and the dual - negative group , respectively . 
 a significant difference was observed in diarrhea duration among the three groups ( p=0.0395 ; figure 2 ) . 
 comparison between each group found a significant difference in diarrhea duration between the positive stool group and the dual - negative group ( p=0.0312 ) . 
 no significant difference was observed between the positive stool group and the discrepant negative / positive group ( p=0.6006 ) ; however , diarrhea duration tended to be longer in the positive stool group . no difference was observed in diarrhea duration between either of the groups with toxin - negative stool specimens ( p=0.3952 ) . 
 a significant difference was observed among the groups in the proportion of patients who received vancomycin or metronidazole treatment for cdad : positive stool group , 82.1% ( n=32 ) ; discrepant 
 negative / positive group , 7.1% ( n=1 ; vancomycin 500 mg three times daily for 13 days ) ; and dual - negative group , 0% ( n=0 ) ; p<0.001 ( figure 3 ) . in positive stool group , 
 27 patients received oral vancomycin , four received oral metronidazole , and one received oral metronidazole ( 250 mg four times daily for 11 days ) followed by oral vancomycin ( 125 mg four times daily for 7 days ) . 
 of the patients receiving oral vancomycin alone in positive stool group , 70.4% received 125 mg four times daily , 18.5% received 500 mg four times daily , 7.4% received 500 mg three times daily , or 3.7% received 250 mg four times daily . 
 of the patients receiving oral metronidazole alone in positive stool group , 25% received 250 mg four times daily and 75% received 500 mg three times daily . 
 the present findings demonstrated that , although significantly fewer patients in the discrepant negative / positive group received treatment compared to the positive stool group , diarrhea duration tended to be shorter . 
 no significant differences were observed among the groups with regard to inflammation findings , specifically wbc count , c - reactive protein levels , alb levels , and body temperature , on the day stool specimens were submitted ( table 3 ) . 
 of the 68 patients investigated in the present study , 39 ( 57.4% ) had cd toxin - positive stool specimens , 14 ( 20.6% ) had toxin - negative stool specimens but toxin - positive isolates , and 15 ( 22.1% ) had both toxin - negative stool specimens and isolates . 
 quinn et al14 investigated 174 stool specimens and found that 133 ( 76.4% ) were negative for both gdh and cd toxins , 18 ( 10.3% ) were positive for both gdh and toxins , and 23 ( 13.2% ) were positive for gdh but negative for toxins . in the present study , 
 similar rates of gdh positivity were found in both toxin - positive and toxin - negative patients with regard to stool specimens . 
 these gdh - positive but toxin - negative patients represent the proportion of cases in which diagnosis and treatment requirements are unclear . 
 symptom duration was significantly shorter in patients with toxin - negative rather than toxin - positive stool specimens , despite the lack of cdad drug treatment in all but one of the 29 toxin - negative stool specimen patients . 
 furthermore , diarrhea duration also tended to be shorter in patients with toxin - negative stool specimens but toxin - positive isolates compared to those with toxin - positive stool specimens , despite significantly fewer discrepant negative / positive group patients receiving treatment . 
 this suggests that patients who tested positive for c. difficile on culture but negative for toxins in their stool had milder symptoms , despite having toxin - positive isolates . 
 polage et al15 found that eia toxin - negative patients had significantly milder diarrhea , shorter diarrhea duration , and a lower mortality rate than toxin - positive patients . 
 patel et al16 also reported that an infection with an eia toxin - negative strain of c. difficile presents less severe clinical course , such as lower white blood cell counts and shorter length of hospital stay , than infection with eia - positive c. difficile . 
 meanwhile , baker et al17 demonstrated that eia toxin - negative patients present with milder clinical symptoms than toxin - positive patients . 
 conversely , other studies have reported no difference in clinical symptoms between toxin - negative and toxin - positive patients,18 and a consensus has yet to be reached . the present analysis supported the reports of polage et al,15 patel et al,16 and baker et al,17 finding that mean diarrhea duration was significantly shorter in patients with toxin - negative ( 5.6 days ) than toxin - positive ( 10.6 days ) stool specimens , even with similar gdh positivity . 
 furthermore , 13 of 14 patients with toxin - negative stool specimens but toxin - positive isolates did not receive cdad treatment , and diarrhea duration in this group did not differ significantly from that in the dual - negative group . 
 the group with toxin - negative isolates may have been infected with nontoxigenic c. difficile , while the toxin concentration in the stool and intestines of the group with toxin - positive isolates but toxin - negative stool specimens was likely too low to be detected by eia , despite c. difficile toxigenicity . 
 akerlund et al19 found that toxin concentrations in vitro from cultured c. difficile did not correlate with the toxin concentration in stool , which correlated with diarrhea frequency and abdominal pain . 
 in addition to low toxin concentration , various intestinal factors , such as primary bile salt and human immunity , may contribute to lack of toxin detection in stool specimens in patients with discrepant negative / positive findings,2026 because primary bile salts inhibit the growth of vegetative cells of c. difficile and human alpha - defensins cause loss of cytotoxicity of c. difficile toxin b. however , whatever the cause , diarrhea is comparatively mild in patients with toxin - negative stool , suggesting that severe symptoms are unlikely to develop at toxin concentrations below detection sensitivity . in this study 
 toxin - negative patients with gdh positivity may be simply colonized with another cause of diarrhea , even if toxigenic c. difficile is isolated from stool . 
 some classes of antibiotics , such as clindamycin , broad - spectrum cephalosporins , vancomycin , carbapenems , -lactamase inhibitors combination antibiotics , and fluoroquinolones , are widely known risk factors for cdad.5,2729 on the other hand , linezolid has been reported to have a potential protective role against the development of c. difficile infection30 and a good in vitro activity against c. difficile isolates in a human gut model of c. difficile infection.31 in this study , 48% patients received -lactamase inhibitors combination antibiotics and 25% patients received carbapenems . 
 although cephamycin and fourth - generation cephalosporin tend to be more frequently used in toxin ( + ) stool group than other groups , there were no significant differences among the groups in antimicrobial drug use within the 30 days prior to toxin testing . 
 furthermore , no clear correlation was observed between type of antimicrobial drug administered prior to cdad onset and the presence of toxins . in the majority of patients , 
 stool testing was conducted within several days of diarrhea onset , but no significant differences were observed among the groups with regard to clinical inflammation during the same period , suggesting no strong correlation between toxin detection and inflammation at the time of symptom onset . 
 these findings indicate that it is difficult to predict toxin negativity or positivity prior to toxin testing based on drug use status or other test results . 
 the sensitivity of eia for toxin detection is far inferior to that of pcr , which amplifies the toxin coding regions.14,32 however , pcr detects the presence of bacteria with toxin genes rather than toxin protein production ; therefore , patients who are identified as toxin - positive on pcr may include carriers with diarrhea due to a cause other than toxin.13 eia directly measures the toxin proteins in stool and is thus more likely to reflect the patient s actual disease status . 
 while eia is often associated with low sensitivity , it has a high positive predictive value,14 making it an important test in determining treatment approach 
 . limitations of the present study include the retrospective nature of the study , the small sample size , and the absence of molecular biological testing . 
 potential differences in the gene sequences related to toxin production between patients with toxin - positive stool and those with toxin - positive isolates were also not investigated . 
 hyper - virulent c. difficile strain , such as bi / nap-1/027 strain , has emerged and causes severe and recurrent diseases . in japan , 
 the nap-1 strain is low prevalence.33 recently , polage et al34 reported that patients with a positive molecular test result and a negative toxin immunoassay test result had outcomes that were comparable to patients without c. difficile by either method and that the prevalence of hypervirulent strain was 107 of 293 isolates . 
 however , further large number analysis is required to determine whether the patient groupings in the present study reflect bacterial genetic characteristics . 
 in this study , around half with toxin - negative stool specimens among the patients with eia gdh - positive stool specimens had toxin - positive isolates . 
 since toxigenic c. difficile was thus present in their stool , these patients are at risk of transferring the bacteria to other patients and require care from the perspective of preventing hospital infection . however , from the perspective of treatment for individual patients , diarrhea duration is shorter in patients with toxin - negative stool specimens compared to those with toxin - positive stool specimens even without treatment . 
 nucleic acid amplification tests are recommended to detect toxinogenic c. difficile in stool for a diagnosis of cdad ; however , toxin production is not evaluated . 
 these results may suggest a limited need for cdad treatment for patients with gdh - positive and toxin - negative stool specimens .","purposethe enzyme immunoassay ( eia ) has lower sensitivity for clostridium difficile toxins a and b than the polymerase chain reaction in the diagnosis of c. difficile - associated diarrhea ( cdad ) . 
 furthermore , toxin positivity with eia performed on c. difficile isolates from stool cultures may be observed even in patients with eia glutamate dehydrogenase ( gdh)-positive and toxin - negative stool specimens . 
 it is unclear whether such patients should be treated as having cdad.methodsthe present study retrospectively compared patient characteristics , treatment , and diarrhea duration among three groups of patients who underwent stool eia testing for cdad diagnosis : a toxin - positive stool group ( positive stool group ; n=39 ) ; a toxin - negative stool / toxin - positive isolate group ( discrepant negative / positive group , n=14 ) ; and a dual toxin - negative stool and isolate group ( dual negative group , n=15 ) . 
 all cases included were confirmed to be gdh positive on eia test.resultspatients backgrounds and comorbidities were not significantly different among three groups . 
 no difference was observed among the three groups with regard to antimicrobial drug use before diarrhea onset . 
 treatment was received by 82.1% of the positive stool group compared to 7.1% of the discrepant positive / negative group and 0% of the dual negative group , while mean diarrhea duration was 10.6 days compared to 7.9 days ( p=0.6006 ) and 3.4 days ( p=0.0312 ) , respectively.conclusioneven without treatment , patients with toxin - negative stool specimens had shorter diarrhea duration than those with toxin - positive stool specimens even with toxin - positive isolates . 
 these findings may suggest a limited need for cdad treatment for gdh - positive patients and toxin - negative stool specimens .",pubmed
"the design and principal findings of the dream trial have been presented in previous publications ( 17 ) . 
 briefly , the dream trial was a large , international , multicenter , double - blind , randomized controlled trial designed to determine whether ramipril and/or rosiglitazone could prevent or delay the development of type 2 diabetes in people with ifg or igt , metabolic states that indicate very high risk for eventual progression to diabetes ( 17 ) . 
 eligibility for the dream trial included a diagnosis of ifg , igt , or both ifg and igt based on a screening 75-g oral glucose tolerance test ( ogtt ) ( 17 ) . 
 a total of 5,269 participants with these disorders were recruited and randomized to either ramipril and/or rosiglitazone using a two - by - two factorial design and followed for a median of 3 years after randomization . 
 participants were assessed at regular intervals to ascertain the occurrence of the primary outcome , which included new - onset diabetes or all - cause mortality . as part of a substudy 
 , 982 dream trial participants attending canadian study centers had ogtts at baseline , after 2 years , and at the end of the study , with blood samples drawn fasting as well as 30 and 120 min after the glucose challenge . 
 the primary outcome variable in the present study was change in -cell function over the course of follow - up . 
 -cell function was assessed using two measures : the insulinogenic index ( igi ) and proinsulin ( pi ) concentration , with igi defined as ( 30-min insulin  fasting insulin)/(30-min glucose  fasting glucose ) . 
 both indexes have previously been validated against gold - standard measures of insulin secretion ( 18,19 ) and have been shown to be significant predictors of incident diabetes in large epidemiological studies . to account for the compensatory response of insulin secretion in relation to background insulin resistance , 
 igi was divided by the homeostasis model assessment of insulin resistance index ( homa - ir ) ( defined as fasting glucose  fasting insulin/22.5 ) ( igi / ir ) for univariate analysis or adjusted for homa - ir in multivariate analysis . 
 similarly , pi concentration was divided by c - peptide concentration ( i.e. , the pi / c - peptide ratio [ pi / c ] ) for univariate analysis or adjusted for insulin secretion using c - peptide as a covariate in multivariate analysis . 
 although the pi - to - insulin ratio is often used to identify disproportionate elevations in pi , c - peptide offers advantages over insulin as a denominator for pi because it is cosecreted with insulin in an equimolar ratio , but unlike insulin it is not extracted by the liver and thus it has a constant peripheral clearance . 
 glucose concentration was determined using an enzymatic reference method on a roche hitachi 917 instrument and a roche reagent kit ( roche diagnostics , indianapolis , in ) . 
 serum insulin and c - peptide were measured on the roche elecsys 2010 immunoassay analyzer using an electrochemiluminescence immunoassay . 
 the insulin assay had a sensitivity of 1.39 pmol / l , an interassay coefficient of variation ( cv ) of < 4.6% at all levels , and < 0.05% cross - reactivity with human c - peptide and pi . 
 the c - peptide assay had a sensitivity of 3.0 pmol / l , an interassay cv of < 3% at all levels , and < 0.005% cross - reactivity with human insulin . 
 pi concentration was measured using a sandwich enzyme - linked immunosorbent assay manufactured by linco research ( linco research , st . 
 this assay had a sensitivity of 2.0 pmol / l , an interassay cv of < 9% at all levels , and no cross - reactivity with human insulin or des ( 31,32 ) split pi , although this assay does cross - react with human des ( 64,65 ) split pi . 
 statistical analyses were conducted using sas version 9.1 ( sas institute , cary , nc ) , and p values < 0.05 were considered statistically significant . 
 the distributions of continuous variables were assessed for normality , and transformations of skewed variables were used in the analysis as appropriate . means and sds for primary -cell function measures ( igi / ir and pi / c ) were calculated for each time point ( baseline , at 2 years , and final visit ) , according to the marginal treatment group . 
 p values for change were based on a t test of the average change being different from zero , while p values for treatment difference were calculated using the wilcoxon 's rank - sum test . 
 similar analyses were conducted within subgroups of isolated ifg ( iifg ) , isolated igt ( iigt ) , and combined ifg and igt ( ifg+igt ) . 
 as there was no significant interaction between ramipril and rosiglitazone 's effect on -cell function , main - effects analyses were conducted according to marginal randomization groups ( i.e. , rosiglitazone versus placebo , ramipril versus placebo ) . 
 longitudinal analyses of the associations between treatments and changes over time in -cell function measures were examined using random - effects models in proc mixed , which provides appropriate options for handling the covariance structure of the repeated - measures ( longitudinal ) data . 
 specifically , we ran four models , which assessed the impact of treatment group on 1 ) igi , with age and homa - ir as covariates ; 2 ) igi / homa - ir , with age as a covariate ; 3 ) pi , with age and c - peptide as covariates ; and 4 ) pi / c , with age as a covariate . 
 finally , using cox proportional hazards regression , we assessed whether the impact of ramipril and/or rosiglitazone treatment on diabetes incidence was independent of baseline levels and changes over the course of the trial in -cell function . 
 the outcome variable in these analyses was diabetes status at the final visit , and the primary exposures were the marginal treatment groups ( rosiglitazone , ramipril ) . in separate models , we assessed the impact of treatment on diabetes incidence , adjusting for either baseline -cell function ( including baseline igi and homa - ir or baseline pi and c - peptide as covariates ) or change over the course of the trial in -cell function ( including changes in the above - mentioned covariates ) . 
 baseline models were also adjusted for age and baseline waist circumference , fasting glucose , triglycerides , and hdl , while change models were also adjusted for age and changes in these covariates . 
 statistical analyses were conducted using sas version 9.1 ( sas institute , cary , nc ) , and p values < 0.05 were considered statistically significant . 
 the distributions of continuous variables were assessed for normality , and transformations of skewed variables were used in the analysis as appropriate . means and sds for primary -cell function measures ( igi / ir and pi / c ) were calculated for each time point ( baseline , at 2 years , and final visit ) , according to the marginal treatment group . 
 p values for change were based on a t test of the average change being different from zero , while p values for treatment difference were calculated using the wilcoxon 's rank - sum test . 
 similar analyses were conducted within subgroups of isolated ifg ( iifg ) , isolated igt ( iigt ) , and combined ifg and igt ( ifg+igt ) . 
 as there was no significant interaction between ramipril and rosiglitazone 's effect on -cell function , main - effects analyses were conducted according to marginal randomization groups ( i.e. , rosiglitazone versus placebo , ramipril versus placebo ) . 
 longitudinal analyses of the associations between treatments and changes over time in -cell function measures were examined using random - effects models in proc mixed , which provides appropriate options for handling the covariance structure of the repeated - measures ( longitudinal ) data . specifically , we ran four models , which assessed the impact of treatment group on 1 ) igi , with age and homa - ir as covariates ; 2 ) igi / homa - ir , with age as a covariate ; 3 ) pi , with age and c - peptide as covariates ; and 4 ) pi / c , with age as a covariate . finally , using cox proportional hazards regression , we assessed whether the impact of ramipril and/or rosiglitazone treatment on diabetes incidence was independent of baseline levels and changes over the course of the trial in -cell function . 
 the outcome variable in these analyses was diabetes status at the final visit , and the primary exposures were the marginal treatment groups ( rosiglitazone , ramipril ) . in separate models , we assessed the impact of treatment on diabetes incidence , adjusting for either baseline -cell function ( including baseline igi and homa - ir or baseline pi and c - peptide as covariates ) or change over the course of the trial in -cell function ( including changes in the above - mentioned covariates ) . 
 baseline models were also adjusted for age and baseline waist circumference , fasting glucose , triglycerides , and hdl , while change models were also adjusted for age and changes in these covariates . 
 the average age and bmi were 54 years and 31.5 kg / m , respectively , and 60% of participants were female . the majority ( 81% ) were of european origin , and substantial proportions had a family history of diabetes or a history of gestational diabetes ( 61 and 16% , respectively ) , characteristics that were consistent with the recruiting strategy for the dream trial ( 17 ) . 
 there were no significant differences between the marginal randomization groups for any of the baseline characteristics other than family history of diabetes in the ramipril versus placebo marginal group ( p = 0.02 ) ( table 1 ) . 
 baseline characteristics of participants with measures of -cell function overall and by allocation data are means  sd or n ( % ) . 
 no significant differences between rosiglitazone versus placebo or ramipril versus placebo other than family history of diabetes in the ramipril versus placebo group ( p = 0.02 ) . 
 changes in markers of -cell function in marginal randomization groups are presented in table 2 . 
 participants receiving rosiglitazone versus placebo had a significant increase in igi / ir during the study ( 25.59 vs. 1.94 , p < 0.0001 ) and a significant decrease in pi / c ( 0.010 vs. 0.006 , p < 0.0001 ) . 
 in contrast , there were no significant changes in igi / ir or pi / c in participants receiving ramipril versus placebo ( 11.71 vs. 18.15 , p > 0.05 , and 0.007 vs. 0.008 , p > 0.05 , respectively ) ( table 2 ) . in the rosiglitazone group , 
 changes in the -cell function measures were more substantial between the baseline and 2-year visits compared with changes that occurred between the 2-year and final visits ( table 2 ) . 
 the impact of rosiglitazone on igi / ir and pi / c was similar within subgroups of iigt and ifg + igt ( iigt , igi / ir : 27.74 vs. 2.76 , p < 0.0001 ; pi / c : 0.009 vs. 0.008 , p < 0.001 ; ifg + igt , igi / ir : 27.39 vs. 0.70 , p < 0.0001 ; pi / c : 0.014 vs. 0.002 , p < 0.0001 ) , although effects were more modest in those with iifg ( igi / ir : 8.95 vs. 2.13 , p = 0.03 ; pi / c : 0.003 vs. 0.001 , p > 0.05 ) ( supplemental table , available at http://care.diabetesjournals.org/cgi/content/full/dc09-1579/dc1 ) . 
 changes in markers of -cell function data are means  sd and are reported for marginal treatment groups . 
 p value for change were based on a t test of the average change being different from zero . 
 we further assessed the impact of treatment on markers of -cell function by utilizing longitudinal data from multiple study time points in mixed - model analyses . compared with placebo , 
 rosiglitazone significantly increased igi after adjustment for age and homa - ir ( p = 0.015 ) ( table 3 ) . 
 in contrast , ramipril did not significantly affect adjusted igi ( p > 0.05 ) . 
 similar findings were seen using pi concentration as a measure of -cell function . specifically , rosiglitazone significantly reduced pi concentrations over time after adjustment for age and c - peptide concentration ( p = 0.0064 ) ( table 3 ) . 
 in contrast , ramipril did not significantly change adjusted pi concentrations ( p > 0.05 ) . 
 longitudinal changes in markers of -cell function in dream trial : analysis of slopes using mixed - model analysis analysis in table are based on full data ; results essentially unchanged when analysis repeated on subjects with information from all visits . 
 we assessed whether the impact of ramipril and/or rosiglitazone on diabetes incidence was independent of baseline levels or changes in indexes of -cell function with time ( fig . 
 1 ) . after accounting for baseline -cell function as measured by either igi or pi in models that adjusted for insulin resistance and other covariates , 
 rosiglitazone significantly reduced the risk of developing diabetes ( hazard ratio 0.32 [ 95% ci 0.220.45 ] , p < 0.001 , and 0.33 [ 0.230.47 ] , p < 0.001 , in igi and pi models , respectively ) . adjusting for the change in -cell function as measured by igi attenuated the preventive effect of rosiglitazone on incident diabetes ( 0.53 [ 0.280.99 ] , p = 0.046 ) . 
 such attenuation was not noted when the change in -cell was measured using pi ( 0.28 [ 0.180.42 ] , p < 0.0001 ) ( fig . 
 association of treatment allocation with risk of diabetes : impact of baseline levels and changes in -cell function . for igi / ir models : model 1 adjusted for age and baseline igi , homa - ir , fasting glucose , waist circumference , triglyceride , and hdl ; model 2 adjusted for age and changes in igi , homa - ir , fasting glucose , waist circumference , triglyceride , and hdl . 
 for pi / c models : model 1 adjusted for age and baseline pi , c - peptide , fasting glucose , waist circumference , triglyceride , and hdl ; model 2 adjusted for age and changes in pi , c - peptide , fasting glucose , waist circumference , triglyceride , and hdl . 
 rosiglitazone , but not ramipril , improved measures of -cell function over time in people with ifg and/or igt . 
 specifically , rosiglitazone increased igi / ir and reduced pi / c by 86 and 42% , respectively . 
 these findings were consistent across glucose tolerance subgroups ( iifg , iigt , and ifg+igt ) , although there was the suggestion of a more modest effect in the subgroup with iifg . 
 finally , rosiglitazone 's effect on diabetes prevention persisted after adjustment for baseline -cell function . 
 the demonstration that an agent that reduces diabetes incidence also improves -cell function invites the hypothesis that measures of change in -cell function in response to therapy are indexes of that therapy 's ability to reduce the incidence of diabetes . 
 the modest attenuation of rosiglitazone 's effect after accounting for the change in -cell function suggests that some but not all of the effect of rosiglitazone on diabetes prevention / delay is mediated through its effects on -cell function . whereas a number of previous studies ( 911 ) have documented improvements in -cell function with tzd treatment in people with diabetes , only four studies ( 1215 ) have investigated this question in people with ifg and/or igt . 
 the results of these studies have been inconsistent , with two studies ( 12,15 ) indicating a significant improvement in measures of -cell function with tzd treatment and two studies ( 13,14 ) reporting no change . 
 these studies all had small sample sizes ( n  30 ) , and the methods used to assess -cell function varied widely from intensive approaches ( insulin responses to glucose infusion ) to simple fasting - based indexes ( homa - b ) . 
 interestingly , the complexity of the method used to assess -cell function did not appear to explain the differences in findings of these previous studies ( 12,14 ) . 
 finally , in a diabetes prevention trial among hispanic women with previous gestational diabetes , 70% of whom had igt at enrollment , treatment with troglitazone resulted in significant improvements in the frequently sampled intravenous glucose tolerance 
 our results suggest a significant improvement in -cell function with tzd treatment in pre - diabetic subjects . 
 first , the sample size of our study ( n = 982 ) was much larger that previous investigations . 
 second , we demonstrated improvements in -cell function under tzd treatment using two validated , widely used proxy measures of -cell function , namely igi and pi . 
 third , in our analysis we accounted for the compensatory impact on -cell function of background insulin resistance . specifically , in analyses of igi we used homa - ir either as a covariate in multivariate analysis or as the denominator in the igi  to  homa - ir ratio . 
 accounting for background insulin resistance is crucial to the interpretation of -cell function measures in this study because tzds improved insulin sensitivity , thus reducing pancreatic -cell demand . 
 the exact mechanisms responsible for the increases in igi / ir and the reductions in pi / c documented in the present study are not known , although a number of possibilities exist . the reduction in insulin resistance with tzds 
 would be expected to reduce insulin secretory demand on the -cells and thus reduce -cell stress . 
 in addition , tzds such as rosiglitazone may improve -cell function indirectly by ameliorating a number of pathogenic processes that have been shown to be detrimental to the -cells , including lipotoxicity , glucotoxicity , and inflammation ( 7,8 ) . 
 tzds lower free fatty acids ( 22 ) , elevated levels of which result in excess deposition of lipid within -cells , which in turn leads to increases in ceremide and the stimulation of nitric oxide 
 mediated -cell apoptosis . as well , the glucose - lowering effect of tzds may reduce the impact of reactive oxygen species on the -cells , which are known to be especially susceptible to oxidative damage ( 23 ) . 
 finally , tzds have been demonstrated to reduce levels of inflammatory cytokines ( 24 ) , which may induce -cell apoptosis when chronically elevated . 
 tzds may also impact -cell function directly by maintaining -cell proliferation and/or reducing -cell apoptosis ( 25 ) . in the hope study , 
 treatment with ramipril was shown to reduce the incidence of diabetes in middle - aged individuals with vascular disease . in the dream trial , although ramipril did not significantly reduce the incidence of diabetes in people with ifg and/or igt , it did significantly increase regression to normoglycemia . 
 the mechanism by which ramipril might reduce glucose levels and/or prevent / delay diabetes is unknown , although vascular and metabolic effects on the muscle and pancreatic -cell have been proposed ( 16 ) . 
 the results of the present study suggest that ramipril does not have significant effects on -cell function compared with placebo in people at high risk for diabetes , and thus its glucose - lowering effects may operate through other metabolic mechanisms . 
 the improvement in -cell function in the placebo arm of the ramipril marginal group analysis may be explained by the fact that under the factorial design of the dream trial , half of the participants on ramipril placebo were receiving active rosiglitazone . the major strengths of the present study include the large sample size , the randomized , double - blind design , and the completeness of follow - up ( 92.6% ) . 
 further , participants were thoroughly characterized in terms of glucose tolerance status and were all in the pre - diabetic range ( either iifg , iigt , or ifg+igt ) . 
 the major limitation of the present study is the lack of detailed measures of -cell function , such as those obtained from the hyperglycemic clamp technique or the frequently sampled intravenous glucose tolerance test . 
 notwithstanding , we used two proxy measures of -cell function that have been extensively validated and used in previous studies including the diabetes prevention program and the american diabetes association genetics of type 2 diabetes study ( in the case of igi / ir ) and the iras study ( in the case of pi ) . in conclusion , 
 rosiglitazone , but not ramipril , resulted in significant improvements in measures of -cell function over time . 
 these findings demonstrate that the diabetes preventive effect of rosiglitazone may in part be a consequence of improved -cell function . 
 ","objectivethe objective of this study was to determine the degree to which ramipril and/or rosiglitazone changed -cell function over time among individuals with impaired fasting glucose ( ifg ) and/or impaired glucose tolerance ( igt ) who participated in the diabetes reduction assessment with ramipril and rosiglitazone medication ( dream ) trial , which evaluated whether ramipril and/or rosiglitazone could prevent or delay type 2 diabetes in high - risk individuals.research design and methodsthe present analysis included subjects ( n = 982 ) from dream trial centers in canada who had oral glucose tolerance tests at baseline , after 2 years , and at the end of the study . 
 -cell function was assessed using the fasting proinsulin  to  c - peptide ratio ( pi / c ) and the insulinogenic index ( defined as 300 min insulin/300 min glucose ) divided by homeostasis model assessment of insulin resistance ( insulinogenic index [ igi]/insulin resistance [ ir]).resultssubjects receiving rosiglitazone had a significant increase in igi / ir between baseline and end of study compared with the placebo group ( 25.59 vs. 1.94 , p < 0.0001 ) and a significant decrease in pi / c ( 0.010 vs. 0.006 , p < 0.0001 ) . 
 in contrast , there were no significant changes in igi / ir or pi / c in subjects receiving ramipril compared with placebo ( 11.71 vs. 18.15 , p = 0.89 , and 0.007 vs. 0.008 , p = 0.64 , respectively ) . 
 the impact of rosiglitazone on igi / ir and pi / c was similar within subgroups of isolated igt and ifg + igt ( all p < 0.001 ) . 
 effects were more modest in those with isolated ifg ( igi / ir : 8.95 vs. 2.13 , p = 0.03 ; pi / c : 0.003 vs. 0.001 , p = 0.07).conclusionstreatment with rosiglitazone , but not ramipril , resulted in significant improvements in measures of -cell function over time in pre - diabetic subjects . although the long - term sustainability of these improvements can not be determined from the present study , these findings demonstrate that the diabetes preventive effect of rosiglitazone was in part a consequence of improved -cell function .",pubmed
"microbial communities differ from monocultures in terms of the toxins they degrade ( pelz et al , 1999 ) , the natural products they produce ( pettit , 2009 ) , and even their capacity to grow at all in certain environments ( schink , 2006 ) . 
 a complete understanding of microbial metabolism must extend from the properties of individual strains in pure culture to the combinatorial interactions supported by complex communities . 
 such strains may exhibit naturally complementary metabolism ( kim et al , 2008 ; rozen et al , 2009 ; hillesland and stahl , 2010 ) , or may be genetically engineered to interact ( shou et al , 2007 ; balagadd et al , 2008 ; gore et al , 2009 ) . 
 interacting partners share metabolites , such as hydrogen ( hillesland and stahl , 2010 ) , acetate ( rozen et al , 2009 ) , amino acids ( shou et al , 2007 ) , fixed nitrogen ( kim et al , 2008 ) or glucose ( kim et al , 2008 ; gore et al , 2009 ) . in each case , it has been possible to extrapolate interactive dynamics from the individual characteristics of the participating strains . 
 a general model of metabolic interaction would account for the variety of metabolites that can be exchanged and the genotypic diversity of the strains that exchange them 
 . ideally , such a model would not require detailed biochemical characterization of each individual participant to be predictive . 
 optimization - based techniques of flux balance analysis have been successful in describing metabolic systems ( feist et al , 2007 ) . 
 these approaches draw on the well - characterized biochemical reaction network of organisms such as escherichia coli ( kanehisa and goto , 2000 ) , and can be easily generalized to more exotic species ( feist et al , 2009 ) . using a model set of e. coli auxotrophs , 
 we extend these approaches to the higher - order system of two strains in concourse . 
 we find that certain pairs of auxotrophs will complement one - another 's growth in minimal media by cross - feeding essential metabolites , a relationship we refer to as synthetic mutualism in trans ( smit ) . 
 the smit interactions constitute a class of synthetic genetic interactions because , similar to other synthetic interactions , they reflect and elucidate the genetic and metabolic network from which they derive . 
 we develop a quantitative and predictive theoretical model that reveals the extent to which complex metabolic interactions emerge from the relatively simple topology of the biochemical reaction network . 
 we selected 46 conditionally lethal auxotrophic e. coli from the keio collection ( baba et al , 2006 ) . 
 each deletion blocks the biosynthesis of an essential metabolite , imparting a strict dependency on an external supply of that metabolite for growth . 
 the selected mutations cover a significant portion of metabolic diversity and include genes required for the biosynthesis of amino acids , nucleotides , and cofactors , as well as genes involved in glycolysis and respiration . 
 each deletion strain exhibits robust growth in rich ( lb ) medium but no measurable growth in minimal ( m9 ) medium . 
 strains were labeled individually with plasmids expressing either venus or mcherry fluorescent proteins and co - cultured in minimal media in all pairwise combinations ( box 1 ) . 
 theoretical and empirical precedents have established that metabolic cooperation is enhanced at intermediate cell densities when the concentration of cross - fed metabolites is sufficiently high to improve cooperative fitness ( shou et al , 2007 ; bull and harcombe , 2009 ) . 
 strains were therefore each inoculated at 10 cells per ml , roughly 1/200th saturation density , and fold growth is reported relative to this initial inoculation level . 
 a subset of mixed pairs showed improved growth relative to either corresponding monoculture ( figure 1 ) . in control cultures of self - same pairs , 
 no strain was observed to grow more than eight - fold . however , 17% of dissimilar pairings showed greater than 50-fold growth , indicating metabolic synergy . 
 growth phenotypes were variable , with alternately one strain , both , or neither benefiting from the interaction . 
 for each strain , we were able to identify at least one partner with which synergistic growth was evident . 
 conversely , no strain exhibited a universally cooperative phenotype , either in the role of nutrient donor or recipient . 
 we first explored how the growth of a given strain relates to its level of cooperation , as inferred from the growth of its partner strain . cooperating strains in this system 
 may show enhanced proliferation through the mechanism of invested benefits ( connor , 1995 ; west et al , 2007 ) . 
 cooperation on the part of strain a will augment a 's growth only if b cooperates in turn . by increasing b 's abundance , 
 a then cultivates the source of a 's own metabolites and therefore a 's own growth . 
 given the relatively low growth rates of the co - cultured strains and the excess availability of nutrients , we do not expect a significant metabolic burden to be associated with the production of shared metabolites . 
 however , we do expect a limit to the invested benefits of cooperative growth . specifically 
 , this should arise when oversharing on the part of a results in b monopolizing the batch culture and therefore necessarily limiting a. the data reflect a significant growth benefit for cooperating strains . 
 we record an overall positive correlation between the growth of strains a and b ( spearman 's =0.2 , p - value : < 10 ) . 
 this correlation was highest when the total growth of a and b was low to moderate . 
 we could also observe a characteristic anti - correlation between the growth of a and b in cultures that approach saturation , when more growth of one strain tended to impede the other ( figure 2a ) . 
 we assign cooperation levels , ca and cb , to quantify cooperation on the part of strain a and b , respectively . 
 growth was allowed to proceed for a fixed and finite amount of time , so our system does not necessarily approach a steady state . 
 although analytical solutions to ( 1 ) are not available , numerically solving the system of two equations for the two variables ca and cb transforms the growth data into biologically relevant parameters that reflect the level of cooperation evidenced by each strain . 
 the measured growth of a increases monotonically with respect to the derived value of cb , indicating that it is always beneficial to be partnered with a cooperating strain . 
 a characteristically also increases with ca for low ca , as strain a reaps the invested benefits of cultivating strain b. however , a declines for high ca , when excess cooperation allows b to dominate the available carrying capacity ( figure 2b ) . 
 we sought to relate the various observed cooperation phenotypes to the properties of individual mutations in the global context of e. coli metabolism . 
 synthetic genetic interactions , the synergistic growth effects of multiple gene deletions , are known to identify epistatic or modular relationships among genes ( segr et al , 2005 ; ooi et al , 2006 ; motter et al , 2008 ) . 
 previous studies emphasize the phenotypic consequences of multiple genetic deletions within the genome of a single strain . 
 we define a new synthetic relationship , the synergistic growth between two otherwise lethal mutants , as smit . similar to other synthetic interactions , smit interactions can be applied to infer properties of the e. coli metabolic network . 
 pairings of strains with metabolic blocks in the same biosynthetic pathway grew less than pairings of strains blocked in distinct pathways . 
 approximately 3.6% of pathway - matched pairs showed growth above 50-fold against 18% of pathway - distinct pairs ( n=55 , n=980 , respectively , p - value : 2  10 ) . 
 however , mutants sharing pathways showed consistency in their set of cooperating partners ( supplementary figure 1 ) . 
 we constructed for each strain a cooperation profile vector consisting of the measured growth of that strain and that of its partner strain in each of the 46 co - cultures . 
 correlations of the cooperation profiles therefore indicate the overall similarity of the cooperative behavior between two strains . 
 we applied an unsupervised hierarchical clustering algorithm to the set of cooperation profiles , using the spearman rank correlation between vectors as a distance metric and a complete linkage function . 
 mutants in the biosynthetic pathways of tryptophan , guanine , histidine , purine , pyramidine , arginine , cysteine , leucine , and pantothenate were recovered as distinct branches in the resulting dendrogram . 
 analysis of smit interactions is therefore capable of identifying biosynthetic modules ( figure 3a ) . 
 more distal correspondence in the metabolic network can also be detected in the analysis of smit interactions . 
 for each pair of interacting mutants , we define a shortest path through the metabolic network connecting the two reactions . 
 the correlation between the cooperation profiles characterizing any two strains declines with the distance spanning their mutations ( =0.34 , p - value : < 10 ) . 
 this suggests that , to a significant extent , the complex emergent phenotype of smit growth is governed by the relatively simple stoichiometries of the metabolic reaction network ( figure 3b ) . 
 metabolic network stoichiometries can be applied to infer biochemical reaction rates through the method of flux balance analysis ( fba ; palsson , 2006 ) . 
 we extend fba modeling from a single e. coli to a system of two interacting strains . beginning with the standard iaf1260 reconstruction ( feist et al , 2007 ) , the stoichiometric matrices and flux bounds for the two strains 
 are concatenated appropriately to produce the joint model , allowing the free exchange of metabolites between the two mutants ( see materials and methods ) . 
 we derived solutions to the joint model by applying the minimization of metabolic adjustment ( moma ) objective function ( segr et al , 2002 ) . 
 the moma methods hypothesize that mutant systems will tend to approximate the wild - type flux distribution , even as mutation precludes an exact match . 
 this objective function is conservative ; it identifies solutions in the joint vector closest , in the euclidean sense , to the wild - type fluxes . 
 total growth of the co - cultures was correlated with predicted growth derived from the joint flux model . 
 we calculated a rank correlation of 0.42 across predictions for all pairs ( p - value : < 10 ) . 
 this model performed less well in comparisons of the subset of pairs with mutations in the nucleotide biosynthesis pathway ( =0.17 , p - value : 0.13 , n=78 ) , and better when both mutants were involved in amino - acid production ( =0.4 , p - value : < 10 , n=378 ) , or in neither of those classes ( =0.72 , p - value : 0.02 , n=10 ) . 
 the joint flux model was effective in reproducing our observation that mutants in shared pathways generally do not supplement one another 's growth ( supplementary figure 2 ) . 
 in general , growth predictions from the joint flux model were higher than those measured . 
 this might be explained by the relatively high levels of cooperation implicit in the objective function . in selecting an optimal joint flux vector , 
 fluxes are weighted equally in their contribution to minimizing the metabolic adjustment of their own strain and of the partner strain . 
 this is theoretically unlike the situation in vivo , in which any flux optimality is expected only within a given strain , and any benefits to the partner strain are only as a byproduct . 
 indeed , our system has no co - evolutionary history and has not been optimized by natural selection in any sense . 
 we reasoned that the likelihood of a given metabolite being shared should depend on the growth value of that metabolite to both the donor and recipient strain . 
 metabolites less valuable to the donor might be more readily shared , and metabolites more valuable to the recipient should effect more growth . in an efficient cooperative interaction , each strain would exchange growth - cheap metabolites for growth - dear metabolites ( boucher , 1988 ) . 
 we can derive explicit predictions for the value of each exchanged metabolite for each mutant strain in our system as shadow prices . 
 a shadow price , in a linear programming model , is defined as the marginal change in the objective function associated with the strengthening or loosening of a particular model constraint . as in standard flux balance models , calculating shadow prices requires only the reaction network stoichiometries and the measured composition of biomass . 
 we solved the iaf1260 model for shadow prices representing the fitness benefit , b , to each strain a in taking up a unit of its required metabolite . 
 for example , a tryptophan auxotroph is associated with a high benefit term because a unit of the rare amino acid tryptophan is sufficient to produce a relatively high amount of biomass . 
 similarly , we derive a cost term , p , representing the loss in growth rate for each strain , b , in secreting a given metabolite . the calculated costs and benefits produced the expected correlation with measured growth . the benefit associated with a metabolite b 
 was positively correlated with the growth of strain a ( =0.11 , p - value : 10 ) , but showed no correlation with the growth of the partner strain . 
 conversely , the production cost of metabolites p , incurred by strain b , was negatively correlated with a 's growth ( =0.26 , p - value:<10 ) but was uncorrelated with b 's growth . 
 thus , strains tended to show the most growth when predicted to require only small quantities of metabolites that were cheap for their partner to produce . 
 we define the predicted efficiency of cooperation ,  , as the ratio b / p . 
 we observed that  was more predictive than either b or p alone in determining the growth of strain a (  : 0.31 , p - value : < 10 ) . 
 we also determined an overall positive correlation between the growth of strain a and  , the efficiency term as calculated for the partner strain , consistent with the invested benefits a receives from the growth of b (  : 0.14 , p - value : 10 ) . 
 indeed , in the regime of low a growth ,  was more strongly correlated with a 's growth than  , emphasizing the strong fitness advantage for efficient cooperators . 
 the cooperation efficiency terms reproduce the characteristic pattern in their correlation with strain growth , predicted in our invested benefits model of cooperation ( figure 3c ) . 
 the term  is positively correlated with a 's growth across all growth levels , as it is always beneficial to be paired with an efficient cooperator . 
 the  correlates positively to a 's growth for low growth , then negatively as the strains approach saturation . only in this narrow range would the less generous show more growth . 
 cooperation efficiencies derive purely from stoichiometric information , and so can be calculated for strains without detailed biochemical characterization . 
 they are independent of the specific physiological mechanisms that effect metabolite production , secretion , and exchange , which in the case of this system remain to be described . 
 intracellular metabolites represent valuable resources , and should therefore be retained by cell membranes in the absence of a specific mechanism for secreting them . 
 such reasoning can explain the behavior of most co - cultures , in which essential metabolites are retained and no cross - feeding occurs . 
 e. coli are known to secrete a number of amino acids , particularly when starved or otherwise stressed ( burkovski et al , 1995 ; kaderbhai et al , 2003 ; valle et al , 2008 ) . 
 alternately , the intracellular metabolite pool also undergoes significant changes during starvation ( tweeddale et al , 1998 ) . 
 the results presented here reflect only cooperation before co - evolution , and do not reveal the evolutionarily stable steady state that may arise for cross - feeding organisms . 
 to persist in natural systems , cooperative behavior must be robust to variations of environment , and to the emergence of exploitative mutants . 
 many natural syntrophic systems seem to be cases of byproduct cooperation , in which one strain feeds on the waste products of another ( marx , 2009 ; rozen et al , 2009 ) . 
 we find that the metabolites most readily exchanged are those of little value to the secreting strain . 
 this is consistent with proposals that byproduct cooperation evolves more readily than other forms , because it requires no active investment of one partner in the other ( connor , 1995 ) . 
 the fitness costs and benefits of biological interactions are often difficult or impossible to quantify ( boucher , 1988 ) , but are required for the application of population dynamic models ( nowak , 2006 ) . 
 the a priori prediction of such parameters in an experimentally tractable model organism brings a new degree of quantitative resolution to theories of metabolic interaction . 
 deletion strains from the keio collection were grown to saturation in rich ( lb ) medium with appropriate antibiotics , washed twice in pbs , and re - suspended in m9 minimal medium at a 1:100 dilution ( 6.8 g / l na2hpo4 , 3 g / l kh2po4 , 0.5 g / l nacl , 1 g / l nh4cl , 20 g / l glucose , 100 m cacl2 , 1 mm mgso4 , 50 g / l ampicillin , and 100 g / l kanamycin ) . 
 strains were cultured overnight in minimal medium to allow depletion of residual nutrients , then re - diluted to the initial 1:100 level . 
 viable cell counts were determined by plating for all strains both before and after starvation , and were observed to be linear with od and similar for all strains . 
 this indicated that total cell density could be accurately measured by od , despite the unique genetic background and metabolic conditions of each co - culture . 
 growth was measured by optical density at 600 nm using a perkin elmer envision plate reader . 
 cell density for a strain a was calculated as (odmodbkg)afrac , where odm is the measured od , odbkg was the background absorbance of blank medium ,  is the linear scaling factor from od to cell density , calculated by plating standard curves , and afrac is the proportion of strain a as measured by flow cytometry . 
 growth experiments were conducted in duplicate and with label - switched pairs , a total of four replicates for each of 1035 possible experimental pairs . 
 growth measurements were reproducible in both biological replicates ( =0.92 ) and label - switched replicates ( =0.9 ) . following data acquisition , a random sample of 50 growing mixed cultures were struck out on rich medium and minimal medium . in no case were single colonies observed to grow on minimal plates , indicating that observed growth was not due to the restoration of prototrophy in a single lineage from any genetic event . 
 metabolic network distances were calculated excluding metabolites with very high connectivities ( > 20 reactions ) , because of their effect of radically shrinking the network radius . a standard metabolic optimization problem with a moma - type objective function identifies a flux vector j in the mutant flux space , which minimizes the euclidean distance to the wild - type flux vector . 
 thus , it satisfies the following : where j denotes a wild - type flux vector , derived from a standard fba formulation ( segr et al , 2002 ) . 
 bounds on the exchange fluxes , corresponding to the media conditions , are set to u and l . 
 finally , js and jx are the subsets of internal and exchange fluxes of j. for the joint problem , we concatenate the flux vectors , bounds , and stoichiometric matrices appropriately to define a joint flux space , thus solving : superscripts a and b denote fluxes , matrices and bounds associated with strain a or b. the exchange matrix , x , is identical for all mutants . 
 the third row of the joint stoichiometric matrix allows the exchange of metabolites between strains . 
 the upper bounds of the exchange reactions , u , are modified to allow the uptake of all shared metabolites . 
 the term 2xu transposes the unmodified bounds from the individual fluxes on problem 2 to apply to the sum of the joint fluxes , maintaining conservation of mass between the two strains . 
 the mean marginal production cost of a metabolite m , pm , we define as the reciprocal of the solution to a linear programming problem : this represents the minimum flux value , that is , the maximum secretion level , which can be attained consistent with a growth rate of at least 90% wild type . 
 the reciprocal of this solution , 1/jm , is proportional to the mean shadow price associated with the bound on the exchange flux of metabolite m , jm , taken over the interval 10090% of wild - type growth rate . 
 we allow pm to take the value of infinity for those cases when jm=0 , meaning a given metabolite is stoichiometrically blocked from being secreted and is therefore not available at any cost . 
 the mean marginal benefit , bm , which a given strain ,  , receives from a metabolite , m , is the reciprocal of the solution to a similar problem : note that although secretion costs are calculated with respect to the wild - type strain background , uptake benefits are defined with respect to a specific mutant strain that can not grow without supplemented metabolites . 
 problem 4 identifies the minimum supplemental flux consistent with growth of at least 10% of the wild - type level . 
 most metabolites are not capable of rescuing growth of most mutant strains , therefore the solution to problem 4 is undefined and we allow bm=0 . otherwise   . as above , 
 the benefit is proportional to the mean shadow price for the uptake bound on metabolite m over the interval 010% of wild - type growth rate . in cases , 
 in which pm and bm are defined for more than one m , because strain a can be rescued by more than one metabolite , we take the values p and b to be the mean values over all m for which they are defined . 
 the efficiency of cooperation for a strain b ,  , for a strain b as the ratio b / p . in cases , in which a can be rescued by more than one metabolite , we take  to be the mean ratio for all such metabolites . 
 deletion strains from the keio collection were grown to saturation in rich ( lb ) medium with appropriate antibiotics , washed twice in pbs , and re - suspended in m9 minimal medium at a 1:100 dilution ( 6.8 g / l na2hpo4 , 3 g / l kh2po4 , 0.5 g / l nacl , 1 g / l nh4cl , 20 g / l glucose , 100 m cacl2 , 1 mm mgso4 , 50 g / l ampicillin , and 100 g / l kanamycin ) . 
 strains were cultured overnight in minimal medium to allow depletion of residual nutrients , then re - diluted to the initial 1:100 level . 
 viable cell counts were determined by plating for all strains both before and after starvation , and were observed to be linear with od and similar for all strains . 
 this indicated that total cell density could be accurately measured by od , despite the unique genetic background and metabolic conditions of each co - culture . 
 growth was measured by optical density at 600 nm using a perkin elmer envision plate reader . 
 cell density for a strain a was calculated as (odmodbkg)afrac , where odm is the measured od , odbkg was the background absorbance of blank medium ,  is the linear scaling factor from od to cell density , calculated by plating standard curves , and afrac is the proportion of strain a as measured by flow cytometry . 
 growth experiments were conducted in duplicate and with label - switched pairs , a total of four replicates for each of 1035 possible experimental pairs . 
 growth measurements were reproducible in both biological replicates ( =0.92 ) and label - switched replicates ( =0.9 ) . following data acquisition , a random sample of 50 growing mixed cultures were struck out on rich medium and minimal medium . in no case were single colonies observed to grow on minimal plates , indicating that observed growth was not due to the restoration of prototrophy in a single lineage from any genetic event . 
 metabolic network distances were calculated excluding metabolites with very high connectivities ( > 20 reactions ) , because of their effect of radically shrinking the network radius . 
 a standard metabolic optimization problem with a moma - type objective function identifies a flux vector j in the mutant flux space , which minimizes the euclidean distance to the wild - type flux vector . 
 thus , it satisfies the following : where j denotes a wild - type flux vector , derived from a standard fba formulation ( segr et al , 2002 ) . 
 bounds on the exchange fluxes , corresponding to the media conditions , are set to u and l . 
 finally , js and jx are the subsets of internal and exchange fluxes of j. for the joint problem , we concatenate the flux vectors , bounds , and stoichiometric matrices appropriately to define a joint flux space , thus solving : superscripts a and b denote fluxes , matrices and bounds associated with strain a or b. the exchange matrix , x , is identical for all mutants . 
 the third row of the joint stoichiometric matrix allows the exchange of metabolites between strains . 
 the upper bounds of the exchange reactions , u , are modified to allow the uptake of all shared metabolites . 
 the term 2xu transposes the unmodified bounds from the individual fluxes on problem 2 to apply to the sum of the joint fluxes , maintaining conservation of mass between the two strains . 
 the mean marginal production cost of a metabolite m , pm , we define as the reciprocal of the solution to a linear programming problem : this represents the minimum flux value , that is , the maximum secretion level , which can be attained consistent with a growth rate of at least 90% wild type . 
 the reciprocal of this solution , 1/jm , is proportional to the mean shadow price associated with the bound on the exchange flux of metabolite m , jm , taken over the interval 10090% of wild - type growth rate . 
 we allow pm to take the value of infinity for those cases when jm=0 , meaning a given metabolite is stoichiometrically blocked from being secreted and is therefore not available at any cost . 
 the mean marginal benefit , bm , which a given strain ,  , receives from a metabolite , m , is the reciprocal of the solution to a similar problem : note that although secretion costs are calculated with respect to the wild - type strain background , uptake benefits are defined with respect to a specific mutant strain that can not grow without supplemented metabolites . 
 problem 4 identifies the minimum supplemental flux consistent with growth of at least 10% of the wild - type level . 
 most metabolites are not capable of rescuing growth of most mutant strains , therefore the solution to problem 4 is undefined and we allow bm=0 . otherwise   . as 
 above , the benefit is proportional to the mean shadow price for the uptake bound on metabolite m over the interval 010% of wild - type growth rate . in cases , 
 in which pm and bm are defined for more than one m , because strain a can be rescued by more than one metabolite , we take the values p and b to be the mean values over all m for which they are defined . 
 the efficiency of cooperation for a strain b ,  , for a strain b as the ratio b / p . in cases , in which a can be rescued by more than one metabolite , we take  to be the mean ratio for all such metabolites . 
 ","mixed microbial communities exhibit emergent biochemical properties not found in clonal monocultures . 
 we report a new type of synthetic genetic interaction , synthetic mutualism in trans ( smit ) , in which certain pairs of auxotrophic escherichia coli mutants complement one another 's growth by cross - feeding essential metabolites . 
 we find significant metabolic synergy in 17% of 1035 such pairs tested , with smit partners identified throughout the metabolic network . 
 cooperative phenotypes show more growth on average by aiding the proliferation of their conjugate partner , thereby expanding the source of their own essential metabolites . we construct a quantitative , predictive , framework for describing smit interactions as governed by stoichiometric models of the metabolic networks of the interacting strains .",pubmed
"brook - spiegler syndrome ( bss ) is a rare genodermatosis characterized by the progressive formation of adnexal skin tumors in the scalp and face , mainly trichoepiteliomas , cylindromas and spiradenomas . 
 it is due to mutations in the tumor suppressor gene cyld ( cylindromatosis gene ) on chromosome 16q12q13 . around 93 mutations 
 the study of cyld gene in patients and their relatives can provide the molecular diagnosis and appropriate genetic counselling . 
 there is a low risk of malignancy and patients require long - term follow - up . 
 brooke  spiegler syndrome ( bss ) is a rare genodermatosis due to mutations in the tumor suppressor gene cylindromatosis ( cyld gene ) on chromosome 16q12q13 . 
 there is a low risk of malignancy and patients require long - term follow - up . 
 a 71-year - old woman was evaluated due to the progressive development of asymptomatic lesions in her head during the last 4 years . 
 the physical examination showed multiple rounded skin - colored or erythematous papules , 28 mm in diameter , located in scalp and forehead [ figure 1 ] . 
 we performed several excisional biopsies and the histopathological diagnosis was spiradenoma in all of the lesions , except for one cylindroma . 
 multiple rounded skin - colored or erythematous papules in scalp and forehead , compatible with spiradenomas and cylindromas regarding her family history , she referred the presence of lesions at the scalp and forehead in her mother , her mother 's brother , and two of her three daughters , some of them very similar to our patient s . 
 next , we performed a physical examination of her two affected daughters showing multiple papules and nodules of 13 cm , some of them painful [ figures 2 and 3 ] . 
 once all the information was collected , we elaborated the family pedigree , which suggested an autosomal dominant inheritance pattern of the disease [ figure 4a ] . above : rounded pink nodule with superficial telangiectasias at the scalp of one of the patient 's daughters . 
 below : multiple skin - colored or yellowish millimetric papules at the forehead and periocular area , suggesting multiple trichoepitheliomas multiple firm pink nodules of variable size in scalp of the second daughter ( a ) family pedigree . 
 ( b ) exon 8-cylindromatosis gene sequencing that shows ( a ) heterozygous mutation c. 1628_1629delct in our patient and her two affected daughters . 
 ( b ) wild - type genotype in the unaffected daughter suspecting a genetic syndrome , we requested the study of the cyld gene in our patient and her two affected offsprings . 
 the exon 8 sequencing of the cyld gene revealed the heterozygous mutation c. 1628_1629delct [ figure 4b ] . 
 it consists of a two base - pair deletion that originates a new frameshift , the first codon is now the stop codon p. ser543xfs*1 . 
 therefore , to complete the familiar study , we performed the genetic test on our patient 's daughter presenting no signs or symptoms suggesting bss . 
 the result was negative , showing the wild - type genotype [ figure 4b ] . 
 her 11 and 5-year - old children did not present any clinical data either . finally , we offered genetic counseling to her first - grade relatives . 
 we provided concise information about the main characteristics of sbs and the 50% probability of the descendants to develop this syndrome , due to its autosomal dominant inheritance . 
 it is characterized by the progressive development of adnexal skin tumors in scalp and face . 
 clinical manifestations start since puberty and its incidence is higher in females without distinction of race . 
 bss has also been associated with increased risk of salivary glands neoplasms and other tumors although with very little evidence . 
 cylindromas appear at the scalp reaching a considerable size , often forming the  turban tumor  . 
 spiradenomas are also multiple and appear in extremities , trunk and less frequently in the scalp ( this is a rare location of single sporadic spiradenomas ) . 
 recent studies demonstrate a common apocrine origin of spiradenomas and cylindromas , with an expression of hair follicle stem cells specific markers , mainly cd200 and cytokeratin . 
 thus , we prefer to avoid the adjective  eccrine  traditionally applied to spiradenomas . 
 this syndrome is due to mutations in the tumor suppressor gene cyld localized on chromosome 16q12q13 . 
 it is a tumor suppressor gene that codifies a protein of the deubiquitinating enzymes family . 
 it negatively regulates activation of factors such as nuclear factor - kappa - b , jnk , and wnt , involved in inflammation and cellular division . 
 the loss of deubiquitinating activity of cyld correlates with tumorigenesis ( apoptosis resistance , uncontrolled cell division ) . 
 germ - line mutations predispose to the disease , and sometimes , a second acquired somatic mutation determines the final phenotype . around 93 different mutations 
 its phenotypic expression has been found so far . the genetic study in our case revealed the existence of the heterozygotic mutation c. 1628_1629delct . 
 there is only one report of this mutation in 2008 found in a family with multiple trichoepitheliomas ; therefore , our case confirm the association of this genetic mutation with bss . 
 two entities have been recognized as phenotypic variations of bss ( mim#605041 ) due to mutations in the cyld gene . 
 multiple familial trichoepithelioma , also known as trichoepithelioma adenoides cystica ( mim#601606 ) , presents with numerous trichoepitheliomas since puberty predominantly located in the face . in familial cyld ( mim#132700 ) 
 , multiple cylindromas appear in face and scalp of the patients . on the other hand , 
 there have been described some cases of multiple familial spiradenomas , predominantly located in the scalp . 
 no genetic study was performed in these cases ; therefore , it could not be confirmed the existence of brss or one phenotypic variation . 
 alarm signs are ulceration , bleeding , fast growth , blue nodules , or pain . 
 malignant spiradenoma is a very aggressive neoplasm with a risk of metastasis around 50% and a mortality of 39% if left untreated . 
 we present a new case of bss in a family due to the heterozygous cyld mutation c. 1628_1629delct . 
 particularities of this case were the late onset in our patient and the relatively low number of cylindromas and trichoepitheliomas found in comparison with the cases found in the literature . 
 thus , our case may support the hypothesis about the influence of the concrete germline mutation and the variable presence of a somatic mutation in the development of the final phenotype . 
 for this reason , it is imperative to perform a correct diagnosis and periodic follow - up of the patients . 
 in addition , the genetic study should be considered individually with the possibility of genetic counseling to the family members . 
 the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) 
 has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . 
 the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . 
 we describe a new case of bss due to the rare heterozygous cyld gene mutation c.1628_1629delct in three women of the same family . 
 the particularities of the clinical manifestations ( late onset , relatively low number of tumors ) may support the hypothesis about the relationship between the germline mutation , variable presence of somatic mutation and the final phenotype . 
 the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) 
 has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . 
 the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . 
 we describe a new case of bss due to the rare heterozygous cyld gene mutation c.1628_1629delct in three women of the same family . 
 the particularities of the clinical manifestations ( late onset , relatively low number of tumors ) may support the hypothesis about the relationship between the germline mutation , variable presence of somatic mutation and the final phenotype . 
 the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) 
 has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . 
 the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . 
  
 we describe a new case of bss due to the rare heterozygous cyld gene mutation c.1628_1629delct in three women of the same family . 
 the particularities of the clinical manifestations ( late onset , relatively low number of tumors ) may support the hypothesis about the relationship between the germline mutation , variable presence of somatic mutation and the final phenotype .","brooke  spiegler syndrome ( bss ) is a rare genodermatosis characterized by the progressive formation of adnexal skin tumors in the scalp and face , mainly trichoepitheliomas , cylindromas , and spiradenomas . 
 it has also been associated with salivary glands neoplasms . 
 it is due to mutations in the tumor suppressor gene cylindromatosis ( cyld gene ) localized on chromosome 16q12q13 . around 93 mutations 
 have been described . 
 the study of cyld gene in patients and their relatives is of vital importance to establish the molecular diagnosis and offer appropriate genetic counseling . 
 there is a low risk of malignancy and patients require long - term follow - up . a case of bss in a family 
 is described . 
 the existence of the genetic mutation at the cyld gene c. 1628_1629delct in three of the women affected was demonstrated . 
 this mutation has only been described once in a previous study .",pubmed
"biological scaffold materials derived from dermis , pericardium , and small intestine submucosa of human , bovine , and porcine origin have been utilized over the last decade for soft tissue repair applications such as hernia repair , breast reconstruction , staple - line reinforcement , and orthopedic applications . 
 these scaffold materials are particularly useful in clean - contaminated or contaminated settings due to their rapid revascularization and clearance of bacteria [ 5 , 6 ] . 
 permanent , synthetic polymer mesh materials are not typically utilized in these settings due to risk of infection . 
 biological scaffolds are also utilized as an alternative to fascial closure when there is excessive tension on the wound , when tissue loss makes closure especially difficult , or in  damage - control/abdominal compartment settings in which the abdomen must be left open until the patient is stabilized . in these settings , scaffolds are utilized to protect the abdominal contents , typically until granulation of the wound occurs and a split - thickness skin graft can be applied . 
 although biological scaffolds present many attractive advantages , these materials are also extremely expensive , exhibit substantial scaffold variability , and provoke patient - specific immunological responses . 
 early designs of absorbable polymer scaffolds included materials such as dexon ( covidien , mansfield , ma ) and vicryl ( ethicon , inc . , 
 dexon is comprised of polyglycolic acid ( pga ) , and it degrades in vivo via hydrolysis . 
 full resorption of the pga is expected to be complete in approximately 6090 days ( 2 - 3 months ) . 
 animal studies have shown that dexon is quickly resorbed and associated with hernia formation after just 14 days due to central mesh failure . by 
 90 days , greater than 80% of the scaffolds were fully resorbed in that particular animal study . 
 clinical studies have shown that dexon has primarily been utilized in damage control situations to cover the open abdomen until granulation tissue is present and a skin graft can be applied . 
 however , subsequent development of a ventral hernia has also been observed in these patients . in one study , 
 the authors concluded that the dexon mesh was useful for providing a temporary support for the abdominal wall but would likely require a ventral hernia repair with placement of a permanent mesh once the contamination was resolved , making this material a less than ideal long - term solution . 
 vicryl is a copolymer of glycolide and lactide which also degrades in vivo through hydrolysis . 
 according to its instructions for use , vicryl loses 77% of its strength in the first 14 days ( 0.5 months ) in a rat model , and it is fully resorbed in approximately 6090 days ( 2 - 3 months ) . 
 clinically , vicryl has been utilized primarily in damage - control applications as a buttress to the abdominal wall   or as a prophylactic measure to prevent incisional hernia formation . 
 animal studies have shown that the tensile strength of the rat abdominal wall at 30 and 60 days is equivalent whether vicryl mesh or biological scaffolds were utilized ( surgisis and alloderm ) . however , collagen deposition and neovascularization were greater for the biological scaffolds than for vicryl . 
 this could be explained by the decreased ph and associated increase in inflammation observed at the wound site as the vicryl mesh degrades . 
 pronounced inflammation , reduced tissue ingrowth , and greater angiogenesis were observed for vicryl mesh compared to permanent prolene and ultrapro meshes in another animal study involving a hamster model . 
 the authors of that study concluded that the more aggressive foreign body response incited by the resorbable vicryl mesh did not lead to better tissue incorporation as predicted . in a similar study , 
 tissue ingrowth was also unsatisfactory for the absorbable vicryl mesh when compared to permanent synthetic materials such as polypropylene and expanded polytetrafluoroethylene in a rabbit model . 
 overall , both dexon and vicryl scaffolds lose mechanical strength and are resorbed fairly quickly , making them less than ideal for hernia repair applications which require more long - term support of the repair site until tissue remodeling is complete . 
 thus , more recent absorbable scaffold designs have been developed which utilize long - lasting polymers that degrade more slowly . 
 scaffolds such as gore bio - a ( w.l . gore and associates , inc . , flagstaff , az ) , tigr matrix ( novus scientific , uppsala , sweden ) , and phasix mesh ( c. r. bard , inc./davol inc . , 
 gore bio - a is a copolymer of poly(glycolide : trimethylene carbonate ) that degrades in vivo through both hydrolytic and enzymatic mechanisms and is fully resorbed within approximately 180 days ( 6 months ) according to the instructions for use . in a rat model with methicillin - resistant staphylococcus aureus ( mrsa ) contamination , 
 bacteria were cleared from the gore bio - a mesh more effectively than either vicryl or tigr matrix at the 10 inoculum . 
 overall , all three scaffolds exhibited reduced tensile strength and increased rate of mesh failure regardless of scaffold composition . 
 gore bio - a has also been utilized in a number of clinical applications including amyand hernia repair , open elective hernia repair , paraesophageal / hiatal hernia repair , suture line reinforcement , pelvic floor reinforcement , and breast reconstruction . 
 the outcomes thus far have been promising with low rates of recurrence , infection , and pain . however , the majority of these studies have been case reports or very small series ( i.e. , less than 10 patients ) . 
 larger , more comprehensive clinical trials are needed to fully understand the long - term capabilities of this scaffold . 
 tigr matrix is knitted from two fibers having different resorption rates . according to the instructions for use 
 , the first fiber makes up approximately 40% of the overall mesh by weight and is a copolymer of polyglycolide , polylactide , and polytrimethylene carbonate . 
 this fiber degrades in vivo through hydrolysis , loses substantial mechanical strength in the first 14 days ( 0.5 months ) , and is fully resorbed in approximately 120 days ( 4 months ) . 
 the second fiber makes up approximately 60% of the overall mesh by weight and is a copolymer of polylactide and polytrimethylene carbonate . 
 this fiber also degrades in vivo through hydrolysis , but it retains its mechanical strength longer than the first fiber . 
 it begins to demonstrate loss of mechanical strength after approximately 270 days ( 9 months ) and is fully resorbed in approximately 1095 days ( 36 months ) . 
 tigr matrix has been evaluated in a long - term animal model , and a clinical trial is currently underway . in the animal study , 
 tigr matrix was compared to permanent polypropylene mesh in sheep with full thickness abdominal wall defects over the course of 4 , 9 , 15 , 24 , and 36 months . 
 the results showed a typical long - term inflammatory response to the permanent polypropylene contrasted with a gradual resorption of the tigr matrix until it was fully resorbed at 36 months . 
 the tigr matrix also exhibited collagen deposition at the repair site that increased over time and eventually resembled native connective tissue . in the clinical trial , 
 forty subjects were enrolled ( n = 40 ) and followed for 1 year after placement of tigr matrix to repair a primary inguinal hernia . 
 pain and recurrence were evaluated at 0.5 , 1 , 3 , 6 , and 12 months , and pain scores were reduced from an average of 17.4 before surgery to 0.3 after just 6 months . 
 p4hb is a natural polymer from the class of polyhydroxyalkanoates . in nature , these polymers are produced by microorganisms for the purpose of regulating energy metabolism . in the case of phasix mesh , p4hb is a naturally derived , fully absorbable polymer produced by escherichia coli k12 bacteria via transgenic fermentation techniques . 
 p4hb has a chemical structure very similar to many of the synthetic polyester polymers , but because it is biologically derived rather than chemically synthesized , p4hb does not contain any residues from metal catalysts that are typically utilized during chemical synthesis of other polyester polymers . 
 p4hb degrades in vivo through both hydrolysis and a hydrolytic enzymatic digestive process and is fully resorbed in approximately 365545 days ( 1218 months ) according to the instructions for use . 
 the resulting by - products ( carbon dioxide and water ) are metabolized very quickly via the krebs cycle and beta - oxidation . unlike absorbable scaffolds such as dexon and vicryl , 
 whose by - products decrease the ph at the wound site , degradation of p4hb is not as acidic , which may reduce the inflammatory response associated with these materials . 
 in addition , p4hb has been shown to degrade more slowly than pga , yielding more gradual loss of mechanical strength . 
 this is advantageous in applications such as hernia repair in which the rate of degradation should ideally match the rate of remodeling and neotissue deposition at the repair site . 
 a gradual change in mechanical properties is also advantageous because it leads to a gradual transfer of the load from the scaffold back to the tissue , which may help to prevent hernia recurrence . 
 p4hb has been evaluated in a number of animal studies over the past decade , particularly those investigating cardiovascular applications such as tissue engineered trileaflet heart valves , artery augmentation patches , and small diameter vascular grafts , as well as development of p4hb as a suture material . however , this is the first study to evaluate this material specifically for hernia repair applications . 
 thus , the purpose of this study was to determine the mechanical properties , resorption profile , and histological characteristics of phasix mesh and p4hb plug compared to the native abdominal wall when utilized to bridge a surgical defect in a porcine model . 
 phasix mesh is comprised of a fully resorbable polymer monofilament ( poly-4-hydroxybutyrate , p4hb ) that is knitted into a flat sheet configuration as shown in figure 1(a ) . 
 the p4hb plug design is also comprised of p4hb monofilament , but it is preformed into a three - dimensional shape with a fluted outer layer and inner layers or  petals  attached at the tip as shown in figure 1(b ) . 
 twelve specimens ( n = 12 ) measuring 2.5  5.1  cm ( 1  2  in ) were prepared from phasix mesh and subjected to suture retention testing at time zero , t0 ( i.e. , prior to implantation ) . 
 six of the specimens ( n = 6 ) were oriented such that the load applied during testing was parallel to the longest dimension of the mesh interstices . 
 the other six ( n = 6 ) specimens were oriented such that the load applied during testing was perpendicular to the longest dimension of the mesh interstices . 
 a custom test fixture was utilized in which the mesh specimen was loaded vertically in the instron machine ( instron , norwood , ma ) with a gauge length of 2.5  cm ( 1  in ) and clamped along the upper edge using pneumatic grips set to 60  psi . 
 a stainless steel wire with a diameter of 0.36  mm ( simulating 0 polypropylene suture material ) was passed through the mesh 1.0  cm from the bottom edge of the mesh . 
 each specimen was tested in tension at a rate of 300  mm / min ( 12  in / min ) until the suture pulled through the mesh . 
 the  suture retention strength  was recorded as the maximum load sustained by the mesh in units of newtons ( n ) . 
 tear resistance testing was based on the american society for testing and materials ( astm ) specification # d2261 - 07a . for this type of testing , twelve mesh specimens ( n = 12 ) measuring 2.5  7.6  cm ( 1  3  in ) were prepared from phasix mesh and subjected to tear resistance testing at time zero , t0 ( i.e. , prior to implantation ) . a 2.5  cm ( 1  in ) slit was cut from the center of the 2.5  cm edge of the specimen toward the center of the mesh to form two tabs . 
 the left tab was clamped in the upper grip of the instron machine using a pneumatic grip set to 60  psi , and the right tab was clamped in an identical fashion in the lower grip . 
 six of the specimens ( n = 6 ) were oriented such that the load applied during testing was parallel to the longest dimension of the mesh interstices . 
 conversely , the other six ( n = 6 ) specimens were oriented such that the load applied during testing was perpendicular to the longest dimension of the mesh interstices . 
 the test was conducted in tension at a rate of 300  mm / min ( 12  in / min ) until the specimen tore in half . 
 the  tear strength  was recorded as the maximum load sustained by the mesh in units of newtons ( n ) . 
 six circular specimens ( n = 6 ) measuring 7.5  cm in diameter ( 3  in diameter ) were prepared from phasix mesh and subjected to ball burst testing at time zero , t0 ( i.e. , prior to implantation ) . 
 for this reason , only one set of mesh specimens was prepared with no indication of the orientation of the mesh interstices during testing . 
 two circular stainless steel rings were utilized to clamp the mesh specimen to prevent slipping during the test . 
 then , a 2.5  cm diameter ( 1  in ) stainless steel ball was applied in compression at a rate of 300  mm / min ( 12  in / min ) until it burst through the mesh . the ultimate tensile stress and the strain at a stress of 16 
 n / cm were recorded in units of n / cm and percent , respectively .   the institutional animal care and use committee ( iacuc ) of the cbset , inc . 
 facility ( lexington , ma ) where the study was conducted approved the experimental protocol prior to the start of the study , and standard operating procedures were followed at all times . 
 twenty ( n = 20 ) castrated male , yucatan minipigs weighing 33.741.8  kg ( at surgery ) were acquired for the study and acclimated to the facility for a minimum of 21 days . 
 animals were fasted for at least 12 hours prior to surgery . on the morning of surgery , buprenorphine ( 0.03 
 mg / kg , i m ) and rimadyl ( 2.2  mg / kg , po ) were administered . 
 following sedation , the animals were intubated and maintained under anesthesia with 0.55% , isoflurane inhalant anesthetic , to effect . 
 the animals were placed in dorsal recumbency , and the ventral abdomen was prepared for aseptic survival surgery by shaving the entire abdominal region , cleaning the operative area with three alternating scrubs of povidone - iodine solution and 70% isopropyl alcohol solution , and applying sterilized surgical drapes over the entire field . following preparation of the abdomen , a midline laparotomy ( ~30  cm ) was performed . 
 two , 3  cm diameter ( 1.2  in diameter ) bilateral muscular defects were created in the anterior abdominal wall using a preperitoneal approach ( i.e. , the peritoneum remained intact ) . 
 the surgical defects were not closed and were bridged with a 10.2  cm diameter ( 4  in diameter ) phasix mesh on the right side ( figure 2(a ) ) or a 7.9  cm diameter ( 3.1  in diameter ) p4hb plug on the left side ( figure 2(b ) ) . 
 the phasix mesh was placed in the preperitoneal plane ( bridging the defect ) and fixated circumferentially with approximately n = 16 sorbafix absorbable fixation device constructs comprised of poly(d , l - lactide ) . 
 the p4hb plug was placed in the preperitoneal plane as an onlay mesh bridging the defect and fixated circumferentially with approximately n = 12 permanent prolene ( 2 - 0 ) sutures . the abdominal midline was then repaired via standard closure techniques , and the skin was tattooed to define the cranial , caudal , medial , and lateral aspects of each device to facilitate identification at the time of explant . 
 the animals were recovered from anesthesia and allowed free access to food and water ad libitum . 
 mg / kg , i m ) was administered every 412 hours , and rimadyl ( 2.2  mg / kg , po ) was administered every 24 hours for 72 hours postoperatively . 
 the abdominal region of each animal was examined daily to assess the condition of the wound and the subcutaneous tissues for evidence of herniation , diastasis , seromas , and/or hematomas . 
 animals were survived for 6 , 12 , 26 , or 52 weeks ( n = 5 in each group ) , followed by euthanasia . 
 humane euthanasia was carried out after sedation with telazol ( 4  mg / kg , i m ) and anesthesia via inhalant isoflurane . 
 euthanasia was achieved by administering an overdose of sodium pentobarbital ( 60150  mg / kg , intravenously , to effect ) in accordance with the american veterinary medical association ( avma ) panel of euthanasia and journal of the american veterinary medical association . following euthanasia , 
 the abdominal skin was dissected from the entire abdomen , and the abdominal wall ( including the 2 surgical defects ) was excised , photographed , and placed in saline solution ( 0.9% nacl ) for subsequent mechanical testing . a representative specimen from each phasix mesh and p4hb plug repair 
 one half was placed in 10% neutral buffered formalin for histological analysis , and the other was placed in an empty specimen jar for molecular weight analysis . 
 the repair sites ( i.e. , abdominal wall tissue plus test device as a composite specimen ) were subjected to ball burst testing to evaluate the peak load and relative stiffness of each repair site compared to native abdominal wall . 
 the peritoneum was carefully removed from both of the repair sites and the native abdominal wall sites prior to testing to eliminate the contribution of the peritoneum and to allow assessment of the strength of the repair site alone . 
 the repair sites and native abdominal wall sites were subjected to burst testing using an instron servohydraulic test frame ( instron , norwood , ma ) . 
 a 0.95  cm ( 0.375  in ) diameter ball was applied in compression at a rate of 25.4  mm / min ( 1  inch / min ) until the ball burst through . the peak load ( ball burst force in units of newtons , n ) sustained by each specimen was recorded , and the relative stiffness was calculated from the slope of the line formed when load was graphed versus elongation ( units = n / mm ) . 
 relative stiffness was taken from the slope of the line in the region of 3080% of the peak load . 
 explanted phasix mesh and p4hb plug specimens were enzymatically digested and manually cleaned to remove residual tissue prior to gel permeation chromatography ( gpc ) analysis . 
 gpc was conducted to quantify the molecular weight of the mesh material remaining at each time point . 
 specimens were placed in 50  ml tubes containing 40  ml collagenase ( type i ) solution ( 1.0  mg / ml ) in tesca buffer ( 50  mm tes , 2  mm cacl2 , 10  mm nan3 , ph 7.4 ) . the tubes were placed on a shaker ( 50  rpm ) and incubated at 37c to digest the tissue . after overnight incubation ( ~17 hours ) , the specimens were removed from the buffer and manually cleaned of tissue . 
 the cleaned specimens were rinsed in distilled water , followed by 70% ethanol , and dried prior to gpc analysis . 
 cleaned mesh specimens were dissolved at 1  mg / ml in chloroform , filtered using a 0.45  m filter to remove undigested particulates , and 95  l of this solution was then injected onto a gpc column for analysis . 
 gpc was performed in chloroform at 1  ml / min using a polymer labs , plgel column ( 5 micron , mixed c , 300  7.5  mm ) with an agilent 1100 series hplc with ri detector . 
 all histological assessments were conducted by a board - certified veterinary pathologist ( cbset , lexington , ma ) . 
 the explanted specimens were immersion - fixed in 10% neutral buffered formalin , cut into cross - section , and paraffin - embedded for further processing . 
 paraffin - embedded sections were microtomed ( 4 - 5  m ) , mounted onto glass slides , and stained with hematoxylin and eosin ( h&e ) , masson 's trichrome , and picrosirius red to characterize the host inflammatory / fibrotic response , collagen deposition / remodeling , and absorption properties associated with each specimen based on a standardized scoring system . 
 specimens were scored for inflammatory cell infiltrates ( neutrophils , eosinophils , macrophages , lymphocytes , giant cells ) , neovascularization , fibroplasia ( granulation tissue ) , hemorrhage , necrosis , and fibrosis using the following scoring system as described previously :  0 = absent / no response  1 = minimal / barely detectable  2 = mild / slightly detectable  3 = moderate / easily detectable  4 = marked / very evident . 0 
 = absent / no response 1 = minimal / barely detectable 2 = mild / slightly detectable 3 = moderate / easily detectable 4 = marked / very evident . 
 newly deposited , type iii collagen appeared green , while mature , type i collagen appeared red or orange under these conditions . 
 a score of  1  was assigned when green ( type iii collagen ) predominated , a score of  2  was assigned when there was a mixture of green , yellow , and yellow - orange , and a score of  3  was assigned when red - orange ( type i collagen ) predominated . 
 san jose , ca ) was utilized to perform all statistical analyses . for continuous data 
 in which three or more groups of data were compared , ( i.e. , peak burst load , relative stiffness , molecular weight ) a one - way analysis of variance ( anova ) was performed followed by a fisher 's lsd post - test as appropriate . 
 for scores such as histological parameters in which three or more groups of data were compared , a nonparametric test ( kruskal - wallis ) was performed followed by a dunn 's posttest as appropriate . 
 all data are reported as mean  standard error of the mean ( sem ) except the histological parameters in which the median is reported . 
 when phasix mesh was evaluated in the direction parallel to the longest dimension of the interstices , the suture retention strength was 59.16    5.7  n , which was greater than the 20  n suture retention strength suggested for hernia repair applications [ 29 , 30 ] . 
 similarly , when tested in the direction perpendicular to the longest dimension of the interstices , the suture retention strength of phasix mesh was again greater than 20  n at 49.10  2.3  n. when phasix mesh was evaluated in the direction parallel to the longest dimension of the interstices , the tear resistance strength was 30.33  3.1  n , which was greater than the 20  n tear resistance strength suggested for hernia repair applications [ 29 , 30 ] . 
 similarly , when tested in the direction perpendicular to the longest dimension of the interstices , the tear resistance strength of phasix mesh was again greater than 20  n at 29.48  2.4  n. when subjected to ball burst testing , the maximum compressive load sustained by the phasix mesh was 486.97  12.6  n , with a tensile strength of 140.70  5.4  n / cm and a strain at a stress of 16  n / cm of 15.43  0.2% . tensile strength greater than 50  n / cm and strain in the range of 1030% are considered suitable properties for hernia repair applications [ 29 , 30 ] . 
 thus , phasix mesh possessed appropriate tensile strength and strain values at time zero , t0 ( prior to implantation ) . 
 one animal died prior to its expected time point for reasons unrelated to the materials being evaluated in this study ( i.e. , sedation - related complication ) . 
 this animal was replaced with an additional animal in order to maintain n = 5 animals in each group . 
 all other animals survived until their expected time points . as shown in figures 3(a)3(d ) both devices ( i.e. , phasix mesh and p4hb plug ) and both types of fixation ( i.e. , sorbafix absorbable fixation device fasteners and prolene sutures ) remained visible and intact at the repair sites at 6 , 12 , 26 , and 52 weeks . 
 no evidence of hernia or diastasis was documented in any of the animals at any of the time points . as shown in figure 4(a ) , the burst strength of the native abdominal wall tissue remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 weeks , the burst strength of the native abdominal wall was 76.9  6.3  n , 62.8  14.7  n , 58.7  9.4  n , and 69.7  13.6  n , respectively . 
 no significant differences were detected between 6 and 12 weeks ( p = 0.788 ) , between 12 and 26 weeks ( p = 0.938 ) , between 26 and 52 weeks ( p = 0.834 ) , or overall between 6 and 52 weeks ( p = 0.891 ) . 
 the burst strength of the abdominal wall repaired with phasix mesh also remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 
 weeks , the burst strength of the phasix mesh repair site was 294.0  31.4  n , 277.0  14.8  n , 217.6  20.3  n , and 260.7  93.8  n , respectively . 
 no significant differences were detected between 6 and 12 weeks ( p = 0.746 ) , between 12 and 26 weeks ( p = 0.262 ) , between 26 and 52 weeks ( p = 0.414 ) , or overall between 6 and 52 weeks ( p = 0.527 ) . 
 the burst strength of the abdominal wall repaired with p4hb plug also remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 
 weeks , the burst strength of the p4hb plug repair site was 215.2  9.3  n , 307.0  36.0  n , 231.0  28.0  n , and 298.5  57.6  n , respectively . 
 no significant differences were detected between 6 and 12 weeks ( p = 0.086 ) , between 12 and 26 weeks ( p = 0.153 ) , between 26 and 52 weeks ( p = 0.204 ) , or overall between 6 and 52 weeks ( p = 0.118 ) . 
 phasix mesh and p4hb plug repairs demonstrated similar burst strengths at 6 , 12 , 26 , and 52 weeks with no significant differences detected between the two devices at any of the time points evaluated ( p = 0.139 , p = 0.568 , p = 0.798 , and p = 0.474 , resp . ) . 
 in addition , porcine abdominal wall sites repaired with phasix mesh or p4hb plug materials both demonstrated significantly greater burst strength compared to the native abdominal wall at 6 , 12 , 26 , and 52 weeks regardless of whether the mesh ( p < 0.001 , p < 0.001 , p = 0.004 , and p = 0.001 , resp . ) or the plug ( p = 0.011 , p < 0.001 , p = 0.002 , and p < 0.001 , resp . ) was utilized to bridge the defect .   as shown in figure 4(b ) , the relative stiffness of the native abdominal wall tissue remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 weeks , the relative stiffness of the native abdominal wall was 5.8  1.2  n / mm , 6.3  1.6 
 n / mm , 5.5  0.7  n / mm , and 6.5  1.8  n / mm , respectively . 
 no significant differences were detected between 6 and 12 weeks ( p = 0.940 ) , between 12 and 26 weeks ( p = 0.910 ) , between 26 and 52 weeks ( p = 0.887 ) , or overall between 6 and 52 weeks ( p = 0.917 ) . 
 the relative stiffness of the abdominal wall repaired with phasix mesh also remained stable throughout the duration of the study with no significant changes over time . 
 at 6 , 12 , 26 , and 52 weeks , the relative stiffness of the phasix mesh repair site was 22.2  4.7  n / mm , 21.9  1.0  n / mm , 28.7  7.8  n / mm , and 30.5  11.1  n / mm , respectively . 
 no significant differences were detected between 6 and 12 weeks ( p = 0.970 ) , between 12 and 26 weeks ( p = 0.329 ) , between 26 and 52 weeks ( p = 0.799 ) , or overall between 6 and 52 weeks ( p = 0.234 ) . 
 the relative stiffness of the abdominal wall repaired with p4hb plug also remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 
 weeks , the relative stiffness of the p4hb plug repair site was 17.9  4.3  n / mm , 22.5  2.4  n / mm , 24.2  2.2  n / mm , and 33.8  6.3  n / mm , respectively . 
 no significant differences were detected between 6 and 12 weeks ( p = 0.508 ) , between 12 and 26 weeks ( p = 0.804 ) , or between 26 and 52 weeks ( p = 0.170 ) . 
 however , there was a slight trend toward greater stiffness overall at 52 weeks compared to 6 weeks ( p = 0.025 ) . 
 phasix mesh and p4hb plug repairs demonstrated similar relative stiffness at 6 , 12 , 26 , and 52 weeks with no significant differences detected between the two devices at any of the time points evaluated ( p = 0.533 , p = 0.940 , p = 0.512 , and p = 0.635 , resp . ) . 
 in addition , porcine abdominal wall sites repaired with phasix mesh or p4hb plug materials both demonstrated significantly greater relative stiffness compared to the native abdominal wall at 6 , 12 , 26 , and 52 weeks regardless of whether the mesh ( p = 0.021 , p = 0.027 , p = 0.001 , and p = 0.001 , resp . ) or the plug ( p = 0.084 , p = 0.023 , p = 0.009 , and p < 0.001 , resp . ) was utilized to bridge the defect . as demonstrated by a p value of 0.084 , 
 the stiffness of the abdominal wall sites repaired with p4hb plug materials at 6 weeks did not quite reach statistical significance and were only trending toward greater relative stiffness compared to the native abdominal wall . 
 as shown in figure 5 , phasix mesh and p4hb plug repairs demonstrated similar molecular weights at 0 , 6 , 12 , 26 , and 52 weeks with no significant differences detected between the two devices at any of the time points evaluated ( p = 0.804 , p = 0.640 , p = 0.268 , p = 0.150 , and p = 0.936 , resp . ) . 
 the molecular weight of the phasix mesh decreased significantly over time with molecular weights of 240 , 510  2 , 018  da , 204 , 282  4 , 457  da , 118 , 884  2 , 821  da , and 44 , 434  879  da at 6 , 12 , 26 , and 52 weeks , respectively . 
 molecular weight decreased significantly at each time point compared to the preimplantation ( i.e. , 0 weeks ) molecular weight of the phasix mesh ( 300 , 397  972  da ) with p < 0.001 in all cases . 
 furthermore , the molecular weight of the phasix mesh decreased progressively between 0 and 6 weeks , between 6 and 12 weeks , between 12 and 26 weeks , between 26 and 52 weeks , and overall between 0 and 52 weeks with p < 0.001 in all cases . similarly , 
 the molecular weight of the p4hb plug decreased significantly over time with molecular weights of 242 , 190  1 , 259  da , 208 , 288  3 , 420  da , 124 , 126  3 , 219  da , and 44 , 148  644  da at 6 , 12 , 26 , and 52 weeks , respectively . 
 molecular weight decreased significantly at each time point compared to the preimplantation ( i.e. , 0 weeks ) molecular weight of the p4hb plug ( 301 , 543  110  da ) with p < 0.001 in all cases . 
 furthermore , the molecular weight of the p4hb plug decreased progressively between 0 and 6 weeks , between 6 and 12 weeks , between 12 and 26 weeks , between 26 and 52 weeks , and overall between 0 and 52 weeks with p < 0.001 in all cases . 
 as shown in table 2 , median scores for inflammation were reported in the range of 2 - 3 for both the phasix mesh and the p4hb plug at the early 6- and 12-week time points , and both consistently scored 2 at the later 26- and 52-week time points . 
 no significant differences in inflammation scores were detected over time for either the phasix mesh or the p4hb plug , and no significant differences in inflammation scores were detected between the mesh and plug designs at any of the time points ( p > 0.05 in all cases ) . 
 macrophage scores of 2 were consistently reported for the phasix mesh at all time points with no significant changes over time ( p > 0.05 ) . 
 interestingly , macrophage scores for the p4hb plug varied slightly over time with a median score of 3 at the 6-week time point , trending toward a significantly lower score of 1 at the 52-week time point ( p < 0.05 ) . again 
 , no significant differences were identified between the mesh and the plug designs at any of the time points evaluated ( p > 0.05 in all cases ) . 
 giant cell scores of 1 were consistently observed for both the phasix mesh and the p4hb plug at all of the time points . 
 no significant differences in giant cell scores were detected over time for either the phasix mesh or the p4hb plug , and no significant differences in giant cells scores were detected between the mesh and plug designs at any of the time points ( p > 0.05 in all cases ) . 
 fibrosis / encapsulation scores for the phasix mesh varied slightly over time with a score of 4 at the 6-week time point that trended toward a significantly lower fibrosis / encapsulation score of 2 at the 12- and 52- week time points ( p < 0.05 ) . 
 no significant changes in fibrosis / encapsulation scores were observed for the p4hb plug with scores of 2 - 3 observed at all time points . 
 in addition , no significant differences in fibrosis / encapsulation scores were detected between the mesh and plug designs at any of the time points . 
 granulation tissue / vascularization scores ranged from 2 - 3 for both the phasix mesh and the p4hb plug at all time points evaluated . 
 the phasix mesh scores alternated between 3 and 2 at 6 and 12 weeks and again at 26 and 52 weeks , respectively , while the p4hb plug scores were 2 at the early 6- and 12-week time points and 3 at the later 26- and 52-week time points . 
 however , no significant differences in granulation tissue / vascularization scores were detected over time for either the phasix mesh or the p4hb plug , and no significant differences were detected between the mesh and plug designs at any of the time points ( p > 0.05 in all cases ) . 
 collagen morphology scores of 2 were consistently reported for the phasix mesh at all time points with no significant changes over time ( p > 0.05 ) . 
 similarly , collagen morphology scores for the p4hb plug were 1 at the early 6-week time point and 2 at all subsequent time points . 
 as with the mesh design , no significant changes in collagen morphology were detected over time for the plug design ( p > 0.05 ) , and no significant differences were detected between the mesh and plug designs at any of the time points ( p > 0.05 in all cases ) . 
 poly-4-hydroxybutyrate ( p4hb ) is a naturally derived , fully absorbable polymer produced by bacteria via transgenic fermentation techniques . 
 this material has been manufactured into a number of configurations including the phasix mesh and p4hb plug , which are designed for soft tissue repair applications such as hernia repair . over the last decade , 
 p4hb materials have been evaluated in a number of animal studies , particularly those investigating cardiovascular applications such as tissue engineered trileaflet heart valves , artery augmentation patches , and small diameter vascular grafts , as well as development of p4hb as a suture material . however , the current study is the first of its kind to evaluate this material specifically for hernia repair applications . 
 the objective of this study was to determine the mechanical properties , resorption profile , and histological characteristics of phasix mesh and p4hb plug designs when utilized to bridge a surgical defect in the porcine abdominal wall over a period of 6 , 12 , 26 , or 52 weeks . to accomplish this , abdominal wall sites repaired with phasix mesh and p4hb plug materials 
 were harvested at the end of each implantation period and subjected to mechanical testing , gel permeation chromatography , and histological analysis to assess the strength of the repair , the amount of p4hb material remaining at the repair site , and the host response to the p4hb material , respectively . 
 mechanical testing revealed that the burst strength and relative stiffness of the phasix mesh and p4hb plug repaired sites were similar to each other at all four time points evaluated with no significant differences detected between the two devices for either of these parameters throughout the course of the study ( p > 0.05 in all cases ) . 
 in addition , the sites repaired with phasix mesh and p4hb plug materials also exhibited significantly greater burst strength and relative stiffness compared to the native abdominal wall at all time points regardless of which device was utilized to bridge the defect ( p < 0.05 in all cases ) . 
 these results indicate that the p4hb material is capable of augmenting the strength of the native porcine abdominal wall regardless of its configuration as a mesh or plug design . 
 it should also be noted that burst strength and relative stiffness of the phasix mesh and p4hb plug repaired sites remained stable and did not change significantly between 6 and 52 weeks , suggesting that scaffolds derived from the p4hb material are durable and capable of maintaining support at the repair site over a 52-week period in a porcine model . 
 gel permeation chromatography was conducted to quantify the molecular weight of the p4hb material remaining at the repair site at each time point . 
 the data showed that phasix mesh and p4hb plug possessed similar molecular weights at all time points with no significant differences detected between the two devices at any time ( p > 0.05 in all cases ) . 
 these results correspond with the mechanical testing data and demonstrate that the configuration of the p4hb material does not significantly impact its resorption profile . 
 in addition , the molecular weight of both the phasix mesh and the p4hb plug decreased significantly at each time point compared to the corresponding preimplantation ( i.e. , 0 weeks ) value and also decreased progressively between each time point ( i.e. , between 0 and 6 weeks , between 6 and 12 weeks , between 12 and 26 weeks , between 26 and 52 weeks , and overall between 0 and 52 weeks ) . 
 these results indicate that the p4hb material was significantly resorbed over time , but as indicated by the mechanical testing data , significant material resorption did not correspond with a significant drop in mechanical strength at the repair site for either the phasix mesh or the p4hb plug design . 
 in addition to mechanical testing and gel permeation chromatography , histological analyses were also performed in order to assess the host response to the p4hb material comprising the phasix mesh and the p4hb plug designs . 
 no statistically significant changes were observed over time for either the phasix mesh or the p4hb plug with respect to histological parameters such as inflammation , granulation tissue / vascularization , giant cells , or collagen morphology . 
 inflammation scores and granulation tissue / vascularization scores ranged from 2 - 3 for both the mesh and the plug design indicating a mild / slightly detectable to moderate / easily detectable inflammatory response and presence of granulation tissue and vascularization for both devices at all time points . 
 in addition , giant cells scores were consistently reported as 1 , indicating a minimal / barely detectable presence of giant cells for both devices at all time points . 
 similarly , collagen morphology scores were consistently reported as 2 , indicating a mixture of both mature ( type i , red and orange - red ) and immature ( type iii , green and yellow - green ) collagen at all time points . 
 it should be noted that a few differences were observed with respect to time of implantation . 
 for instance fibrosis / encapsulation scores for the phasix mesh varied slightly over time with a score of 4 at the 6-week time point that trended toward a significantly lower fibrosis / encapsulation score of 2 at the 12- and 52- week time points ( p < 0.05 ) . 
 these scores correspond to marked / very evident fibrosis / encapsulation at the 6-week time point that was ultimately improved to mild / slightly detectable fibrosis / encapsulation at the later time points . 
 some degree of fibrosis / encapsulation is expected in the presence of an implanted material such as the phasix mesh , and this response is expected to vary over time with the tissue remodeling / wound healing process . 
 similarly , the macrophage scores for the p4hb plug varied slightly over time with a score of 3 at the 6-week time point , trending toward a significantly lower score of 1 at the 52-week time point ( p < 0.05 ) . 
 these scores correspond to moderate / easily detectable presence of macrophages at the 6-week time point that ultimately improved to only minimal / barely detectable presence of macrophages at the 52-week time point . in general 
 , no statistically significant differences were detected between the histological scores reported for the phasix mesh design versus the p4hb plug design for any of the parameters evaluated . 
 these results suggest that the host tissue response was most influenced by the chemical composition of the device ( i.e. , poly-4-hydroxybutyrate ) rather than the structure of the device ( i.e. , mesh versus plug ) . 
 inflammatory infiltrates associated with both devices were predominantly mononuclear with an overall mild response commonly associated with the implantation of a foreign material . 
 similarly , both devices resulted in comparable , mild to moderate granulation tissue / vascularization that is expected in association with wound healing and tissue remodeling , suggesting that the p4hb material performed appropriately in terms of overall foreign body response regardless of its configuration as a mesh or a plug . 
 phasix mesh and p4hb plug provided durable scaffolds for soft tissue repair in a porcine model . 
 both repairs demonstrated significant mechanical strength compared to native abdominal wall over a 52-week period , which remained elevated despite significant material resorption over time with no evidence of herniation and/or diastasis . 
 in addition , histological assessment revealed a comparable and mild inflammatory response and mild to moderate granulation tissue / vascularization associated with the p4hb material regardless of its configuration as a mesh or a plug .","purpose . poly-4-hydroxybutyrate ( p4hb ) is a naturally derived , absorbable polymer . 
 p4hb has been manufactured into phasix mesh and p4hb plug designs for soft tissue repair . 
 the objective of this study was to evaluate mechanical strength , resorption properties , and histologic characteristics in a porcine model . methods . 
 bilateral defects were created in the abdominal wall of n = 20 yucatan minipigs and repaired in a bridged fashion with phasix mesh or p4hb plug fixated with sorbafix or permanent suture , respectively . 
 mechanical strength , resorption properties , and histologic characteristics were evaluated at 6 , 12 , 26 , and 52 weeks ( n = 5 each ) . 
 results . 
 phasix mesh and p4hb plug repairs exhibited similar burst strength , stiffness , and molecular weight at all time points , with no significant differences detected between the two devices ( p > 0.05 ) . 
 phasix mesh and p4hb plug repairs also demonstrated significantly greater burst strength and stiffness than native abdominal wall at all time points ( p < 0.05 ) , and material resorption increased significantly over time ( p < 0.001 ) . 
 inflammatory infiltrates were mononuclear , and both devices exhibited mild to moderate granulation tissue / vascularization . 
 conclusions . 
 phasix mesh and p4hb plug demonstrated significant mechanical strength compared to native abdominal wall , despite significant material resorption over time . 
 histological assessment revealed a comparable mild inflammatory response and mild to moderate granulation tissue / vascularization .",pubmed
"glycopeptide resistance was detected in enterococci more than 25 years ago , ; since then , vancomycin - resistant enterococci ( vre ) have become important nosocomial pathogens associated with high mortality and costs , , , . 
 glycopeptide resistance in enterococci is mediated by van genes , with vana and vanb being the most prevalent . 
 several european countries have reported a rising incidence of vre in recent years , , . in denmark , a dramatic increase since 2012 has been due to multiclonal outbreaks of vana - positive enterococcus faecium 
 , . 
 it has been estimated that for every patient with a vre infection detected in a clinical sample , up to ten patients are intestinal carriers of vre . 
 therefore , current guidelines recommend screening of patients at risk and implementation of isolation precautions for vre carriers , . screening relies on selective culturing and/or detection of the resistance genes vana or vanb by pcr . 
 however , substantial indirect savings can be achieved by rapidly available results , as highlighted by a report from france , which compared the management of two simultaneous vre outbreaks on two different wards . 
 several studies have examined the diagnostic accuracy of various assays to detect vana and vanb with or without a previous enrichment step , , , , , , , , . 
 however , sensitivities ranging from 43.5% to 100% and specificities from 79.2% to 99.6% have been reported . 
 in contrast , the specificity of the detection of the vanb gene is considerably lower , , , which is attributed to the presence of vanb - positive anaerobic bacteria , . in our laboratory 
 , we have used a culture - based method using overnight enrichment and subsequent plating on chromogenic agar to screen for vre . in autumn 2014 , 
 prompted by long - lasting vre outbreaks , we implemented the xpert vana / vanb assay performed directly on rectal swabs . 
 this fully automated system integrating dna extraction and real - time pcr provides results within an hour . 
 we used the vana part of the assay to guide rapid implementation of infection control measures . here , we evaluate the diagnostic accuracy of the xpert vana / vanb assay and the impact on infection control decision making . 
 our laboratory serves the primary and secondary sectors of a population of 800 000 inhabitants in the northern part of the capital region of denmark , including four university hospitals with a total number of 1577 beds and covering all internal medicine subspecialties and three intensive care units . 
 the infection control units at these four hospitals are part of the clinical microbiology department and employ six infection control nurses . 
 diagnosis of vre in a clinical sample results in roommate screening and isolation precautions are implemented until results are available . 
 if any roommate is found positive , an outbreak is declared and all patients in the ward are screened weekly until no new patient is diagnosed with vre . during weekly screenings , 
 isolation precautions are implemented as soon as a positive vre result is available . for roommate screening and weekly screening of wards , 
 the routine culture - based screening is supplemented by vana pcr . before transfer of patients from an outbreak ward , 
 pcr is performed and the implementation of isolation precautions is based on the pcr result . for vre - positive patients readmitted within 6 months after the last positive screening sample , isolation precautions are implemented at admission independently from screening results . in the case of readmission 
 > 6 months after the last positive sample , screening is advised and the implementation of isolation precautions is individualized based on screening results and risk factors . in these cases , 
 for phenotypic screening , eswab transport medium was transferred to an enrichment broth ( brain  heart infusion supplemented with 4  mg / l vancomycin and 60  mg / l aztreonam ) either by shaking the flocked swab several times in the enrichment medium or by pipetting 100 l . 
 after overnight incubation , 100 l of the enrichment medium was plated on selective chromogenic agar plates ( chromagartm vre , chromagar , paris , france ) and read after 1 and 2 days of incubation . for vana pcr - positive , but culture - negative samples , the culture protocol was thoroughly repeated . 
 species identification was performed by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( biotyper , bruker , bremen , germany ) and a preliminary positive result was given if there was abundant growth of e.  faecium on the selective chromogenic agar . 
 susceptibility testing for vancomycin and teicoplanin was performed by the eucast disc diffusion method   and determination of the mic used a gradient strip ( etest , biomrieux , solna , sweden ) to confirm the vana or vanb phenotype . 
 pcr - based screening was undertaken by the automated system genexpert using the xpert vana / vanb assay ( cepheid ab , solna , sweden ) . 
 one hundred microlitres of the eswab transport medium was added to the sample reagent and further handled as recommended by the manufacturer . in case of invalid or error results ( pcr inhibition or technical error ) , the assay was repeated once . 
 only vana pcr results were reported to the requesting wards as a preliminary result and used for guidance of infection control measures . 
 pcr was performed on the same day if the sample arrived in the laboratory before 16.00  h , all days of the week . 
 sample arrival date , laboratory results , dates of preliminary and final results and sample indications were prospectively collected . for final analysis 
 the impact of the vana pcr result on infection control measures was estimated by calculation of  isolation days saved ( number of days from negative pcr result to final negative culture result ) and  transmission risk days saved ( number of days from positive pcr result to preliminary or final positive culture result ) . the computer program 
 , version 3.0.2 ( r foundation for statistical computing , vienna , austria ; http://www.r-project.org/ ) was used for statistical analysis . 
 for comparison of categorical data , fisher s exact test was used . for comparison of numerical data , unpaired groups , 
 sensitivity , specificity , and positive and negative predictive values with 95% ci were calculated using the r package dtcompair . 
 3 - 3013 - 1217/1/ ) and the danish data protection agency ( record no . 2012 - 58 - 0004 ; heh-2015 - 066 ) . 
 from 30 december 2014 to 15 august 2015 , 1110 vre screening samples from 804 patients were examined both by the culture - based method and by pcr . 
 the samples were from 24 different departments from four different hospitals . in 1067 ( 96.1% ) samples , 
 pcr was performed and reported the same day , the sample was received in the laboratory . 
 the median turnaround time for culture results was 3 days ( mean 3.2 ; range 39 days ) . in 35 ( 3.2% ) samples , 
 invalid or error results were obtained after the first pcr analysis and for 11 ( 1.0% ) samples , repeated analysis did not give a result either . 
 of the 1099 samples where a pcr result was obtained , 152 ( 13.8% ) were positive for the vana gene and 167 ( 15.2% ) were culture - positive for an enterococcus species ( all e.  faecium ) with the vana phenotype ( vancomycin - resistant with mic 256  mg / l and teicoplanin - resistant with mic 8256  mg 
 the vana pcr results in relation to culture results are given in table  1 . 
 the vanb pcr was positive in 247 ( 22.5% ) samples , but only one was culture - positive for enterococcus sp . ( enterococcus faecalis ) with the vanb phenotype ( vancomycin - resistant with mic 128  mg / l and teicoplanin - susceptible with mic 0.5 
 discrepant results between culture and vana pcr were further investigated . in 12 of the 22 pcr - negative samples , from which a vana phenotype e.  faecium was cultured , pcr was repeated on the eswab transport medium and two were found positive with high circles of threshold ( ct ) of 36.9 and 38.2 . 
 pcr was also performed on the isolates from the 20 remaining samples and they were all vana pcr - positive . 
 of the 145 culture - positive samples that had been vana pcr - positive , most ( n  =  135 ) were culture positive after 1 day of incubation of the chromogenic agar , whereas the remaining ten samples ( 6.9% ) were only positive after 2 days of incubation . in comparison , eight of the 22 culture - positive samples that had been pcr - negative ( 36.4% ) only turned positive on the second day of incubation ( fisher 's exact test , p  <  0.001 ) . on the other hand , 
 ct values of the culture - negative sample were significantly higher ( median 32.9 ; range 24.138.3 ) than ct values of the culture - positive samples ( median 22.9 ; range 12.837.8 ) ( wilcoxon rank sum test ; p  <  0.001 ) . 
 to calculate the test accuracy of the xpert vana / vanb assay , culture results complemented with information from the patient s earlier and later samples were used as a reference standard . for the analysis of vana pcr , four of seven 
 true positives : three samples from the same patient from which phenotypically vancomycin - susceptible ( mic 1  mg / l ) , but vana - positive e.  faecium was isolated by non - selective culturing ( unpublished data , holzknecht bj ) and one sample taken under linezolid therapy from a patient with vre culture - positive samples 15 days earlier and 28 days later . 
 the sensitivity of the vana pcr assay was 87.1% ( 95% ci 82.1%92.2% ) , the specificity was 99.7% ( 95% ci 99.3%100% ) and the positive and negative predictive values were 98.0% ( 95% ci 95.8%100% ) and 97.7% ( 95% ci 96.7%98.6% ) , respectively . 
 the specificity and positive predictive value of the vanb part of the assay were low at 77.6% ( 95% ci 75.1%80.1% ) and 0.4% ( 95% ci 0.00%1.2% ) , respectively . 
 the majority of the samples ( n  =  1033 ; 93.1% ) were taken as weekly screening samples from 12 different outbreaks during the study period . 
 thirty - seven ( 3.3% ) were roommate screening samples , 14 ( 1.3% ) were taken at readmission , 13 ( 1.2% ) at transferral from outbreak wards and 13 ( 1.2% ) for other indications ( fig .  1 ) . 
 a total of 141 isolation days could be saved as a consequence of faster available negative pcr results . 
 the saving of time due to rapidly available positive pcr results corresponded to a total of 292 saved transmission risk days . 
 on the other hand , false - negative or false - positive pcr resulted in six additional transmission risk days and 13 additional isolation days ( fig .  1 ) . 
 this study describes the performance and impact of the xpert vana / vanb assay , when performed directly on rectal swabs , in the setting of vana e. faecium outbreaks . 
 of the 1099 samples with available pcr and culture results , only 29 showed discordant results : seven were vana pcr - positive , but culture - negative and 22 were culture - positive , but vana pcr - negative . 
 vancomycin - resistant isolates cultured from samples that had been pcr - negative , were vana pcr - positive , which confirms that false - negative pcr results , when performed directly on the sample , were not due to alterations of the primer binding sites . 
 ct - values of vana pcr - positive , culture - negative samples were significantly higher than ct - values in culture - positive samples . likewise , the culture - positive , but vana pcr - negative samples were significantly more often positive after 2 days of incubation of the chromogenic plate than pcr - positive samples , which might also indicate a lower bacteria count . in the implementation phase of the xpert vana / vanb assay , investigation of vana pcr - positive , culture - negative samples had actually led to a change in the culture protocol from plating 10 l to 100 l of enrichment broth on the chromogenic plates to increase sensitivity ( data not shown ) . 
 using culture results complemented with information from patients follow - up samples as a reference standard , the diagnostic accuracy of the vana part of the xpert vana / vanb assay was excellent and in accordance with previous studies , , . however , one previous , but far smaller , study found both a lower sensitivity and a lower specificity of the assay . as there was only one sample that was culture - positive for vanb - positive vre , we could not fully analyse the diagnostic accuracy of the vanb part of the assay . however , the large proportion ( 22.3% ) of false - positive vanb pcr results is in accordance with previous studies , , , , . 
 first , investigation of vana pcr - positive , culture - negative samples from the same patient led to the isolation of phenotypically vancomycin - susceptible , but vana pcr - positive e.  faecium . 
 an outbreak with a similar strain has been described and infection control management similar to vre seems prudent , . 
 the strengths of the diagnostic accuracy study are the large sample size and the prospective and thorough comparison of the xpert vana / vanb assay with the selective culture method , including further investigation of discordant samples by follow - up samples to define a robust reference standard . 
 however , it has been shown that several outbreak clones and multilocus sequence types are circulating in our region . a systematic study of genetically diverse strains would though be necessary to finally conclude on the generalizability of our results . using the xpert vana / vanb assay , 
 96.1% of screening results were available within 1 day , whereas the median turnaround time for selective culturing was 3 days . 
 based on the time interval between available pcr and culture results , we calculated saved isolation days and saved transmission risk days as a consequence of earlier suspension or implementation of isolation measures . 
 however , these numbers might be overestimated , as patients might have been isolated for other reasons or dismissed from the hospital before results were available . 
 besides saved transmission risk days , earlier available positive screening results from roommates also led to faster outbreak declaration and implementation of additional infection control measures such as screening of all patients , enhanced cleaning , enforcement of hand hygiene and staff teaching . 
 we have seen a faster control of vre outbreaks after the implementation of pcr - based screening . however , as this is part of a bundle approach we can not determine the isolated value of the pcr assay ( mogensen and midttun , infection prevention and control canada 2016 national education conference , poster 84 ) . 
 detailed health economics calculations are beyond the scope of this study . however , considering the assay s official price in denmark of 661 dkk ( approx . 
 89 ) per sample and estimated additional costs per isolation day of 3350 ddk ( approx . 
 451 ) , we feel that saving 141 isolation days and 292 transmission risk days as a result of 1110 pcr analyses is a considerable impact and worth the costs . 
 this is more important when the psychosocial consequences of a vre outbreak situation and isolation precautions for patients and staff members are also taken into account . 
 the vana pcr of the xpert vana / vanb assay had a high diagnostic accuracy and considerable impact on infection control decision making in our setting . 
 ln received non - financial support from roche diagnostics a / s and from msd , outside the submitted work . 
 bjh contributed to conception and design of the study , acquisition and analysis of data and drafting of the manuscript . 
 dsh and joj contributed to conception and design of the study and critical revision of the manuscript . 
 ak contributed to conception of the study , acquisition of data and critical revision of the manuscript .","vancomycin - resistant enterococci ( vre ) are increasingly important nosocomial pathogens and screening for colonization status is a mainstay in infection control . 
 we implemented pcr - based screening during vana - positive enterococcus faecium outbreaks in four university hospitals in copenhagen , denmark . 
 xpertvana / vanb was performed directly on rectal swabs and the vana pcr result was used to guide infection control measures . 
 concurrently , all samples were selectively cultured including an overnight enrichment step . 
 diagnostic accuracy was calculated as well as turnaround time and the impact of the earlier available pcr results on infection control decision making . in all 
 , 1110 samples were analysed . 
 the vana pcr positivity rate was 13.8% and culture positivity rate was 15.2% . 
 the diagnostic accuracy of the vana part of the assay was high with a sensitivity of 87.1% , a specificity of 99.7% , and positive and negative predictive values of 98.0% and 97.7% , respectively . 
 the vanb pcr had a considerably lower specificity of 77.6% and a positive predictive value of 0.4% . in 1067 ( 96.1% ) samples , 
 pcr results were reported within 1 day , whereas median culture turnaround time was 3 days . 
 the saving of time to available results corresponded to 141 saved isolation days and 292 saved transmission risk days . 
 false - negative or false - positive pcr results led to six additional transmission risk days and 13 additional isolation days , respectively.the vana pcr had high diagnostic accuracy and the prompt availability of results gave a considerable benefit for infection control decision making .",pubmed
"in addition to the circulatory renin - angiotensin - aldosterone system ( raas ) , there is now a significant body of evidence supporting the concept of a  local tissue or cellular raas  that has important roles in the pathology of cardiovascular diseases . 
 the local production of aldosterone and the discovery of mineralocorticoid receptor ( mr ) expression in the heart have led to a greater understanding of the role of aldosterone / mineralocorticoid receptor activation in the cardiovascular diseases , including hypertension and heart failure [ 2 , 3 ] . 
 aldosterone activates its mineralocorticoid receptor ( mr ) in the nondiabetic heart and can cause structural and electrical remodelling , fibrosis , oxidative stress , inflammation , and arrhythmias [ 1 , 47 ] . 
 mr antagonists have shown significant benefit in patients with left ventricular dysfunction and myocardial infarction . 
 for example , the recent eplerenone in mild patients hospitalization and survival study in heart failure ( emphasis - hf ) study has shown that eplerenone , an mr antagonist , has beneficial effects in patients with moderate heart failure ( nyha class ii ) . 
 however , beneficial effects of mr blockade in pathological states such as diabetes are unclear . 
 it is well established that signaling network alterations in diabetes are such that potential therapies will need to be tailored for this pathological state [ 1012 ] . 
 inhibitors of the renin - angiotensin - aldosterone system ( raas ) , such as angiotensin converting enzyme inhibitors ( aceis ) and angiotensin type-1 receptor ( at1 ) blockers ( arbs ) , have been shown to protect against hypertension and/or diabetes - induced end - organ damage [ 13 , 14 ] . 
 for example , in a group of 99 patients with severe heart failure , van de wal et al .   demonstrated that 45% had elevated plasma angiotensin ii 
 ( ang ii ) levels independent of serum ace activity despite long - term ace inhibitor use . 
 acei and/or arbs do not completely block end - organ damage in diabetes and/or hypertension , and clinical trials of acei and arbs in combination have generally shown that they do not offer added benefits but rather lead to greater adverse effects such as electrolyte imbalance and renal complications [ 1 , 15 ] . 
 in addition , inhibition of ang ii does not reliably suppress aldosterone production , with the aldosterone escape phenomenon occurring in up to 40% of patients with heart failure [ 17 , 18 ] . 
 thus , the need for additional raas inhibition in these individuals would be a logical consequence such as combinations of acei and/or arbs with mr antagonist . 
 the randomized aldactone evaluation study ( rales ) and the eplerenone postacute myocardial infarction heart failure efficacy and survival study ( ephesus ) suggested that an mr antagonist on top of an acei or an arb can reduce mortality in patients with severe congestive heart failure and left ventricular dysfunction after mi [ 15 , 18 ] . 
 thus , there is an important need to identify the best possible treatment option for normal and diabetic patients with cardiac dysfunction . 
 important issues that still need to be addressed include identifying the optimal combination of drugs to use and their timing , either administration before or after ischemic injury , in both normal and diabetic states . 
 hence , the aim of the present study was to characterize and compare the effects of ru28318 ( ru , a selective mr antagonist ) , captopril ( capt , an acei ) , and losartan ( los , an arb ) , alone or in double and triple therapy combinations administered either before or after ischemia on ischemia / reperfusion- ( i / r- ) induced cardiac dysfunction in isolated hearts obtained from normal and diabetic rats . 
 17-week - old male wistar rats were divided into 2 groups ( n = 6 per studied group ) . 
 group 1 was control animals and group 2 was streptozotocin- ( stz- ) treated diabetic animals . 
 all animal experiments in this study were approved by the research administration at kuwait university and conformed to their ethics guidelines for the care and use of laboratory animals that are based on those published by the us national institute of health ( nih publication no . 
 diabetes was induced by a single intraperitoneal injection of 55  mg / kg body weight stz . 
 basal glucose levels were determined prior to stz injection and 48  h after stz injection . 
 rats with a blood glucose concentration above 250  mg / dl were declared diabetic and any not meeting this criterion were excluded from the study . 
 the animals ' diabetic state was re - assessed after 4 weeks just before sacrificing the animals . 
 rats were anesthetized with intraval sodium ( 40  mg / kg body weight ) , and hearts were rapidly removed after intravenous heparinization ( 1000  u / kg body weight ) . 
 the excised hearts were immediately mounted on the langendorff perfusion assembly ( hugo sachs electronics , freiburg , germany ) and were perfused initially with a constant pressure perfusion of 50  mmhg with the oxygenated ( 95% o2 + 5% co2 ) krebs - henselit buffer ( 37c ) of the following composition ( in mm ) : nacl 117 ; kcl 4.39 ; cacl2 2.5 ; nahco3 20.0 ; kh2po4 1.21 ; mgcl26h2o 1.2 ; glucose 12.0 ; osmolarity 300 mosm / l , ph 7.35 . 
 a water - filled balloon was introduced into the left ventricle and connected to a statham pressure transducer ( p23db ) and balloon volume was adjusted to give the baseline end - diastolic pressure of 5  mmhg . left ventricular developed pressure ( pmax ) and its positive and negative derivatives ( + dp / dt and dp / dt , resp . ) and left ventricular end - diastolic pressure ( lvedp ) were continuously monitored . coronary flow ( cf ) 
 was measured by means of an electromagnetic flow probe positioned in the inflow tubing immediately above the aortic perfusion cannula . 
 this system permits accurate adjustment of perfusion pressure between 5 and 300  mmhg to an accuracy of 1  mmhg . 
 hearts removed from animals were perfused for 30  min and then subjected to 30  min of global ischemia ( i ) followed by a period of 30  min of reperfusion ( r ) . 
 drugs were administered for 30  min either during perfusion before ischemia or during reperfusion after ischemia . 
 drug regimens tested were ru28318 ( ru ; 10 m ) , captopril ( capt ; 3.6  10 m ) , losartan ( los ; 3  10 m ) , ru + capt , ru + los , capt + los , and ru + capt + los ( triple ) . the doses used gave maximal responses in preliminary experiments and were similar to those used in previous studies [ 2123 ] . 
 percent recovery ( % r ) was calculated using the following formula : ( reperfusion value)/(baseline value )  100 . 
 the baseline value is the value recorded at 30  min perfusion before exposure to global ischemia . 
 mean values were compared using analysis of variance followed by bonferroni 's post hoc test . 
 hyperglycaemia persisted throughout the study period and was 306    18  mg / dl at four weeks in stz - treated animals compared to 96  4  mg / dl in the nondiabetic ( control ) animals . 
 there were no significant differences in body ( 275  17  gm ) and heart weights ( 0.95  0.03  gm ) corrected to tibia length in these animals . 
 the effects of various acute drug treatments ( ru , capt , los , capt + los , capt + ru , and los + ru , capt + los + ru ) administered during perfusion ( before ischemia ) or during reperfusion ( after ischemia ) on recovery of each cardiac function parameter following i / r in normal and diabetic hearts were recorded . 
 the percent ( % ) recovery in pmax , lvedp , + dp / dt , dp / dt , and cf as a function of reperfusion time for nondiabetic control hearts is shown in figure 1 ( before ischemia ) and figure 2 ( after ischemia ) and for diabetic hearts in figure 3 ( before ischemia ) , and figure 4 ( after ischemia ) . 
 in general , drug treatments resulted in a very rapid rate of cardiac function recovery within the first 10  min of reperfusion followed by a more gradual rate of recovery ( figures 14 ) . 
 ru resulted in the most rapid recovery in cf within the first 10  min of reperfusion for cf when given before ischemia and in + dp / dt and pmax when administered after ischemia in control hearts ( figures 1 and 2 ) . in diabetic hearts , ru , ru + los , and triple therapy similarly gave the most rapid recovery within 10  mins of reperfusion for lvedp when given before ischemia . 
 interestingly , cap + los therapy given before ischemia in diabetic hearts resulted in a very rapid improvement in pmax and cf within the first 10  min of reperfusion that was almost the maximal recovery obtained ( figures 3(a ) and 3(e ) ) . 
 ru + capt and triple therapy given before ischemia to diabetic hearts resulted in the most rapid recovery in + dp / dt . 
 ru + los yielded the most rapid recovery in lvedp when given before ischemia in nondiabetic hearts . 
 other significantly rapid recoveries in function were observed for + dp / dt for capt alone given after ischemia and for dp / dt when ru was given after ischemia in diabetic hearts ( figures 4(c ) and 4(d ) ) 
 .   in untreated controls , % recovery ( % r ) of pmax at the end of the 30  min reperfusion period was around 50% . 
 administration of ru either before ischemia ( during perfusion ) or after ischemia ( during reperfusion ) led to a similar but significant improvement in pmax of between 14 and 18% ( see figure 5(a ) ) . 
 of the individual ( single ) drug treatments administered before ischemia , the improvement ( % change in function relative to untreated controls ) in pmax with ru was the highest compared to capt alone ( 8% ) , whereas los showed no significant improvement ( figure 5(a ) ) . 
 however , when single drugs were administered after ischemia , the % improvement in pmax was the highest for capt at around 25% , and again the least improvement was seen with los at around 14% . when drugs were administered as combinations , triple therapy ( capt + los + ru ) yielded the best recovery in pmax of about 74%an improvement over controls of 48%irrespective of whether it was administered before or after ischemia ( figure 5 ) . 
 of the double combinations , only capt + ru was significantly ( p < 0.05 ) better over either of the single agents alone when administered either before or after ischemia ( figure 5 ) .   recovery in pmax of diabetic hearts ( % r of 9  3  mmhg ) from i / r was significantly ( p < 0.05 ) impaired compared to nondiabetic controls ( % r of 50  2  mmhg ) . 
 administration of all drug treatments to diabetic hearts before ischemia led to significant improvement in pmax of between 122 and 222% ( see figure 5(c ) ) . again in general , the triple therapy appeared to be better than double therapies which appeared to be better than single therapies with some notable exceptions . 
 capt + los was equally as effective as the triple combination , and capt + ru double combination was not significantly ( p < 0.05 ) better than any of the single therapies ( figure 5 ) . 
 however , when single drugs were administered after ischemia in diabetic hearts , interestingly , there was no significant improvement in pmax with ru ( figure 5(d ) ) . 
 this is in marked contrast to what was observed in the nondiabetic controls ( figures 5(a ) and 5(b ) ) . 
 however , all other drug treatments gave comparable improvements in pmax with capt + los being the best treatment option ( figures 5(c ) and 5(d ) ) . 
 % r in lvedp of around 250% was observed in nondiabetic controls that could be significantly reduced ( p < 0.05 ) ( i.e. , improved ) by all drug treatments ( figures 6(a ) and 6(b ) ) . in the case of single therapies for control ( nondiabetic ) hearts , 
 acute treatment with capt gave the best improvement in lvedp when given before ischemia ( figure 6(a ) ) and was joint best with ru when given after ischemia ( figure 6(b ) ) . 
 double therapies gave no advantage in lvedp improvement over single therapies , whereas triple therapy ( capt + los + ru ) yielded the best recovery in lvedp of about of 27% and 38% , respectively , when given before or after ischemia ( figures 6(a ) and 6(b ) ) . 
 capt given before ischemia gave similar improvement of about 23% to the triple therapy ( i.e. , % improvement values were not significantly different ; p < 0.05 ; see figure 6(a ) ) . in diabetes , lvedp was significantly ( p < 0.05 ) elevated compared to nondiabetic control hearts by over 3-fold . 
 capt + los double therapy yielded the best improvement of around 30% in lvedp when given either before or after ischemia ( figures 6(c ) and 6(d ) ) . 
 capt alone was equally effective as capt + los when administered after ischemia ( figure 6(d ) ) . in the case of lvedp , all other therapies gave similar % improvements in function ( figures 6(c ) and 6(d ) ) . 
 interestingly , los given alone in diabetic hearts was relatively more effective at improving lvedp than in nondiabetic control hearts ( figures 5(a ) and 5(b ) ; figures 6(c ) and 6(d ) ) . 
 in contrast to pmax where greater improvements in function were observed for diabetic hearts ( figure 5 ) , in the case of lvedp , drug - induced improvements appeared similar for diabetes and controls when presented as % change ( figure 6 ) . 
 the reasons for this apparent anomaly are that in diabetes lvedp significantly ( p < 0.03 ) increases by about 3-fold , and thus in calculating a % change , the denominator is now a large number and thus yields modest changes as a percentage . 
 however , when considering the actual numerical changes induced by the different drug treatments in terms of % r for lvedp , this suggests that the degree of change induced by drugs was considerably higher ( about 25 fold ) in diabetic hearts compared to control hearts . in untreated controls , % recovery of + dp 
 / dt following i / r was around 47% and similar to that observed for pmax ( figure 5 ) . 
 drug treatments significantly ( p < 0.02 ) improved function when administered either before or after ischemia ( figures 7(a ) and 7(b ) ) . 
 in general , triple therapy ( around 60% improvement ) was better than double therapy which in turn was more effective than single therapies in improving recovery of + dp / dt ( figures 7(a ) and 7(b ) ) . 
 of the single therapies , los appeared to be the least effective in improving + dp / dt ( figures 7(a ) and 7(b ) ) . 
 ru was the most effective when given before ischemia whereas capt was the best when given after ischemia in improving + dp / dt ( figures 7(a ) and 7(b ) ) . diabetes ( 8  2% ) led to about a 6-fold reduction in % r for + dp / dt as compared to controls ( 47  2% ) . 
 drug treatments generally led to marked and significant improvement in function . when given before ischemia 
 , drugs gave similar improvements in function at the end of perfusion , but triple therapy as well as ru + capt gave the most rapid improvement within the first 10  min of perfusion . 
 in the case of capt + los , approximately 250% improvement was attained which was similar to that obtained with triple therapy when given before ischemia . when given after ischemia , surprisingly capt alone ( approx 300% improvement ) was the best therapy ( figure 7 ) . 
 all drug treatments generally showed greater % improvement in diabetes compared to controls where less than 60% improvement in + dp / dt was noted ( figure 7 ) . in untreated controls , % recovery ( % r ) of 
 drug treatments significantly ( p < 0.05 ) improved dp / dt by about 40100% when administered either before or after ischemia ( figures 8(a ) and 8(b ) ) . 
 all the single therapies when administered before ischemia gave similar improvements in recovery of dp / dt ( figure 8(a ) ) . with the exception of los + ru , double therapies were better than single therapies and showed similar effectiveness as the triple therapy ( figure 8(a ) ) . when drugs were administered after ischemia , with the exception of los alone which exhibited the least improvement of about 40% , all other treatment regimens were similarly effective exhibiting improvements in the range 6080% ( figure 8(b ) ) . 
 diabetes ( 11  3% ) led to a about a 3.4-fold reduction in % r for dp / dt as compared to controls ( 37  2% ) . 
 drug treatments in diabetic hearts generally led to marked and significant improvement in function from about 100% to 200% and this was generally higher compared to that seen following drug treatments in control hearts ( see figures 8(c ) and 8(d ) ) . 
 all drug treatments were equally effective in improving dp / dt when administered after ischemia ( figure 8(c ) ) , whereas combination therapies was more effective than single therapies when administered before ischemia ( figure 8(d ) ) . 
 administration of capt + ru before ischemia and ru alone after ischemia were the most effective treatment in improving dp / dt in diabetic hearts ( figure 8(c ) ) . 
 in untreated controls , % recovery ( % r ) of cf following i / r was around 43% . 
 drug treatments significantly ( p < 0.05 ) improved function by about 2060% when administered either before or after ischemia ( figures 9(a ) and 9(b ) ) . 
 when drugs were administered before ischemia , ru gave the most rapid recovery within the first 10  mins of reperfusion . 
 triple therapy was generally better than double therapies which in turn were more effective than single agents ( figure 9(a ) ) . 
 triple therapy ( 60% improvement ) was also more effective when administered after ischemia , and with the exception of los alone ( around 10% improvement ) , all other treatments yielded similar improvements in the range 3040% ; see figures 9(a ) and 9(b ) . 
 diabetes ( 12  3% ) led to about a 3.8-fold reduction in % r for cf as compared to controls ( 46  3% ) . 
 drug treatments in diabetic hearts generally led to marked and significant improvement in function from about 100% to 200% and this was generally higher ( figures 9(c ) and 9(d ) ) , compared to that seen following drug treatments in control hearts ( figures 9(a ) and 9(b ) ) . 
 all other drug treatments were equally effective in improving cf when administered before ischemia except for capt + los which was the best treatment ( about 200% improvement ) ; see figures 3(c ) and 9(c ) . 
 given after ischemia , capt , los , capt + los , and triple therapy were similarly effective in improving cf in diabetic hearts , whereas ru alone was the least effective ( figure 9(d ) ) . 
 a major goal of this study was to identify the optimal treatment strategy of raas blockade to prevent or treat ischemia - reperfusion injury in normal and diabetic hearts [ 2426 ] . 
 although the use of acei or arbs has been well studied , the effects of aldosterone antagonism alone or in combination with these other raas blockers especially in diabetes are not well understood . 
 this study showed that treatment with ru was generally better in preventing or reversing ischemia - induced cardiac dysfunction in normal hearts compared to treatment with an acei ( capt ) or arb ( los ) alone . in the case of diabetic hearts , 
 ru was generally similarly effective as capt or los treatment . also , dual therapies involving ru were similarly effective as capt + los therapy , whereas triple combination was generally equal to or the most effective strategy in preventing or reversing ischemia - induced cardiac dysfunction in normal or diabetic hearts . 
 for example , irrespective of whether drugs were administered before or after ischemia , combination therapies appeared more effective , whereby triple > double > single therapy in most of the study scenarios ( i.e. , 18 out of the 20 experimental scenarios of looking at 5 different cardiac parameters studied for both normal and diabetic hearts for when drugs were given either before or after ischemia ) . 
 in contrast to nondiabetic hearts , capt + los dual therapy was the most effective therapy ( alongside triple therapy ) in diabetic hearts which is consistent with the known diabetes - induced overactivity of the ace / ang ii / at1 signaling cascade [ 16 , 27 , 28 ] . in this study , looking at an mr antagonist in diabetic hearts , we show that indeed the diabetic pathology leads to altered cardiac response to treatments with ru as well as arbs and acei . in general , with the exception of pmax for ru given after ischemia , the relative improvement in cardiac function or effectiveness of raas blockers was greater in diabetes than in control hearts . 
 thus , in nondiabetic hearts , ru alone was highly effective at preventing and treating i / r - induced cardiac dysfunction , whereas in diabetes , in terms of pmax , it was only effective at preventing ( i.e. , when given before ischemia ) i / r - induced cardiac injury . 
 it was ineffective , in terms of pmax , when given after ischemia in diabetes implying a differential role of aldosterone mr signaling in the diabetic pathology . 
 these data suggest that mr signaling is detrimental for all cardiac parameters in nondiabetic hearts during ischemia as well as reperfusion phases , whereas in the diabetic hearts , at least for pmax , it may be detrimental only during the ischemia - induced phase of i / r injury . the reasons for the differential response in pmax in diabetes are not understood but clearly require further study . 
 indeed , the effectiveness of drugs generally appeared to be cardiac parameter specific with some drugs being better at improving one cardiac function parameter over others . 
 for example , although ru was ineffective at improving pmax , it was the most effective at improving dp / dt when administered to diabetic hearts after ischemia . 
 this may imply that ru may not improve systolic function but may show marked improvements in diastolic function in diabetic hearts when given after ischemia . 
 this assertion is further supported by the fact that the ru significantly ( p < 0.03 ) improved lvedp in diabetic hearts ( figures 3 , 4 , and 6 ) . 
 ru exhibited greater beneficial effects in lvedp for diabetic compared to control hearts when considering the actual numerical changes in terms of % r for lvedp . 
 although ru - mediated improvement in cardiac function generally appears to be as good as arbs and aceis , differences exist between ru and other drugs as to effectiveness when given before or after ischemia . in nondiabetic hearts , 
 ru was the best or one of the best single agents when given before or after ischemia , whereas in diabetes , it generally provided minimal or least benefit when given after ischemia with the exception of dp / dt where ru was the best treatment option . 
 this finding may be clinically relevant as diabetes patients generally present with compromised diastolic function as an early indicator of cardiac dysfunction and is detected in about 75% of asymptomatic diabetic patients [ 29 , 30 ] . 
 our results show that ru importantly improves diastolic function ( as measured by dp / dt ) in diabetes especially when given after ischemia and thus early - stage ru treatment may represent a novel strategy for treating diabetes - induced diastolic dysfunction . 
 our study also showed that irrespective of whether drugs were administered before or after ischemia , their effectiveness in recovering cardiac function appeared to be time dependent ; typically , drugs rapidly improved cardiac function within the first 10  min of reperfusion followed by a period of steady improvement thereafter up to the 30  min study period ( figures 14 ) . 
 however , there were some interesting differences as to which cardiac function parameters were improved during this initial rate of recovery . for example , significantly rapid recoveries in function were observed for + dp / dt for therapy with captopril alone when administered after ischemia and for dp / dt when ru was given after ischemia in diabetic hearts ( figures 4(c ) and 4(d ) ) . 
 these data further imply that in patients with diabetes where diastolic function is often the only early indicator of cardiac dysfunction , these treatments offering rapid improvements in dp / dt should be considered as potential therapeutic options .   the possible mechanisms by which ru may be acting beneficially in normal and diabetic hearts subjected to i / r are not known but these may include blockade of aldosterone - induced oxidative stress , endothelial dysfunction , and inflammation in the heart and in the coronary vasculature that generally contribute to abnormal calcium homeostasis and cardiac dysfunction . thus , ru - mediated blockade of these important processes likely also improves calcium handling and overload in hearts subjected to i / r . 
 this assertion is supported by previous findings that aldosterone / mr activation induces cardiomyocyte ionic remodelling by modulating potassium and l - type calcium channel activity [ 31 , 32 ] , t - type calcium channel expression , and ryanodine receptor activity . 
 changes in these calcium handling proteins have been reported to lead to important consequences in the control of calcium signaling , modulation of calcium transients , sarcoplasmic reticulum diastolic leaks , and promotion of rhythm disorders that can be corrected by aldosterone / mr antagonism [ 3537 ] . 
 aldosterone is also known to induce increased na / h exchanger-1 ( nhe-1 ) activity via transactivation of the epidermal growth factor receptor ( egfr ) and subsequent reactive oxygen species ( ros ) formation which is thought to be an important signaling cascade in the genesis of many cardiac pathologies . 
 hence , another possible mechanism by which mr antagonism with ru may be exerting its beneficial effects is via inhibition of the detrimental cardiac egfr / nhe-1 signaling network . 
 however , we have recently shown that egfr signaling via pathways involving erk1/2 , p38 map kinase , and akt / foxo appears to be an important beneficial survival mechanism , and its inhibition leads to worsening of cardiac function following i / r . 
 thus , targeting egfr inhibition with mr antagonists such as ru may have beneficial and detrimental consequences in the heart and thus their net therapeutic advantage will depend on the relative importance or contributions of these different egfr - driven mechanisms in a given pathological state . 
 indeed , we have recently shown that by removing the egfr inhibitory effects of los , which blocks ang ii - mediated transactivation of egfr , by coadministering an egfr ligand , significant improvement in cardiac function over that achieved by los alone was attained . 
 whether the beneficial effects of ru may be via antagonism of aldosterone - mediated effects in cardiac muscle and/or vasculature is not clear but recent studies suggest the involvement of both . 
 it appears that cardiomyocyte aldosterone / mr participates in the crosstalk between cardiomyocytes and coronary blood vessels , such as increased aldosterone synthesis by the cardiomyocytes resulting in coronary dysfunction [ 1 , 41 ] . 
 interestingly , signaling by egfr , the transactivation target of aldosterone , is known to be elevated in the diabetic vasculature and is detrimental to vascular function [ 40 , 4244 ] . 
 thus , ru - mediated blockade of aldosterone - induced egfr transactivation may be beneficial in improving diabetes - induced vascular dysfunction in the heart but this requires further study . in diabetic patients , in addition to cardiac dysfunction , there is also significant risk of renal damage where ang ii blockade is contraindicated as it will lead to attenuation of ang ii - driven glomerular filtration rate ( gfr ) and renal shutdown particularly in patients with renal artery stenosis . 
 however , ru which does not affect gfr may be particularly useful for such patients . 
 further , addition of ru as a combination strategy with acei and arbs may allow for a reduction in the dose of the latter agents such that they have minimal disruption on gfr and electrolyte balance whilst retaining their ability to reduce diabetes - induced proteinuria and their beneficial effects on cardiac function as highlighted in the present study . 
 importantly , mr antagonists when combined with acei but not arbs have shown significant reduction in total mortality in patients with chf . but here in i / r injury , we show generally that combination approaches are better where capt + los + ru ( triple ) therapy is mostly the best treatment option in normal and diabetes . however , combination therapies with acei and arbs are rarely employed in the clinic possibly due to fear over accumulating adverse effects in patients with heart disorders [ 15 , 20 , 46 ] . 
 whether the inclusion of ru in the highly effective triple therapies described herein can lead to dose reduction of acei and/or arbs to minimize or eliminate these adverse effects requires further clinical study .   in diabetic patients , therapies involving aldosterone / mr antagonists 
 they may oppose aldosterone - mediated detrimental effects on structural and functional integrity of the pancreatic [ beta]-cell resulting from islet cell inflammation and oxidative stress as well as aldosterone - induced insulin resistance [ 47 , 48 ] . 
 if our findings reported here are reproduced in clinical studies , our study may have important clinical implications in the way these drugs should be administered in cardiac dysfunction . 
 firstly , our data implies that in normal patients , ru alone could be an effective therapy for prevention of cardiac dysfunction because as a single agent , it yields the best improvements in cardiac function when given before ischemia . 
 furthermore , for postischemic injury , although ru appears to be an effective therapy , aceis and/or arbs appear to be the drugs of choice for diabetics as they yielded the best improvements in cardiac function when administered after ischemia . 
 we also suggest that mr antagonists , since they act through a non - ace / ang ii / at1r pathway , may represent a novel class of raas inhibitor that potentially could overcome the limitations observed with the acei and arb combinations of raas inhibitors . 
 our study by selecting to administer drugs acutely in isolated hearts is advantageous in that it examines the effects of these therapeutic agents directly on the heart and avoids noncardiac contributions of these agents . 
 furthermore , this study highlights that in addition to the benefits observed by mr antagonists when administered systemically , these agents can also be beneficial when administered locally . 
 thus , our data implies that in the clinic ru treatment locally might be considered as an effective therapy or preventative measure in cardiac i / r injury for susceptible patients and possibly also preoperatively for patients undergoing aortic cross - clamping or other cardiac surgeries such as cardiopulmonary bypass or coronary artery bypass grafting . 
 our study also suggests that optimal usage of drug(s ) alone or in combination may require their selection based on several criteria including their relative benefit in the normal versus pathological state , whether being considered for prevention or treatment strategy , on the specific cardiac parameter that might need to be improved ( e.g. , diastolic function in diabetes ) and on the optimal rate of cardiac function recovery required for a given condition .","aims . we evaluated the effects of ru28318 ( ru ) , a selective mineralocorticoid receptor ( mr ) antagonist , captopril ( capt ) , 
 an angiotensin converting enzyme inhibitor , and losartan ( los ) , an angiotensin receptor blocker , alone or in combination with ischemia / reperfusion- ( i / r- ) induced cardiac dysfunction in hearts obtained from normal and diabetic rats . 
 methods . 
 isolated hearts were perfused for 30 min and then subjected to 30 min of global ischemia ( i ) followed by a period of 30 min of reperfusion ( r ) . 
 drugs were administered for 30 min either before or after ischemia . 
 drug regimens tested were ru , capt , los , ru + capt , ru + los , capt + los , and ru + capt + los ( triple ) . 
 recovery of cardiac hemodynamics was evaluated . 
 results . 
 recovery of cardiac function was up to 5-fold worse in hearts obtained from diabetic animals compared to controls . 
 treatment with ru was generally better in preventing or reversing ischemia - induced cardiac dysfunction in normal hearts compared to treatment with capt or los alone . in diabetic hearts , ru was generally similarly effective as capt or los treatment . conclusions . 
 ru treatment locally might be considered as an effective therapy or preventative measure in cardiac i / r injury . 
 importantly , ru was the most effective at improving dp / dt ( a measure of diastolic function ) when administered to diabetic hearts after ischemia .",pubmed
"modern nasal surgery takes into account the cosmetic appearance of the nose but also seeks to improve the natural functions of the nose . 
 furthermore , in modern respiratory medicine , objective measurement of airway patency is a must . 
 measurement of nasal cavity geometry has proven to be a great challenge for researchers in modern rhinology . 
 the most common method used in these measurements is acoustic rhinometry ( ar ) , which is based on analysis of reflected acoustic impulses [ 3 , 4 ] . 
 it has demonstrated that the cross - sectional area of the nasal airway measured by acoustic rhinometry and the area found by calculations using data from rhinomanometry has close correlation [ 3 , 5 ] . 
 furthermore , several significant associations between nasal cavity dimensions and nasal airflow have been reported . 
 and the most important benefit of acoustic rhinomanometry is that is easy to perform and requires less cooperation from subject . 
 however , nose geometry may be influenced by various factors including race , age , sex , height , weight , and smoking habit . 
 we evaluate the changes in nasal dimensions of healthy iranian volunteered for cosmetic rhinoplasty after surgery using acoustic rhinometry and comparing differences in length , minimal cross sectional area ( mca ) , and volume of each nasal cavity before and after surgery . 
 the cases were all healthy , without any prior history of nose surgery , fracture , allergy or asthma , and sinus disorders , and they had no complaint about nasal obstruction . 
 all cases were iranian aged at least 18 who were visited in ent clinic of rassul - e - akram teaching hospital between 2006 and 2008 . 
 the surgeries according to the structure of the nose were open rhinoplasty with or without septoplasty . 
 t - type of osteotomy ( single versus double ) , placement of columellar strut , or other variations of surgery were dependent on the surgeons preference . 
 all operations were performed by a single experienced surgeon , and septoplasty was performed in the case of moderate - to - severe septal deviation . 
 the device was consists of a sound source ( loudspeaker ) distally positioned in relation to a 24  cm tube equipped with a microphone for acquisition in its proximal portion . 
 a sound pulse was generated with a peak power of 146  db sound pressure level and a 50-ms duration . 
 all measurements were performed after a short period of acclimatization and in a relatively quiet room at normal temperature ( mean = 21.4c ) to minimize artifacts from physical stress , environmental noise , and temperature changes . 
 the measurements were performed during a breathing pause while patients were in a sitting position . 
 the nosepiece used in the measurements was 5  cm in length and was anatomically sculptured . to ensure a tight connection between the nosepiece and tip of the nose , 
 a small amount of ultrasound transmission gel was applied to the edge of the nosepiece . 
 care was taken not to obstruct the nasal vestibule with gel or deform the nose during testing . 
 the angle of the incident acoustic impulse was about 45 with respect to a line joining the base of the piriform aperture of the nose to the tragus . 
 measurement performed in either of two conditions of without a decongestant and with a decongestant , respectively . 
 the first measurement performed before surgery and the second measurement performed 3 months after surgery . 
 the technique we used allowed us to analyze two local minimum points giving us minimum cross - sectional areas ( mca1 and mca2 ) and distance ( d1 and d2 ) to the points from the nostril and volumes of nostrils . to examine the geometrically difference in pre - and postoperative nasal cavities , paired t test used for repeated measurements . 
 mean age ( sd ) of cases were 24.63 ( 4.4 ) years , minimum age was 18 , and maximum age was 40 . 
 none cases had complaints of breathing problems before and after the surgery , and all of our cases were satisfied with their surgery result . 
 mean ( sd ) geometric values of cases preoperatively with and without decongestant are demonstrated in table 1 . by the pair wise comparison of cases without decongestant before and after surgery , 
 right ( r ) d1 , bilateral d2 , and left ( l ) mca2 did not change significantly after surgery . 
 however , pairwise comparison of nasal dimensions with decongestant revealed that bilateral d2 and v did not changed significantly postoperatively . 
 bilateral d1 and both mca2 decreased significantly , while significant increase was observed in mca1 postoperatively ( table 2 ) . in order to assess the result of septoplasty , patients divided into 2 groups of rhinoplasty plus septoplasty and rhinoplasty alone . 
 comparison of the finding showed that cases with septoplasty would experience more increase in mca1 ( increase in mca1 was significant and much more in decongested state of group with septoplasty ) and less constriction in mca2 postoperatively ( significant constriction were observed in both groups . however , amount of this constriction was more in group without septoplasty ) ( table 3 ) 
 . in cases of rhinoplasty without septoplasty , increase in mca1 was significant only in group of single osteotomy without decongestant . 
 although changes in mcv2 were significant in both with and without decongestion groups of double osteotomy , but the net effects were not clinically significant comparing single osteotomies . 
 table 4 demonstrates that cases with rhinoplasty alone would benefit from double osteotomy in mca1 . 
 effect of osteotomy on patency of nose in cases with septoplasty is shown in table 5 ; it is more prominent at the level of mca1 . 
 placing a columellar strut in group of cases of rhinoplasty plus septoplasty has significant effect both at mca1 and mca2 levels ( table 6 ) . in cases of rhinoplasty alone , placing a columellar strut caused greater amount of increase in mca1 . 
 mca2 after decongestion significantly increased in group without columellar strut , but it did not changed in group with replacing a columellar strut . 
 regression analysis of difference after surgery in mca1 , mca2 , and v revealed no fix significant effect for septoplasty , osteotomy , and placing a columellar strut . 
 advanced equipment for these methods is now available , and most devices are reliable provided that care is taken to calibrate the device properly . in the current study , we used an ar to measure the dimensions of the nose . 
 ar evaluation of the nasal airway has been reported to be accurate when compared with mri2 and ct [ 15 , 16 ] . 
 the closest correlations have been noted after decongestant use and in the most anterior 5  cm of the nasal cavity . 
 it is believed that acoustic rhinometry provided less variability of results than those obtained with rhinomanometry . in the study of corey 
 he concludes that the anatomic correlate of csa1 ( cross - sectional area ) is not as clearly delineated ; however , it likely corresponds to an area at or near the nasal valve . 
 furthermore , csa2 correspond to the general areas of the anterior portions of the inferior turbinate . 
 he claimed that in conjunction with a careful clinical exam , ar can provide objective documentation and diagnosis for better treatment of nasal airway blockage . 
 acoustic rhinometry also seems very suitable for evaluation of the nasal cavity in cases where septoplasty and turbinoplasty is considered as well as for the postoperative objective evaluation . 
 studied the changes in nasal airways after le fort i osteotomy and functional rhinosurgery with both ar and rhinomanometry techniques . 
 he declare that rhinomanometry and acoustic rhinometry are reliable and objective methods of determining functional and geometric changes in the nasal cavity after le fort i osteotomy . in this paper 
 we described reference values in normal and decongested noses of normal iranian population with acoustic rhinometry . 
 all of our cases were normal people without significant symptoms of nasal dysfunction in their nose , and surgery was performed just because of aesthetic reasons . comparing our finding with mohebbi et al . 
 , in iranian population , no significant difference was observed in mcas and v. this finding supports the original idea of study to enroll cases with normal nose geometry .   by comparing nose geometry before and 
 after surgery in both normal state and decongested state , considering presence of inflammation after surgery and more precise estimation in decongested estate , the main findings were a reduction in d1 , an increase in mca1 , a decrease in mca2 and no change in v and d2 . 
 these findings are consistent with findings of greymer that reported reduction in internal dimensions of the nasal cavity especially the anterior dimensions . 
 this finding highlight the fact that second constriction is a critical place during the surgery and adequate attention should be paid to this point . 
 the anatomic correlate of this point is anterior portion of the inferior turbinate , hence decreasing the resistance in this region could be beneficial for patients . 
 operations on the nasal septum are frequently performed with the intention of improving the nasal airway . 
 for this reason , they form much of the routine workload in most otolaryngology departments . a meta - analysis performed by singh et al . demonstrated that an overall significant improvement following surgery is present in patient undergoing septal surgery .   in our study , 
 comparison of cases of rhinoplasty with and without septoplasty revealed that the cases with septoplasty would experience more increase in mca1 and less constriction in mca2 . 
 as a conclusion , even in the cases which mild septal deviation is present , performing the septoplasty may increase patient nose patency . 
 more increase in mca1 and no significant effect on mca2 were observed in patients in double osteotomy group comparing group of single osteotomy . 
 it seems as if double osteotomy positively affects nose function by more increase in mca1 . 
 double osteotomy is positively affecting mca1 considering the cases without septoplasty or cases with septoplasty . in either group of rhinoplasty with and without septoplasty , placing a strut was beneficial for patients . in the study of oeken and kiefer on 52 cases of septorhinoplasty total nasal airflow 
 it should be mentioned that in the above study , all cases suffered from a deviated septum and some degree of obstruction . 
 anselmo - lima et al . in a study of 30 cases of rhinoplasty with lateral 
 osteotomy observed that there is a reduction in nasal valve area ( significant reduction in mca1 ) as well as in anterior volume of the nose . 
 they propose more criteria to indicate rhinoplasty , because the procedure might lead to nasal obstruction . in the study of kemker 
 patients in the septoplasty - only group showed a statistically significant increase in volume as measured by ar . 
 patients who had septoplasty plus other sinonasal procedures showed significant increases in volume and cross - sectional area ( csa ) 3 , whereas csas 1 and 2 increased also , but not significantly . shemen and hamburg studied 24 patients undergone septal surgery , and skouras et al . studied 16 patients , the improvement in nasal patency was seen in both studies . 
 however , cases in both studies have complained of nasal obstruction before surgery [ 25 , 26 ] . in a study by can et al . on 26 children with septal deviation 
 the results showed that the surgery was successful ; however , they claim that surgery should be restricted to only the pathologic area and should be conservative . 
 as we know , ar has some limitations , as it can not measure the columella width in nasal geometry and also differentiate the nostrils shape that has effect on the turbulence of air delivered to nasal cavity . 
 as we know , the cross - sectional area of the nose is a major factor in the determination of airflow , with airflow increasing as the cross - sectional area increases . 
 hence , according to the above findings , cosmetic rhinoplasty may generate a mix effect on nose function . 
 comparison of our finding to other studies shows that condition of the patient before surgery is the principal factor for the prediction of surgery results . 
 putting all together , surgeons should be aware of common pattern of change in nose geometry ; care must be taken in intervention in the region of second constriction . 
 performing osteotomy may better help patients to save nasal patency , septoplasty is beneficial even in mildly deviated septum , and placing a strut may be beneficial in most of the cases .","objective . to evaluate the changes in nasal dimensions of healthy iranian volunteered for cosmetic rhinoplasty after surgery using acoustic rhinometry 
 . methods . pre- and postoperative nasal dimension of 36 cases undergoing cosmetic rhinoplasty 
 were compared using acoustic rhinometry ( ar ) , and the measured variables were distance to first and second constriction ( d1 , d2 ) , first and second minimal cross - sectional area ( mca1 , 2 ) , and volume . 
 results . 
 mean age ( sd ) of cases were 24.63 ( 4.4 ) years . 
 septoplasty was performed in 12 cases ( 33.3% ) . 
 after surgery , bilateral d1 and both mca2 decreased significantly , while significant increase was observed in mca1 postoperatively using decongestant . 
 cases with septoplasty experienced more increase in mca1 and less constriction in mca2 postoperatively . in cases with rhinoplasty alone , they received benefit from double osteotomy in mca1 . in either group of rhinoplasty with and without septoplasty , placing a strut was beneficial for patients . 
 discussion . 
 the cross - sectional area of the nose is a major factor in the determination of airflow . 
 cosmetic rhinoplasty may generate a mix effect on nose function . performing osteotomy may better help patients to save nasal patency , septoplasty is beneficial even in mildly deviated septums , and placing a strut may be beneficial in most of the cases .",pubmed
"cardiovascular disease is the leading cause of mortality in many economically developed nations accounting for about 30% of all deaths   and its incidence is still increasing . 
 ongoing research aims to investigate and prevent the early development of cardiovascular risk factors such as atherosclerosis , hypertension , dyslipidemia , chronic inflammation , and insulin resistance . 
 the beneficial effects of n-3 polyunsaturated fatty acids ( n-3 pufas ) were proved in several observational and experimental studies . 
 the lipid lowering action of n3-pufas was detected at the beginning , so these nutrients were used for the treatment of dyslipidemic disorders . 
 their anti - inflammatory , antithrombotic , antiatherosclerotic , and antiarrhythmogenic effects were observed later . 
 low - grade chronic inflammation is now recognized as a prominent process in the development of atherosclerosis and coronary heart disease . 
 the induction of inflammation may well provide a link between hyperlipidemia and atherogenesis [ 2 , 3 ] . 
 atherosclerosis is now considered a  systemic disease  featured by low - grade arterial inflammatory lesions that can develop through the disease progression .   in physiological conditions , endothelial cells 
 synthesize and release adequate amounts of nitric oxide ( no ) and prostaglandins ( such as pge2 and pge3 ) and maintain a downstream balance between pro- and anti - inflammatory molecules . 
 however , in the presence of atherosclerosis this balance disrupts leading towards an increase production of proinflammatory cytokines as interleukins 1 , 2 , and 6 ( il-1 , 2 , and 6 ) and tumor necrosis factor  ( tnf- ) , with further progression of the disease . 
 these pro - inflammatory cytokines can induce oxidative stress by enhancing the production of reactive oxygen species ( ros ) by monocytes , macrophages , and leukocytes . 
 pufas and their eicosanoid derivatives may play a significant role modulating the inflammatory response ( figure 1 ) . 
 unsaturated fatty acids are referred to as pufas when two or more double bounds are present . 
 there are two pufas families , omega-3 ( n-3 ) and omega-6 ( n-6 ) fatty acids . 
 they differ in location of the last double bond relative to the terminal methyl end of the molecule . 
 the human body can produce almost all fatty acids , except linoleic acid ( la , c18:2 n-6 , precursor to the n-6 series of fatty acids ) and -linoleic acid ( ala , c18:3n-3 , precursor to the n-3 series of fatty acids ) . 
 these two pufas are named  essential fatty acids  because the body can not synthesize them . 
 endogenous conversion ( elongation and desaturation ) of the initial c18 pufa precursors results in the synthesis of longer - chain counterparts such as eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) in the n-3 family and dihomo--linoleic acid ( dgla ) and arachidonic acid ( aa ) in the n-6 family . in humans 
 the biochemical pathways converting ala to epa and epa to dha are limited : 0.28% of ala is converted to epa ( generally more in women ) and 04% of ala to dha [ 812 ] . 
 results from pilot studies suggest that ala conversion is also limited to function as a surrogate for fish consumption . 
 thus , tissue and circulating epa and dha levels are primarily related to their dietary intake . 
 fish is the major food source of long - chain n-3 pufas , including epa , dha , and docosapentaenoic acid ( dpa ) , while ala is a plant n-3 fatty acid mainly found in seeds , nuts , and their oils . 
 thus , plant sources of n-3 fatty acids can not currently be considered as a replacement for seafood - derived n-3 pufas . 
 this suggests that n-3 fatty acids derived from different sources might have their own specific effects on cardiovascular risk markers . 
 linoleic acid is thought to decrease the conversion of ala into epa and dha by competing for the n-6-desaturase enzyme . 
 previous studies showed that genetic variations in this enzyme may be related to cardiovascular disease . furthermore , because the two fatty acid pathways are mutually exclusive ( i.e. , n-3 fatty acid can not become n-6 fatty acid and vice versa ) , a balanced intake of ala and la as precursors or of their longer - chain products epa , dha , and aa is required . 
 there is a vast amount of epidemiologic evidence of a cardioprotective effect of fish oil - derived epa and dha and it was confirmed in randomized controlled trials [ 1720 ] . 
 several intervention studies , such as the  gruppo italiano per lo studio della sopravvivenza nell'infarto miocardico  ( gissi)-prevenzione trial and the cardiovascular health study , have shown that an increased intake of eicosapentaenoic acid ( epa , c20:5n-3 ) and docosahexaenoic acid ( dha , c22:6n-3 ) lowers the risk of coronary heart disease ( chd ) [ 21 , 22 ] . 
 the first gissi - prevenzione trial , a randomized open label study in 11324 italian patients with recent myocardial infarction , demonstrated that patients had a 15% lower combined risk of mortality , nonfatal myocardial infarction , and stroke upon supplementation for 3.5 years with 850  mgday-1 of lc n-3 pufa . 
 the relative risk of cardiovascular mortality was also decreased by 30% and that of sudden death by 45% . 
 the gissi - prevenzione trial demonstrated that a significant protective effect could be obtained with doses much lower than those previously considered necessary for significant beneficial effects . 
 the second gissi - hf study , a randomized double - blind placebo - controlled trial in 6975 italian patients with chronic heart failure , revealed a moderate decrease in both all - cause mortality admissions to hospital for cardiovascular disease upon supplementation for an average of 3.9 years with 1  g of lc n-3 pufa daily . 
 again , the beneficial effects were seen in a population already treated with recommended therapies . 
 the  japan eicosapentaenoic acid ( epa ) lipid intervention study ,  ( jelis ) trial , was performed on 18645 japanese men and women with hypercholesterolaemia treated with statins . 
 supplementation with 1.8  g epa daily decreased major coronary events by 19% over 4.6 years . 
 this may be evident in japanese population where a consistent reduction of chd is observed probably because of a high background seafood intake [ 25 , 26 ] . in summary , a modest intake of lc 
 however , higher doses and longer duration of intervention , as reported for the jelis study , have the potential to protect from non - fatal chd events . 
 presently , there is evidence to suggest that the two lc n3-pufas  epa ( eicosapentaenoic acid , 20:5n3 ) and dha ( docosahexaenoic acid , 22:6n3)exert pleiotropism ( akin to those effects which have been reported for certain cardiovascular drugs ) by modulating a range of diverse target mechanisms involved in cardiovascular disease development . [ 2730 ] . 
 since the appearance of purified forms of dha in the market in the 1990s , researchers have started to investigate the differential effects of epa and dha on cardiovascular health . however , the number of human studies is still limited in this field and the independent effects of epa and dha on various cardiovascular outcomes are yet to be firmly established . 
 furthermore , the incorporation of epa and dha into the cell membrane influences its organisation , fluidity , and permeability , as well as the activity of transmembrane proteins , including receptors , enzymes , and ion channels . 
 both epa and dha modulate k , na , and ca channel activities in myocardial cells , regulating myocyte electrical excitability and contractility [ 3234 ] . 
 these effects are concentration dependent and appear to be mediated by the action of epa and dha on membrane fluidity , although other mechanisms , such as a direct binding of n-3 lcp to the channel , may be involved . 
 furthermore , animal studies support the increasing evidence that dha rather than epa is preferentially incorporated into the myocardial cell membrane . 
 collectively , these findings help to explain the antiarrhythmic and heart rate ( hr)-lowering effects observed by dha but not by epa in humans . in addition , incorporation of dha into the membrane of cardiomyocytes influences the beta adrenergic system to a greater extent than epa , that is , potentially another important mechanism involved in the hypotensive and anti - arrhythmic action of dha . 
 furthermore , dha incorporation into the membrane of endothelial cells stimulates atp release from the endothelium , increasing vasodilation by stimulating nitric oxide ( no ) release . 
 the induction of no release , together with the decrease in noradrenaline levels , may also account for the bp - lowering effect of dha . a recent meta - analysis including thirty randomized controlled trials showed that prolonged fish oil intake may reduce hr , especially in populations with a high baseline hr . in dyslipidemic males and postmenopausal women 
 , this decrease appears to be mediated by dha rather than epa [ 37 , 41 , 42 ] . 
 in contrast , no significant effect of either epa or dha on hr was observed in healthy males for similar dosage and treatment duration . 
 hr variability ( hrv ) is a strong predictor of cvd , including sudden cardiac death , arrhythmic chd , and atrial fibrillation . 
 fish oils have shown anti - arrhythmic properties in animal studies , and several clinical and epidemiological studies have reported an association between hrv and n-3 lcp blood cell levels and/or fish oil intake [ 4446 ] . 
 short - term trials ( 612 week ) in healthy individuals showed no or low effect of a daily intake of 1.23.6  g of ala on blood lipids , ldl - oxidation , lipoprotein ( a ) , and apolipoproteins a - i and b [ 8 , 47 ] . 
 long - term treatment with high - dose ala ( 40  g / daily ) showed a beneficial effect by reduction of body weight and blood ldl / total cholesterol ratio [ 47 , 48 ] . 
 previous evidence favored recommendation for modest dietary consumption of ala ( 2 - 3  g daily ) in the primary and secondary prevention of chd . 
 the recently published case - control study by campos et al .   shows a strong inverse associations between ala status - intake and nonfatal mi . 
 recent data support the assumption that ala may protect against atherosclerosis [ 50 , 51 ] . 
 however , data from two recent epidemiologic studies suggest that high tissue ala is related to an increased rather than decreased risk of fatal cardiovascular events   and sudden death . 
 also , n-3 pufas may interfere with cvd with other physiological effects : the reduction of triglyceride ( tg ) synthesis is well recognized . moreover 
 , the reduction of hepatic vldl synthesis may contribute to this protective effect reducing the availability of fatty acids for triglyceride synthesis due to the decreased 
 de novo  lipogenesis ( dnl ) , increasing the beta - oxidation of fatty acids , reducing the delivery of nonesterified fatty acids to the liver , reducing the activity of tg synthesis by hepatocytes , and finally increasing the hepatic synthesis of phospholipids [ 5459 ] .   in both experimental models and human studies , 
 a different effect was reported in some studies between epa and dha : both epa and dha produced a significant reduction of tgs and ldl - cholesterol , raising effects of fish oil [ 61 , 62 ] . 
 recently , buckley and coworkers   showed that a four - week supplementation with dha significantly reduced tg levels in normolipidemic human subjects by 22% , while epa decreased tg levels by 15% without reaching statistical significance . in another four - week interventional study , 
 both epa and dha reduced postprandial tg without affecting fasting tg levels in healthy human subjects . 
 however , epa and dha seemed to reduce triglyceridaemia to the same extent when given for a long - enough period [ 60 , 61 , 6772 ] . 
 studies in humans show that an average intake of n-3 pufas 3 - 4  g / daily decreases serum triacylglycerol concentration by 2530% in a dose - dependent manner . 
 the same intake does not affect total cholesterol but increases ldl cholesterol by 510% and hdl cholesterol by 13% . 
 the increase in ldl cholesterol is mainly due to a rise of the larger and the potentially less atherogenic ldl particles . 
 hypertension ( ah ) and dyslipidemia ( dlp ) are the most important and frequent risk factors for cvd in the general population , with a prevalence of 5080% according to different authors [ 7579 ] . 
 on the other hand almost half of the patients with increased total cholesterol ( tc ) have systo - diastolic hypertension [ 8083 ] . 
 modifications of erythrocyte - membrane fatty acids ( fa ) composition are an early indicator of the development of ah and lipid disorders . 
 thus , modifications of erythrocyte - membrane fa are fairly subtle indicators of lipid metabolism pathology which manifest themselves much earlier than changes in plasma lipoproteins . 
 modifications of fatty acids composition of the cell membrane lipid matrix play an important role in the ah pathogenesis . 
 changes of the fatty acids ( fa ) composition with decrease of essential pufas may result in increase of membrane microviscosity , activation of pro - inflammatory eicosanoids synthesis and increased sensitivity of the smooth muscle cells in artery walls to the influence of vasoconstrictors . 
 that is the reason of the great interest in the study of the spectrum of erythrocyte lipids in hypertensive dyslipidemic patients . 
 deficiency of n-3 pufa is a distinctive feature of the modification of erythrocyte - membrane fa in hypertensive dyslipidemic patients . 
 deficiency of endogenous n-3 pufas may lead to changes in physicochemical properties of cell membranes , activation of the synthesis of proinflammatory and vasoconstrictive eicosanoids , and finally induction of a systemic inflammatory syndrome . 
 thus , structural and functional changes in erythrocyte cell membranes develop in hypertension as a part of the common systemic disorder of lipid metabolism . 
 the fa disorganization of the cell membrane may cause the development of hyperlipidemia and the progression of hypertensive disease . 
 thus , an important role of fa in the pathogenesis of cardiovascular disease seems clear . 
 a large part of the cardioprotective actions of lc n-3-pufas is likely to be mediated by the vascular endothelium [ 28 , 8688 ] . 
 this thin monolayer of cells plays a central role in cardiovascular homeostasis and function via the production of a range of potent autocrine and paracrine biochemical mediators that control the tone of vascular smooth muscle cells [ 31 , 89 , 90 ] . 
 vascular endothelium is also the site for the inception , progression , and clinical manifestations of atherosclerosis [ 9193 ] . 
 multifactorial endothelial dysfunction induced by dlp , toxins , cigarette - smoking , and so on may be the early event in the development of atherosclerosis . 
 the endothelium becomes  proadhesive  and induces an increased adhesion of circulating monocytes that subsequently infiltrate the arterial intima . at this level endothelial cells or macrophages 
 release reactive oxygen species that oxidize circulating ldl into oxidized - ldl and create the  lipid streak  . in the evolution from the lipid streak to the atherosclerotic plaque , 
 numerous cytokines that cause infiltration of smooth muscle layer by leukocytes and fibroblasts and promote platelet adhesion are involved in turn   ( figures 2 and 3 ) . during the migration over the vessel wall 
 , monocytes are exposed to various proinflammatory stimuli that contribute to their differentiation into macrophages . 
 other macrophages induce a local inflammatory compartment within the vassel wall , that is , continuously powered by cytokines , activated t - helper cells and scavanger receptor activators . 
 the inhibition of this self - maintaining process may reduce atherogenesis as demonstrated in mutant mice deficient for macrophage colony stimulating factor ( m - csf ) or macrophage migration inhibitory factor ( mif ) [ 95 , 96 ] . in more advanced lesions , cell debris , apoptotic cells , and free cholesterol particles 
 accumulate in the lipid - rich necrotic core of the plaque , are sometimes separated from the blood only by a thin fibrous cap . 
 the number of inflammatory cells within the lesion is strongly associated with the risk of rupture of the so - called  vulnerable plaque .  at last , the process may propagate intraluminal thrombosis , thereby distal ischemia of heart or brain tissue [ 97 , 98 ] . through their positive antithrombotic , lipid - lowering , proendothelial functional activities , n-3 
 in fact supplementation of n-3 pufas improves flow - mediated arterial dilatation by an increased endothelial synthesis of nitric oxide [ 86 , 99111 ] . 
 several , although not all , clinical trials have also found that consumption of n-3 pufas lowers circulating markers of endothelial dysfunction , such as e - selectin , vascular cell adhesion molecule-1 , and intracellular adhesion molecule-1 [ 74 , 112 , 113 ] . 
 thus , normalization of endothelial function could partly mediate the protective effects of n-3 pufa against cvd . a recent randomized clinical trial established the role of pufas on plaque stability . 
 patients with carotid artery atherosclerosis were randomized to take placebo , fish oil ( n-3 pufas ) , or seed oil ( n-6 pufas ) . 
 subjects treated with n-3 pufas showed higher epa and dha concentration , reduction of monocytes and macrophages , and a thicker fibrous cap compared to controls and group treated with n-6 pufas , all changes that can enhance the stability of atherosclerotic plaques . 
 by contrast , increased assumption of n-6 pufas did not affect carotid plaque fatty acid composition or stability . 
 plaques stability may explain the reductions in nonfatal and fatal cardiovascular events , associated with increased n-3 pufas intake . 
 the triglyceride - lowering , antiatherogenic , antithrombotic , and anti - inflammatory effects of n-3 pufas were clearly demonstrated in human studies . 
 conversely , their anti - arrhytmogenic effect was showed only in vitro and in animal experiments , as confirmation in humans has been limited by the absence of reliable physiological measures or biomarkers to quantify antiarrhythmic potential . 
 inflammation is a physiological response to tissue trauma or infection , but leukocytes , which are the effector cells of the inflammatory process , have powerful capabilities of tissue remodelling . 
 thus , the passage of leukocytes from the bloodstream into inflamed tissue is tightly regulated to ensure their precise localization . 
 recruitment of circulating neutrophils into the tissue stroma occurs during the early phases of inflammation . in this process 
 , peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells , through the vessel wall and out into the tissue stroma . 
 although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells , stimulated by the inflammatory cytokine ( tnf- ) , transit across the endothelial monolayer is regulated by additional stimuli . a new step in the neutrophil recruitment process that relies upon a lipid - mediated signal to regulate the migration of neutrophils across endothelial cells 
 this signal is supplied by the metabolism of arachidonic acid into the eicosanoid prostaglandin - d2 ( pgd2 ) by cyclooxygenase ( cox ) enzymes . 
 this step in the neutrophil recruitment process was revealed when the epa was used as an alternative substrate for cox enzymes , leading to the generation of prostaglandin - d3 ( pgd3 ) [ 117120 ] . 
 this alternative eicosanoid inhibited the migration of neutrophils across endothelial cells competing for the pgd2 receptor [ 121 , 122 ] . 
 pgd2 signalling is subordinate to the chemokine - mediated activation of neutrophils , but without the sequential delivery of this signal , neutrophils fail to penetrate the endothelial cell monolayer . the ability of dietary epa to inhibit this process reveals an unsuspected level of regulation in the migration of inflammatory leukocytes . 
 this may contribute to clarify the interactions between pufas and the inflammatory system and direct research on novel therapeutic agents that target the inflammatory system with greater affinity and/or specificity than dietary supplements of n-3-pufas . 
 the inflammation healing mechanisms are of main interest , and in recent years , new endogenous anti - inflammatory and proresolving lipid mediators generated from pufas ( lipoxins , resolvins , protectin , and maresin ) were uncovered . 
 lipid mediator metabolomics of self - resolving inflammatory exudates recently highlighted a new family of potent anti - inflammatory and proresolving mediators . 
 serhan and coworkers identified families of novel bioactive mediators derived from arachidonic acid ( aa - derived lipoxins ) , eicosapentaenoic acid ( epa - derived e - series resolvins ) , and docosahexaenoic acid ( dha - derived d - series resolvins , protectin , and maresin ) [ 124135 ] . 
 these lipid mediators promote resolution through enhanced clearance of apoptotic pmns , chemokines , cytokines , and microbial products by macrophages [ 136 , 137 ] . 
 n-3 pufas may act through several mechanisms , for instance , by preventing conversion of the n-6 pufa arachidonic acid to pro - inflammatory eicosanoids and by their conversion to potent anti - inflammatory mediators such as resolvins [ 124126 , 138 ] . 
 a possible biological mechanism underlying the beneficial effects of lc n-3 pufas on inflammation and endothelial function is that they may compete with n-6 fatty acids for prostaglandin and leukotriene synthesis at the cyclooxygenase and lipoxygenase levels . 
 lc n-3 pufas from fish or fish oil modulate prostaglandin metabolism by increasing the active vasodilator and inhibitor of platelet aggregation prostaglandin e3 , the weak platelet aggregator and vasoconstrictor thromboxane a3 , and the weak inducer of inflammation leukotriene b5 . otherwise , the production of thromboxane a2 , a potent platelet aggregator and vasoconstrictor , and leukotriene b4 , an inducer of inflammation and a powerful inducer of leukocyte chemotaxis and adherence is reduced . another suggested mechanism is that lc n-3 pufas may bind reactive oxygen species because of their multiple double bonds and lead to a decreased production of hydrogen peroxide . 
 hydrogen peroxide is a critical activator of the nuclear factor-b system of transcription factors that controls the coordinated expression of adhesion molecules and of leukocyte - specific chemoattractants upon cytokine stimulation . 
 he and coworkers   found independent inverse associations of lc n-3 pufas and nonfried fish with il-6 , crp and mmp3 . 
 the relevance of these biomarkers of inflammatory and endothelial activation in the atherogenic process is well recognized . 
 previous investigations suggest that both crp and il-6 , two systemic inflammatory markers , are independent predictors of cvd and may play an important role in atherogenesis . 
 in addition , mmp3 is suggested as an independent prognostic factor in stable coronary artery disease . 
 soluble intercellular adhesion molecule-1 ( sicam-1 ) is thought to be a key factor in the adherence of monocytes to the endothelium and subsequent transmigration into the intima ( figure 2 ) . 
 the role of sicam-1 in the pathogenesis of inflammation and atherosclerosis has been confirmed in experimental models . 
 previous investigations also indicate that sicam-1 is an independent predictor of cvd apart of other traditional risk factors . 
 .   observed that fried fish but not non - fried fish consumption was significantly inversely related to sicam-1 . 
 this finding is not expected since the frying process may reduce the content of lc n-3 pufas and produce trans - fatty acids . 
 one possible explanation is that fried fish consumption may be a marker of relatively unhealthy lifestyle ; those who had high fried fish consumption were more likely to suffer from dyslipidemia and were likely to be under treatment with medications ( e.g. , statins ) , which may lower sicam-1 . however , the inverse association between fried fish intake and sicam-1 level remained significant when patients taking cholesterol - lowering medications were excluded . 
 lc n-3 pufas are believed to affect inflammatory processes mainly through two pathways : endothelial activation and changes in eicosanoid production , or a combination of the two . 
 the first , predicated on the observation that n-3 pufas regulate the transcription of endothelial cell inflammatory genes by downregulating the activity of the nuclear factor - kb , predicts changes in the levels of adhesion receptor and chemokine expression after supplementation with epa [ 149151 ] . 
 the second hypothesis proposes that upon endothelial cell activation , epa may compete with the n-6-pufa arachidonic acid ( aa ; 20:4n-6 ) for cyclooxygenase enzymes ( cox1 and cox2 ) after both fatty acids are released from membrane phospholipids by endogenous phospholipases . 
 a large amount of investigations suggest , the cardioprotective effects of lc n-3 pufas epa and dha intake in human subjects , because of their lipid lowering , hypotensive , anti - arrhythmic , and anti - thrombotic properties . 
 moreover , studies performed in the last twenty years showed heterogeneous effects of different n-3 pufas on various cardiovascular outcomes , which may be of paramount relevance in primary and secondary prevention of cardiovascular disease . 
 recent in vitro investigations as well as clinical studies also demonstrated that lc - n3-pufas significantly interact with inflammation - related mechanisms , such as endothelial activation , modification of eicosanoid metabolism , and resolution of the inflammatory process . 
 low - grade chronic inflammation is present in several diseases and is characterized by abnormal circulating levels of pro- and anti - inflammatory cytokines . 
 n-3 pufas may modulate inflammation , that is , suggested by the reduction of plasma inflammatory cytokines ( tnf- , il-6 ) and inflammatory markers as high sensitive c reactive protein , observed after the intake of epa and dha . 
 low - grade chronic inflammation plays a key role in the induction and progression of atherosclerosis and consequently of cardiovascular disease . 
 taking into consideration the pleiotropic nature of their actions , we suggest that dietary intake of lc n-3 pufas may lead to improvements in cardio - metabolic health parameters of their antioxidant , anti - inflammatory , and antiarrhytmic actions .","phospholipids play an essential role in cell membrane structure and function . 
 the length and number of double bonds of fatty acids in membrane phospholipids are main determinants of fluidity , transport systems , activity of membrane - bound enzymes , and susceptibility to lipid peroxidation . 
 the fatty acid profile of serum lipids , especially the phospholipids , reflects the fatty acid composition of cell membranes . 
 moreover , long - chain n-3 polyunsatured fatty acids decrease very - low - density lipoprotein assembly and secretion reducing triacylglycerol production . n-6 and n-3 polyunsatured fatty acids are the precursors of signalling molecules , termed 
 eicosanoids ,  which play an important role in the regulation of inflammation . 
 eicosanoids derived from n-6 polyunsatured fatty acids have proinflammatory actions , while eicosanoids derived from n-3 polyunsatured fatty acids have anti - inflammatory ones . 
 previous studies showed that inflammation contributes to both the onset and progression of atherosclerosis : actually , atherosclerosis is predominantly a chronic low - grade inflammatory disease of the vessel wall . 
 several studies suggested the relationship between long - chain n-3 polyunsaturated fatty acids and inflammation , showing that fatty acids may decrease endothelial activation and affect eicosanoid metabolism .",pubmed
"our understanding of h pylori  induced inflammation leading to cancer is built on the work of 3 pioneering pathologists ( figure  1 ) . 
 rudolf virchow , a 19th century prussian physician - scientist , is widely regarded as the father of modern pathology . among his many contributions to outlining the scientific basis of disease was the idea , based on many of his own observations , that cancer arose from initially normal cells in response to chronic irritation , or inflammation . 
 numerous examples of inflammation - induced cancers are now appreciated , including many of the common gastrointestinal tract and hepatobiliary malignancies , such as acid reflux  induced esophageal adenocarcinoma , inflammatory bowel disease 
 associated colon cancer , and hepatocellular neoplasms associated with chronic viral hepatitis . throughout the 20th century , evidence accrued that gastric cancers tended to arise in stomachs already affected by chronic inflammation , especially atrophic gastritis with its accompanying hypochlorhydria , and that gastric cancer was a consequence and not a mere accompaniment of the gastritis . 
 these ideas set the stage for pelayo correa , a pathologist from colombia , a country with a particularly high gastric cancer prevalence . 
 after training in pathology in colombia and in the united states ( emory university ) , correa devoted his professional career at home and subsequently in the united states ( at the national cancer institute , louisiana state university , and now vanderbilt university ) to understanding the etiology of gastric cancer . in 1975 , correa et  al , from the national cancer institute , massachusetts institute of technology , and from his home country , published a  model for gastric cancer development .  in this landmark report , it was hypothesized that the development of the more common intestinal subtype of gastric cancer resulted from a stepwise process , beginning with chronic atrophic gastritis and progressing to intestinal metaplasia and cancer over the next 3050 years . 
 the initial changes were postulated to occur in the first decade of life , which we now know to be when h pylori colonization occurs . 
 a more detailed model , published in 1988 , included what was known of the phenotypic markers accompanying these sequential changes . 
 correa initially thought that the agent(s ) responsible for promoting this slow progression from gastritis to cancer were environmental , based on studies of migrants from high gastric cancer risk areas . 
 for example , japanese immigrants to hawaii and european immigrants to the united states had been shown to have lower gastric cancer rates than their parents and grandparents , more similar to those of the native population where they settled.7 , 8 the prime environmental culprit originally was thought to be a diet high in salt and n - nitroso - compounds and low in micronutrients from fresh fruits and vegetables . 
 it was postulated that this led to the promotion of gastric mutagenesis and , together with hypochlorhydria , bacterial overgrowth , thereby contributing to further nitrosamine formation . 
 interestingly , in his 1988 publication , correa briefly discussed a possible role for campylobacter pylori , a newly discovered gastric bacterium , in the initiation of the disease . 
 correa s subsequent work has focused primarily on the role of h pylori in gastric cancer , and this model has stood the test of time ( figure  2 ) . for his outstanding contributions to the field of gastric carcinogenesis , 
 the third pathologist of note is robin warren from australia who , together with barry marshall , was awarded the nobel prize in physiology or medicine in 2005 for the discovery of h pylori and its role in gastritis and peptic ulcer disease . in the 1970s , the widespread use of gastrointestinal endoscopy allowed pathologists 
 the opportunity to view gastric tissue that had been removed during a biopsy and fixed rapidly , without the artifacts inherent to the ischemia and autolysis of surgical specimens . 
 he then recruited marshall , a medical resident looking for a research project , to correlate the pathologic findings with the endoscopic features . 
 together , they discovered that these bacteria ( initially termed campylobacter pyloridis , then c pylori , and , subsequently , h pylori ) were very common in peptic ulcer patients . eventually , they successfully cultured these formerly elusive bacteria and showed that they caused gastritis and ulcer disease.11 , 12 , 13 , 14 in retrospect , other investigators also had observed such bacteria over the preceding century , but their clinical significance had not been appreciated and they had even been proven to be a post mortem artifact before their rediscovery in australia . although warren and marshall did not investigate the role of h pylori in gastric cancer directly , they were aware of the relationship between gastritis and cancer . 
 indeed , marshall speculated with amazing prescience in their very first publications ( in unusual side - by - side , single - author letters in 1983 ) that  if these bacteria are truly associated with antral gastritis , as described by warren , they may have a part to play in other poorly understood , gastritis associated diseases ( ie , peptic ulcer and gastric cancer ) . 
 initial investigations into a possible association of h  pylori with gastric cancer that were conducted in the 1980s and early 1990s provided only weak evidence for a link between h pylori and gastric cancer . a positive correlation between h pylori seroprevalence and gastric cancer in cross - sectional sampling 
 was reported in a study conducted among 13 european nations . however , although some other publications reported similar trends,17 , 18 several others did not find a positive correlation between the presence of h pylori antibodies and either gastric cancer or precancerous gastric lesions.19 , 20 , 21 , 22 around this time , clinicopathologic studies performed to look for evidence of h pylori infection ( by serum antibodies ) or in gastric tissue directly in cases of gastric cancer also yielded inconsistent results . 
 some studies showed h pylori infection rates no higher than those observed in noncancer controls . 
 this is most likely because by the time gastric cancer has developed , extensive intestinal metaplasia and hypochlorhydria had rendered the stomach less hospitable to persistent h pylori colonization , thus also explaining some of the negative findings in the cross - sectional studies discussed earlier . 
 much more convincing evidence for the role of h pylori in gastric cancer came from 3 large cohorts with nested case - controls , in which serum had been banked from cancer - free subjects and the cohort had been followed up for approximately a decade.24 , 25 , 26 in each cohort , evidence of prior h pylori infection ( evaluated by enzyme - linked immunosorbent assay [ elisa ] in the banked serum ) was found to be significantly more common in those subjects who subsequently developed gastric cancer compared with a sample of those who had not ( table  1 ) . in a meta - analysis , these 3 studies provided an overall odds ratio for gastric cancer development in h pylori  infected vs -uninfected persons of 3.8 ( 95% confidence interval [ ci ] , 2.36.2 ) . based on the compelling results of these cohort studies , the world health organization s international agency for research on cancer declared in 1994 that there was sufficient evidence to classify h pylori as a definite ( group 1 ) carcinogen . 
 this was despite the fact that at that time there was no significant supporting evidence from either animal models or mechanistic basic science research . 
 subsequent research worldwide has confirmed and expanded upon the early epidemiologic studies , with the key additional clinical findings as follows:1.cardia cancers are not associated strongly with h  pylori infection , supporting the idea that cardia cancers arise via alternative mechanisms , with risk factors more similar to cancers of the gastroesophageal junction and lower esophagus.2.in pooled meta - analysis , the odds ratio for h pylori in gastric cancer was no different when considering intestinal vs diffuse histologic cancer subtypes . 
 thus , in addition to promoting the well - established correa cascade of intestinal - type cancers , h pylori is just as great a risk factor for the much less well - defined pathway leading to diffuse - type gastric cancer.3.measuring h pylori exposure by elisa underestimates prior exposure and , therefore , the attributable h pylori risk in noncardia cancer , because of false - negative results . 
 for example , in a retrospective swedish case - control study , measuring prior exposure through a western blot against h pylori cytotoxin - associated gene a ( caga ) , rather than a conventional igg elisa , increased the odds ratio from 2.2 ( 95% ci , 1.43.6 ) to 21.0 ( 95% ci , 8.353.4).4.in noncardia gastric cancers , caga - positive h pylori strains ( which are the more common strains worldwide ) increase the risk of gastric cancer to a greater extent than do infections by the more rare caga - negative strains . 
 this is consistent with the oncogenic effects of the caga protein that have been evaluated mainly in  vitro.5.h pylori eradication can reduce the risk of developing gastric cancer ( see later ) . 
 cardia cancers are not associated strongly with h  pylori infection , supporting the idea that cardia cancers arise via alternative mechanisms , with risk factors more similar to cancers of the gastroesophageal junction and lower esophagus . in pooled meta - analysis , 
 the odds ratio for h pylori in gastric cancer was no different when considering intestinal vs diffuse histologic cancer subtypes . 
 thus , in addition to promoting the well - established correa cascade of intestinal - type cancers , h pylori is just as great a risk factor for the much less well - defined pathway leading to diffuse - type gastric cancer . 
 measuring h pylori exposure by elisa underestimates prior exposure and , therefore , the attributable h pylori risk in noncardia cancer , because of false - negative results . 
 for example , in a retrospective swedish case - control study , measuring prior exposure through a western blot against h pylori cytotoxin - associated gene a ( caga ) , rather than a conventional igg elisa , increased the odds ratio from 2.2 ( 95% ci , 1.43.6 ) to 21.0 ( 95% ci , 8.353.4 ) . in noncardia gastric cancers , caga - positive h pylori strains ( which are the more common strains worldwide ) increase the risk of gastric cancer to a greater extent than do infections by the more rare caga - negative strains . 
 this is consistent with the oncogenic effects of the caga protein that have been evaluated mainly in  vitro . 
 h pylori eradication can reduce the risk of developing gastric cancer ( see later ) . 
 in a prolonged observational study following patients who had attended a japanese endoscopy unit , uemura et  al showed that those who were h pylori  infected ( and  in whom the infection was not treated ) had a much higher rate of progression to gastric cancer over the  subsequent 12 years than the patients who had no  initial evidence of h pylori . 
 however , to prove conclusively that h  pylori is responsible for the increased gastric cancer risk , prospective interventional studies were necessary . 
 recruitment into such studies was hampered by the 1994 declaration of h pylori as a definite carcinogen , after which it became ethically problematic to enter patients into the control arms of eradication studies designed to determine the gastric cancer risk reduction . 
 consequently , an appropriately powered , definitive study of a large number of subjects followed up for many years to the end point of gastric cancer was never completed . nevertheless , recent meta - analyses of the many underpowered studies that were conducted are conclusive , indicating that h pylori eradication decreases the risk of gastric cancer development by approximately 40% in studies of primary prevention ( asymptomatic individuals ) , and by 54% as a tertiary prevention strategy ( preventing the occurrence of a second gastric malignancy after endoscopic resection of an early gastric cancer).36 , 37 it is not known whether or not there is a point of no return along the correa pathway , beyond which h pylori eradication will not prevent progression to gastric cancer . in the analysis by lee et  al , which included 24 publications of more than 48,000 individuals followed up for more than 340,000 person - years ( 14 studies of primary prevention , 10 studies of tertiary prevention ) , the benefit of h pylori eradication was , not surprisingly , most evident in subjects living in areas with the highest gastric cancer prevalence . 
 because all investigations were conducted in parts of the world where gastric cancer is most prevalent ( mostly in south - east asia ) , it is uncertain if similar reductions can be extrapolated to regions where noncardia gastric cancer is less common , such as the united states . 
 however , it may be reasonable to suppose that high - risk populations in low - risk countries , including immigrants from central and south america and  south - east asia to the united states who remain h  pylori  infected from childhood , should benefit greatly from h pylori eradication too . 
 however , with the exception of endoscopic screening in south korea , and radiologic and endoscopic programs in japan , there had been little effort made previously to control this burden with public health programs . 
 the results of the recent intervention studies reported earlier have now prompted renewed interest in screening for h pylori and eradicating the organism when found as a potentially cost - effective strategy , especially in parts of the world that have a high prevalence of h pylori and gastric cancer.39 , 40 , 41 a pilot study of mass population screening and eradication started in the matsu islands of taiwan in 2004 , and the initial results are very promising . gastric cancer incidence has decreased by approximately 25% , peptic ulcers by two thirds , and gastric atrophy by 77% compared with historical data . 
 several other population - based trials are underway , including one with almost 100,000 chinese subjects from linqu county , a region of high gastric cancer prevalence . 
 the eradication of h pylori from a community should help prevent much gastric cancer , as well as greatly diminish the morbidity of peptic ulcers too . 
 it has been calculated that the number needed to treat to prevent a case of gastric cancer may be as low as 15 in chinese men , to as high as 245 in women in the united states . although there is a concern regarding the ecologic consequences of using multiple antibiotics in a large proportion of the population to prevent h pylori  related disease , the recent exciting results of vaccination against h pylori with an oral urease b  construct suggest that a nonantibiotic strategy may be possible in the future , with potentially less impact on the general human microbiome . 
 further investigation of this vaccine should be a priority , particularly because promising preclinical models with a variety of vaccine candidates since the 1990s had never resulted previously in impressive results in clinical trials . 
 if preventive or therapeutic vaccination emerges as a viable alternative for h pylori eradication , it will help avert widespread antibiotic administration and the consequent problems of microbiome perturbation and the generation of microbial antibiotic resistance among h pylori and other innocent bystander bacteria . 
 in the midst of growing optimism for implementing h  pylori eradication strategies in high gastric cancer risk regions , it is important to recognize that there is considerable hesitation to adopt similar approaches in the developed world , mainly out of concern for a beneficial effect of h  pylori on esophageal diseases . 
 the results of multiple studies have indicated that h  pylori is significantly less common in patients with esophageal adenocarcinoma than in matched controls , although h pylori infection has no association , positive or negative , with esophageal squamous cancer47 , 48 ( figure  3 ) . 
 this places h pylori in a unique position among the world health organization s list of biological agents that cause cancer , in that its presence is associated positively with certain cancers ( gastric noncardia adenocarcinoma , gastric marginal zone b - cell lymphoma ) although related inversely to another cancer type ( esophageal adenocarcinoma ) . 
 the absence of h pylori may promote more severe esophageal mucosal damage , barrett s metaplasia , and the consequent development of esophageal adenocarcinoma . 
 a postulated mechanism is through a greater gastric acid secretory capacity from a pristine , uninflamed , parietal cell mass in the absence of h pylori gastritis , although there is little evidence that this actually leads to enhanced esophageal acid exposure . 
 an inverse relationship between h pylori and esophageal adenocarcinoma does not necessarily imply that h pylori is protective any more than a positive association can be taken to be evidence of causation . 
 h pylori has been an almost universal inhabitant of the human stomach from the dawn of mankind until the 1980s . 
 its recent disappearance from the 21st human microbiome , one of the  missing microbes ,  has been linked to extragastric immune dysregulation ( for which there is good evidence from animal models ) and a multitude of other contemporary maladies ( eg , asthma , obesity , and inflammatory bowel disease ) , for which the evidence is questionable and the biological plausibility equally uncertain.55 , 56 , 57 the decrease in noncardia gastric cancer in north america and western europe over the past 100 years that coincided with ( or maybe even preceded ) decreasing h  pylori prevalence , was famously described as the  epidemiology of an unplanned triumph .  the rapid increase in esophageal adenocarcinoma in the same population more recently ( such that it is now approaching the prevalence of noncardia gastric cancer ) perhaps may be considered the unplanned downside of the disappearance of h pylori from western stomachs . 
 it is this unexpected accompaniment of h pylori eradication that largely has tempered enthusiasm for population - based h pylori eradication efforts in the developed world . whether to pursue population - based h  pylori screening and treatment strategies 
 therefore will need to be individualized according to the current and projected future medical needs of each community , and these differ quite markedly around the globe . 
 the declaration of h pylori as a definite gastric carcinogen in 1994 and the subsequent realization that the vast majority of noncardia cancers are attributable to h pylori infection has led to a fundamental change in the way that we view gastric cancer pathogenesis in the funderburg era . considering gastric cancer as the consequence of an infection has led to enthusiasm for screening for and eradicating h pylori in areas of high gastric cancer prevalence , but the fact that gastric cancer remains a rare consequence of h  pylori infection provides plenty of opportunity for investigating the co - factors that promote gastric neoplastic development after h pylori colonization . 
 recent research in this field has focused on genetic susceptibility ( such as polymorphisms in genes governing gastric inflammatory responses ) , h pylori heterogeneity , and on other environmental influences , such as dietary salt or the presence of non - helicobacter species within the gastrointestinal microbiome that may explain why only a small proportion of individuals who are colonized by h  pylori go on to develop gastric cancer . 
 this knowledge ultimately may have clinical utility in stratifying individuals with h pylori infection into high vs low gastric cancer risk , and to the creation of personalized surveillance , chemoprevention , and dietary intervention programs focused on patients at highest risk . 
 as an example , an early study reported enormous differences ( up to 90-fold ) in gastric cancer susceptibility in h pylori  infected cases from portugal , when stratified by polymorphisms in interleukin 1 and the interleukin 1receptor antagonist , together with h pylori vacuolating cytotoxin a ( vaca ) and caga typing . 
 investigating the precise molecular and cellular mechanisms of gastric cancer development associated with h pylori infection is likely to provide additional insights into gastric cancer , as well as stimulate ideas about the pathogenesis of other cancers that are known to be associated with inflammation , although not necessarily with specific constituents of the microbiome . 
 since 1994 , considerable progress has been made on dissecting the role of h pylori role in the molecular pathogenesis of inflammation - associated gastric cancer ( as outlined in the review by crowe in this issue ) . 
 the multiplicity of animal models to study h pylori  associated gastric carcinogenesis ( reviewed in tsukamoto et  al and krueger et  al , as well as by wang in this issue ) also has provided many important mechanistic insights that were not conceivable back in 1994 , when rodent infection studies were in their infancy and the evidence for h pylori as a carcinogen was based on a limited number of clinical studies . 
 support from the funderburg family has been instrumental in promoting new ideas about gastric cancer and its origins , as well as opening new therapeutic possibilities . 
 the work that started in our laboratory in 2002 as a result of this funding has allowed us to investigate how h pylori misregulates the expression of p27 , a cyclin - dependent kinase inhibitor that is an important constitutive endogenous cell - cycle regulator.67 , 68 we also subsequently explored the p27-deficient mouse as a model with which to study the mechanisms of h pylori  induced gastric carcinogenesis,69 , 70 and to examine its reversibility after h  pylori eradication . 
 our understanding of gastric carcinogenesis continues to grow rapidly . for many years , pathologists were at the cutting edge of this knowledge , based on their meticulous observations of the unfortunate patients who died of this largely untreatable disease . 
 the realization that a single bacterial species was the inciting cause of the majority of gastric cancers went against the prevailing clinical and scientific dogma . as a result of this initially uncomfortable discovery , gastric cancer , which was a disease that formerly was principally the focus of pathologists and surgeons , 
 has become widely embraced by the scientific and clinical community as a model of bacterial carcinogenesis and a window to inflammation - associated cancers in general .","gastric cancer has long been recognized to be accompanied and preceded by chronic gastritis , lasting decades . 
 arguably , the most important development in our understanding of gastric cancer pathogenesis over the past 50 years has been the realization that , for most cases of gastric cancer , helicobacter pylori is the cause of the underlying gastritis . 
 gastritis can promote gastric carcinogenesis , typically via the correa cascade of atrophic gastritis , intestinal metaplasia , and dysplasia . 
 nested case - control studies have shown that h pylori infection increases the risk of gastric cancer significantly , both of the intestinal and diffuse subtypes , and that h pylori is responsible for approximately 90% of the world s burden of noncardia gastric cancer . based largely on randomized studies in high gastric cancer prevalence regions in east asia 
 , it appears that primary and tertiary intervention to eradicate h pylori can halve the risk of gastric cancer . 
 some public health authorities now are starting screening and treatment programs to reduce the burden of gastric cancer in these high - risk areas . 
 however , there is currently much less enthusiasm for initiating similar attempts in the united states . 
 this is partially because gastric cancer is a relatively less frequent cause of cancer in the united states , and in addition there are concerns about theoretical downsides of h pylori eradication , principally because of the consistent inverse relationship noted between h pylori and esophageal adenocarcinoma . nevertheless , 
 establishing a link between chronic h pylori infection and gastric cancer has led to novel insights into cancer biology , the gastrointestinal microbiome , and on individual and population - based gastric cancer prevention strategies .",pubmed
"neovascular age - related macular degeneration ( namd ) is a leading cause of visual loss among elderly population [ 1 , 2 ] . before the introduction of intravitreal antivascular endothelial growth factor ( anti - vegf ) agents for the treatment of namd , only prevention from visual acuity loss 
 might have been achieved in a limited number of patients with different treatment options like laser photocoagulation , photodynamic therapy , and vitreoretinal surgery [ 39 ] . 
 intravitreal treatments with bevacizumab ( full length antibody against vegf - a ) and ranibizumab ( fab part of antibody against vegf - a ) have led the majority of the patients to prevent the baseline visual acuity ( va ) and even to achieve visual improvement in some of the patients [ 10 , 11 ] . 
 the multicenter studies with ranibizumab , like marina , anchor , pronto , and excite , and the comparative study of ranibizumab and bevacizumab , the catt study , showed that ranibizumab and bevacizumab were effective to prevent va loss up to 95% of the patients and is effective to make an improvement in va up to 40% of the patients [ 1115 ] . 
 today , the catt trial answered the questions about the efficacy of bevacizumab versus ranibizumab and showed that both drugs had similar effects in the treatment of namd . 
 however , the safety of the drugs still remains unclear , and there is an ongoing debate about this issue . in this study , we aimed to compare the functional and anatomical outcomes of bevacizumab therapy to ranibizumab therapy on as - needed treatment regimen for namd in turkish patients . 
 the clinical data of the patients who were treated with either bevacizumab or ranibizumab between 2009 and 2011 were retrospectively evaluated for this study . 
 inclusion criteria were age of 50 years or more , newly diagnosed namd , no treatments received other than bevacizumab alone or ranibizumab alone , and a minimum follow - up period of 12 months . 
 patients were not included if they had any of the following criteria : retinal diseases other than namd , previous intravitreal injections , or a history of photodynamic therapy or laser photocoagulation . 
 the tenets of the declaration of helsinki were followed throughout the study , and written informed consent was obtained from all patients before the treatments were started . 
 data collected from the patients ' records included age , gender , type of choroidal neovascularization ( cnv ) ( predominantly or minimally classic or occult ) , bcva , and central retinal thickness ( crt ) before treatment , at months 3 , 6 , 9 , and 12 during treatment , and at the most recent follow - up examination . 
 all patients underwent a standardized examination including measurement of bcva via the early treatment diabetic retinopathy study ( etdrs ) chart at 4 meters , slit - lamp biomicroscopy and fundus examination and measurement of intraocular pressure ( iop ) via applanation tonometry . 
 fundus photography , fluorescein angiography ( hra-2 , heidelberg engineering , heidelberg , germany ) , and optical coherence tomography ( oct ) imaging ( stratus oct tm , carl zeiss meditec inc . , 
 all examinations were repeated monthly , except fluorescein angiography , which was repeated only when the cause of visual acuity deterioration could not be clarified with the clinical examination and other imaging methods . 
 oct was used for detecting subretinal fluid and measurement of crt , the latter being defined as the mean thickness of the neurosensory retina in the central 1  mm diameter region , computed via oct mapping software provided with the device . 
 all injections were performed under sterile conditions after topical anesthesia , and 10% povidone - iodine scrub ( betadine , purdue pharma , stamford , ct , usa ) was used on the lids and lashes , and then 5% povidone - iodine was administered on the conjunctival sac . 
 intravitreal bevacizumab ( avastin , genentech , south san francisco , ca , usa ) or ranibizumab ( lucentis , novartis , basel , switzerland ) was injected with a 30-gauge needle through the pars plana at 3.5  mm to 4  mm posterior to the limbus . 
 patients were then instructed to consult the hospital if they experienced decreased vision , eye pain , or any new symptoms . for the first three months of treatment , 
 all patients received monthly doses of bevacizumab , or ranibizumab ( 1.25  mg/0.05  ml for bevacizumab , 0.5  mg/0.05  ml for ranibizumab ) . 
 the patients were then examined monthly and were retreated if they met any of the following criteria : visual loss of 1 or more lines , newly developed macular hemorrhage , evidence of cnv enlargement on examination or fluorescein angiography , any amount of persistent subretinal fluid one month after an injection . 
 visual loss of 1 or more lines , newly developed macular hemorrhage ,   evidence of cnv enlargement on examination or fluorescein angiography , any amount of persistent subretinal fluid one month after an injection . 
 visual acuity was converted to logarithm of minimum angle of resolution ( logmar ) for statistical analysis . 
 the changes in bcva and crt over time were analyzed with paired sample t - test . 
 chi - square test was used to compare nominal parameters between the groups , and independent sample t - test was used for continuous parameters . 
 the statistical analysis was performed using spss version ( version 15.0 , spss inc . , 
 the bevacizumab group consisted of 79 patients , and the ranibizumab group consisted of 74 patients . 
 the general characteristics of the patients were similar between the two groups ( table 1 ) . 
 the mean bcva of the patients in the bevacizumab group at baseline , months 3 , 6 , 9 , 12 , and at the most recent followup was 1.02  0.46 logmar ( range 0.12.1 logmar ) , 0.86  0.46 logmar ( range 0.22.1 logmar ) , 0.83  0.43 logmar ( range 0.22.1 logmar ) , 0.82  0.43 logmar ( range 0.22.1 logmar ) , 0.82  0.42 ( range 0.22.1 logmar ) , and 0.83  0.43 ( range 0.22.1 logmar ) , respectively . 
 the mean bcva of the patients in the ranibizumab group at baseline , months 3 , 6 , 9 , 12 , and at the most recent followup was 0.97  0.46 logmar ( range 0.12.1 logmar ) , 0.83  0.47 logmar ( range 0.11.8 logmar ) , 0.81  0.47 logmar ( range 0.01.8 logmar ) , 0.81  0.42 logmar ( range 0.11.8 logmar ) , 0.82  0.43 ( range 0.01.8 logmar ) , and 0.82  0.47 ( range 0.02.1 logmar ) , respectively ( figure 1 ) . 
 the change in mean bcva from baseline to months 3 , 6 , 9 , 12 , and the most recent followup was statistically better in both of the groups ( p < 0.05 , for all ) . 
 however , there was not a statistically significant difference between the two groups in regards of change in bcva at all of the time points ( p = 0.7 for month 3 , p = 0.6 for month 6 , p = 0.5 for month 9 , p = 0.5 for month 12 , and p = 0.6 for the most recent followup ) ( table 2 ) . at the most recent followup , 34 of the 79 patients ( 43.0% ) in the bevacizumab group and 30 of the 74 patients ( 40.5% ) in the ranibizumab group gained va  3 lines ( p = 0.4 ) . 
 sixty - eight of the 79 patients ( 86.1% ) in the bevacizumab group and 64 of the 74 patients ( 86.5% ) in the ranibizumab group had stable or improved vision ( loss of < 3 lines , remained stable , or gained  1 lines ) ( p = 0.5 ) . 
 eleven of the 79 patients ( 13.9% ) in the bevacizumab group and 10 of the 74 patients ( 13.5% ) in the ranibizumab group had va loss  3 lines ( p = 0.5 ) . 
 the mean crt in the bevacizumab group at baseline , and at months 3 , 6 , 9 , and 12 , and at the most recent followup was 335  120  m ( range 180758  m ) , 289  118  m ( range 106796  m ) , 266  79  m ( range 140558  m ) , 260  93  m ( range 148713  m ) , 266  100 ( range 150789  m ) , and 262  124 ( range 103979  m ) , respectively . 
 the mean crt in the ranibizumab group at the baseline , and at months 3 , 6 , 9 , and 12 , and at the most recent followup was 315  88 ( range 139681  m ) , 250  68  m ( range 133487  m ) , 243  63  m ( range 135410  m ) , 254  72  m ( range 135540  m ) , 247  60 ( range 140415  m ) , and 256  73 ( range 130483  m ) , respectively ( figure 2 ) . 
 the mean crt at months 3 , 6 , 9 , and 12 , and at the most recent followup was statistically different compared to baseline in both of the groups ( p < 0.05 for all ) . 
 however , the change in mean crt from the baseline to months 3 , 6 , 9 , and 12 , and at the most recent follow - up was not statistically different between the two groups ( p = 0.2 , p = 0.8 , p = 0.3 , p = 0.9 , and p = 0.2 , resp . ) ( table 3 ) . 
 the mean number of injections at month 12 was 4.8  1.2 ( range 37 ) in the bevacizumab group and 4.7  1.4 ( range 38 ) in the ranibizumab group , and the difference was not statistically significant between the two groups ( p = 0.8 ) . 
 the mean baseline intraocular pressure ( iop ) was 15.5  2.3  mm  hg ( range 919  mm  hg ) and 15.4  1.7 ( range 1220  mm  hg ) in the bevacizumab and ranibizumab groups , respectively ( p = 0.7 ) . 
 the mean iop at the most recent followup was 15.3  2.4  mm  hg ( range 1022  mm  hg ) and 15.6  2.1 ( range 1122  mm  hg ) , in the bevacizumab and ranibizumab groups , respectively ( p = 0.4 ) . 
 no serious ocular or systemic complications were observed in any of the patients . only mild complications like punctate keratitis , subconjunctival hemorrhage , and transient mild anterior uveitis 
 functional and anatomical outcomes of namd patients treated with bevacizumab and ranibizumab in a population of turkish patients were reported in this study . in both of the groups , 
 the mean bcva and crt were significantly better than that in baseline at every time point . 
 the two groups were parallel to each other with respect to change in bcva and crt , injection numbers , and complications .   in previous clinical studies , these two drugs , bevacizumab or ranibizumab , 
 have been found to provide similar improvements in visual and anatomical outcomes to our study [ 13 , 1620 ] .   a short term efficacy and safety study by landa et al . 
 reported that bevacizumab and ranibizumab treatments resulted in similar improvements in visual and anatomical outcomes one month after an intravitreal injection in patients with namd . 
 they also stated that bevacizumab was as safe as ranibizumab .   in a retrospective study by feng et al . 
 , 371 namd patients who were treated with intravitreal bevacizumab or ranibizumab on an as - needed treatment regimen were evaluated . 
 they reported that , after a median follow - up time of 12 months , 24.5% of patients in the bevacizumab group and 25.8% of patients in the ranibizumab group had a gain of 15 or more letters ; in addition , 79.5% of the patients in the bevacizumab group , and 84.9% of the patients in the ranibizumab group lost fewer than 15 letters in visual acuity . 
 they stated that there was not a statistical difference in regards of visual acuity changes between the two groups . 
 we also did not find any difference in visual acuity change at different time points between the bevacizumab and ranibizumab groups . 
 and the rate of the patients who were stable in regards of visual acuity results was very similar to the study by feng et al . 
 reported that bevacizumab and ranibizumab were both effective in stabilizing visual acuity in namd patients after 12 months of followup . in the aforementioned study 
 , it was reported that visual acuity outcomes were similar between the two drugs ; 27.3% of the patients in the bevacizumab and 20.2% of the patients in the ranibizumab group were reported to have an improvement of three or more lines in visual acuity , and the difference between the groups was not found to be statistically significant . 
 the mean injection numbers at month 12 were 4.4 in the bevacizumab group and 6.2 in the ranibizumab group which were statistically different between the groups . 
 the mean injection numbers at month 12 were similar to our study ; however , we did not find a difference between the two groups . in a retrospective study concerning the efficacy of bevacizumab and ranibizumab in namd by bellerive et al . 
 , the mean visual acuity improvement at month 12 was greater in the ranibizumab group than the bevacizumab group . 
 they stated that the proportion of occult lesions were greater in the bevacizumab group than the ranibizumab group and postulated that this trend might be explained by this variability . 
 also the proportion of the patients who lost fewer than 0.3 logmar was reported to be 83% in the bevacizumab group and 92 in the ranibizumab group , with a mean number of injections of 4.7 in the bevacizumab group and 4.9 in the ranibizumab group at month 12 . 
 the visual acuity outcomes and the mean injection numbers of the study were comparable with our study . 
 however , the visual and anatomical outcomes were similar between the groups in our study . 
 the catt study was designed as a multicenter , noninferiority trial to compare the efficacy and safety of bevacizumab and ranibizumab . in the study , 
 both monthly and as - needed treatment regimens were evaluated within and between the two drugs . at first year , it was reported that there was not a significant difference between the two drugs in regards of visual acuity and anatomical outcomes and the mean injection numbers . at month 12 
 , as - needed bevacizumab was found to be equivalent to as - needed ranibizumab with a gain of 5.9 etdrs letters and 6.8 etdrs letters in visual acuity , respectively . at month 12 , the eyes treated with as - needed bevacizumab and ranibizumab showed a significant decrease in crt , and the difference was not statistically significant between the bevacizumab groups and ranibizumab groups . 
 the mean injection numbers of as - needed bevacizumab group and as - needed ranibizumab group were 7.7 and 6.9 at month 12 , respectively . 
 the two - year results of the study were similar to the first - year results ; however the mean gain in visual acuity was found to be greater in monthly groups than as - needed groups . 
 the study also addressed the safety issue and adverse events between the two drugs . at 2 years , it was reported that 6.1% of the patients in the bevacizumab group and 5.3% of the patients in the ranibizumab group had died . 
 the proportion of the patients with atherothrombotic events and venous thrombotic events were similar between the two groups . 
 the rate of serious adverse events was found to be greater in the bevacizumab groups than the ranibizumab groups with a cumulative risk ratio of 1.30 ; however , this difference was not found to be statistically significant . 
 in addition , the studies which addressed the safety issues of bevacizumab and ranibizumab showed that this issue still remains as a debate [ 2123 ] . 
 strengths of the study include the fact that patients in the bevacizumab and ranibizumab groups had received no other forms of treatment and similar baseline characteristics of the two groups . in summary , in the present study we found that 86.1% of the patients in the bevacizumab group and 86.5% of the patients in the ranibizumab group had a stable or improved visual acuity after a mean follow - up time of approximately 18 months . 
 the visual and anatomical outcomes and the number of injections were similar between the two drugs . 
 the patients with namd treated with as - needed bevacizumab or as - needed ranibizumab showed significant improvements in visual acuity throughout at least 1.5 years compared to baseline visual acuity .","purpose . to compare the efficacy of intravitreal bevacizumab versus ranibizumab in the treatment of neovascular age - related macular degeneration ( namd ) 
 . 
 methods . 
 retrospective , comparative study . 
 the newly diagnosed namd patients who were treated with intravitreal bevacizumab or ranibizumab on an as - needed treatment regimen were included in the study . 
 main outcome measures were the change in best corrected visual acuity ( bcva ) , and central retinal thickness ( crt ) . 
 secondary outcome measures were the number of injections , and complications . 
 results . 
 a total of 154 patients were included in the study . 
 bevacizumab group consisted of 79 patients , and ranibizumab group consisted of 74 patients . 
 mean follow - up time was 18.9 months , and 18.3 months in the bevacizumab and ranibizumab groups , respectively . 
 there was not a significant difference between the two groups regarding the change in bcva and crt at all time points ( p > 0.05 for all ) . 
 the mean number of injections at month 12 was 4.8 and 4.7 in bevacizumab and ranibizumab groups , respectively ( p > 0.05 ) . 
 no serious complications were detected in any of the groups . 
 conclusion . both of the bevacizumab and ranibizumab found to be effective in the treatment of namd in regards of functional and anatomical outcomes with similar number of treatments and similar side effects .",pubmed
"preeclampsia ( pe ) is a leading cause of maternal and neonatal mortality and morbidity , complicating 25% of all the pregnancies . 
 classically , the presence of proteinuria in women with gestational hypertension is clinically diagnosed as pe and is associated with increased maternal or neonatal morbidity and mortality compared with gestational hypertension . 
 however , several studies have shown that proteinuria is not a significant predictor of adverse maternal and neonatal outcomes . 
 the incidence of the disease increased in china , as the opening of the two - child policy , but there was not an indicator of the severe pe . 
 severe pe is often combined with hypoalbuminemia ; we hypothesize that hypoalbuminemia may serve as an indicator of the severity of pe . 
 hypoalbuminemia occurs mainly due to systemic small vessel spasm , increased secretion of angiotensin and damaged and increased permeability of vascular endothelial cells , thereby leading to a large number of proteins and liquid leaking in tissue clearance and a loss of a large number of plasma proteins ( especially serum albumin ) , causing intravascular dehydration . 
 this intravascular dehydration may accelerate the appearance of intravascular lesions , which is thought to be a predisposing factor of the hemolysis , elevated liver enzymes and low platelet ( hellp ) syndrome and acute fatty liver of pregnancy . 
 in addition , the reduction of hepatic blood flow due to the production of albumin is decreased . in two recent studies , hypoproteinemia during pregnancy 
 was observed in the pe group in the second trimester before the onset of clinical symptoms , and the authors suggest the possibility of preventing pe by maintaining adequate blood protein levels during early pregnancy . in the present study 
 , we asked whether hypoproteinemia was effective at predicting perinatal risk in women with pe . 
 the aim of our study was to evaluate maternal and perinatal outcomes in women with pe , according to the different value of albumin ( 25 g / l and < 25 
 in this retrospective cohort study , 299 women from the maternal and child health hospital of anhui province were enrolled between january 2014 and june 2015 . 
 the anhui medical university ethics committee provided ethics approval , and written informed consent was obtained from all participants . 
 g / l ( mild hypoproteinemia [ mhp ] , n = 220 ) , pe with an albumin value of 25 
 g / l ( severe hypoproteinemia [ shp ] , n = 79 ) . for the study 
 serum albumin was measured at the laboratory of the maternal and child health hospital of anhui province , using the unicel dxc800 of beckman coulter ( the united states ) . 
 pe was defined according to the guidelines of the international society for the study of hypertension in pregnancy : ( 1 ) hypertension : blood pressure 140/90 mmhg on at least two occasions and at least 46 h apart ; ( 2 ) the severe features , such as severe hypertension , platelet count below 100,000/l , serum creatinine concentration of 1.1 mg / dl , or doubling of the serum creatinine concentration in the absence of other renal disease , levels of liver transaminases elevated to twice the normal concentration , pulmonary edema , new - onset cerebral or visual disturbance ; ( 3 ) proteinuria : more than 300 mg protein in a 24-h urine collection , or a urinary protein - to - creatinine ratio of 0.3 , or a urine dipstick protein grade of 1 + or greater if other methods are unavailable . 
 pe was defined by ( 1 ) + ( 2 ) or ( 1 ) + ( 3 ) . 
 moreover , exclusion criteria were : kidney diseases , preexisting proteinuria , essential hypertension or other maternal chronic conditions ( autoimmune diseases , diabetes , etc . ) , and twin pregnancies . 
 maternal outcomes were evaluated : delivery mode ( spontaneous vaginal , instrumental vaginal , cesarean section [ cs ] ) , eclampsia , hellp syndrome , hypertension , abnormal liver function , abnormal renal function , ascites , oligohydramnios , thrombocytopenia , and abruptio placenta . for these complex cases , 
 the trial management team reviewed the case and a diagnosis was made by two senior deputy chief physicians or chief physicians , acting independently . 
 neonatal outcomes included : perinatal deaths , preterm birth ( < 37 and 28 weeks gestation , spontaneous or iatrogenic ) , neonatal asphyxia , and fetal growth restriction ( fgr , defined as actual birth weight below 10% for gestational age ) . in the meantime , birth weight was also assessed . 
 the comparisons of the continuous variables between the two groups were estimated using the t - test or mann  whitney test . 
 the comparisons of the categorical variables between the groups were estimated using the chi - square test or fisher s exact test . 
 a univariate logistic regression was used to analyze the associations between shp pe with poor maternal and neonatal outcomes . 
 pe was defined according to the guidelines of the international society for the study of hypertension in pregnancy : ( 1 ) hypertension : blood pressure 140/90 mmhg on at least two occasions and at least 46 h apart ; ( 2 ) the severe features , such as severe hypertension , platelet count below 100,000/l , serum creatinine concentration of 1.1 mg / dl , or doubling of the serum creatinine concentration in the absence of other renal disease , levels of liver transaminases elevated to twice the normal concentration , pulmonary edema , new - onset cerebral or visual disturbance ; ( 3 ) proteinuria : more than 300 mg protein in a 24-h urine collection , or a urinary protein - to - creatinine ratio of 0.3 , or a urine dipstick protein grade of 1 + or greater if other methods are unavailable . 
 pe was defined by ( 1 ) + ( 2 ) or ( 1 ) + ( 3 ) . 
 moreover , exclusion criteria were : kidney diseases , preexisting proteinuria , essential hypertension or other maternal chronic conditions ( autoimmune diseases , diabetes , etc . ) , and twin pregnancies . 
 maternal outcomes were evaluated : delivery mode ( spontaneous vaginal , instrumental vaginal , cesarean section [ cs ] ) , eclampsia , hellp syndrome , hypertension , abnormal liver function , abnormal renal function , ascites , oligohydramnios , thrombocytopenia , and abruptio placenta . 
 for these complex cases , the trial management team reviewed the case and a diagnosis was made by two senior deputy chief physicians or chief physicians , acting independently . 
 neonatal outcomes included : perinatal deaths , preterm birth ( < 37 and 28 weeks gestation , spontaneous or iatrogenic ) , neonatal asphyxia , and fetal growth restriction ( fgr , defined as actual birth weight below 10% for gestational age ) . in the meantime , birth weight was also assessed . 
 the comparisons of the continuous variables between the two groups were estimated using the t - test or mann  whitney test . 
 the comparisons of the categorical variables between the groups were estimated using the chi - square test or fisher s exact test . 
 a univariate logistic regression was used to analyze the associations between shp pe with poor maternal and neonatal outcomes . 
 a total of 309 women with singleton pregnancies were identified ; ten women were excluded due to incomplete outcome data . 
 there was no significant difference in age and body mass index between the two groups of pregnant women ( p = 0.17 and 0.23 , respectively ) , shown in table 1 . maternal characteristics in mild hypoproteinemia and severe hypoproteinemia preeclampsia the analysis shows that women with shp were delivered almost 1.5 weeks earlier than the mhp group ( p  0.01 ) . in the shp group , 
 the percentage of cs was higher , and the rate of spontaneous delivery was lower ( p = 0.04 and 0.04 , respectively ) . 
 severe hypertension was more frequent in the shp group than in the mhp group ( p = 0.01 and < 0.01 , respectively ) . 
 women with shp were more likely to present with abnormal liver or renal function , ascites , and abruptio placenta than women in the mhp group ( p < 0.05 ) . 
 pregnancy outcome in mild hypoproteinemia and severe hypoproteinemia preeclampsia neonatal outcomes are presented in table 3 ; the short - term neonatal outcome was worse in the shp group . 
 the average birth weight was significantly lighter ( 2498.1  866.5 g ) in the shp group than in the mhp group ( 2940.1  768.0 g ) . 
 neonates of the women in the shp group were more likely to have fgr , neonatal intensive care unit admission , and preterm deliveries ( p < 0.01 ) . 
 short - term neonatal outcome in mild hypoproteinemia and severe hypoproteinemia preeclampsia to further clarify the associations between shp pe with cs , poor maternal and neonatal outcomes , a univariate logistic regression was performed to analysis . 
 the results show that shp was a significant risk factor for cs ( odds ratio [ or ] 2.99 , 95% confidence interval [ ci ] 1.137.91 ) , poor maternal outcomes ( or 5.83 , 95% ci 3.3210.24 ) , and poor neonatal outcomes ( or 4.43 , 95% ci 2.577.62 ) in pe women [ table 4 ] . 
 a total of 309 women with singleton pregnancies were identified ; ten women were excluded due to incomplete outcome data . 
 there was no significant difference in age and body mass index between the two groups of pregnant women ( p = 0.17 and 0.23 , respectively ) , shown in table 1 . maternal characteristics in mild hypoproteinemia and severe hypoproteinemia preeclampsia 
 the analysis shows that women with shp were delivered almost 1.5 weeks earlier than the mhp group ( p  0.01 ) . in the shp group , 
 the percentage of cs was higher , and the rate of spontaneous delivery was lower ( p = 0.04 and 0.04 , respectively ) . 
 severe hypertension was more frequent in the shp group than in the mhp group ( p = 0.01 and < 0.01 , respectively ) . 
 women with shp were more likely to present with abnormal liver or renal function , ascites , and abruptio placenta than women in the mhp group ( p < 0.05 ) . 
 neonatal outcomes are presented in table 3 ; the short - term neonatal outcome was worse in the shp group . 
 the average birth weight was significantly lighter ( 2498.1  866.5 g ) in the shp group than in the mhp group ( 2940.1  768.0 g ) . 
 neonates of the women in the shp group were more likely to have fgr , neonatal intensive care unit admission , and preterm deliveries ( p < 0.01 ) . 
 short - term neonatal outcome in mild hypoproteinemia and severe hypoproteinemia preeclampsia to further clarify the associations between shp pe with cs , poor maternal and neonatal outcomes , a univariate logistic regression was performed to analysis . 
 the results show that shp was a significant risk factor for cs ( odds ratio [ or ] 2.99 , 95% confidence interval [ ci ] 1.137.91 ) , poor maternal outcomes ( or 5.83 , 95% ci 3.3210.24 ) , and poor neonatal outcomes ( or 4.43 , 95% ci 2.577.62 ) in pe women [ table 4 ] . the associations between severe hypoproteinemia preeclampsia with cesarean section , poor maternal and neonatal outcomes 
 the pathophysiologic mechanisms of pe are unclear ; the leading theory is that defects in placental implantation and function ultimately display as the development of pe . 
 scholars believe that gestational hypertension and preeclampsia are isolate disease processes with different mechanisms , research evidence includes the differential risk factors between hypertension and pe , specific histologic changes in the placenta and kidneys in pe only , the levels of antiangiogenic peptides of placental origin are elevated in pe but not in gestational hypertension , and a far lower irculating volume in pe compared with gestational hypertension . 
 pe patients often complicated with multiple organ hypoperfusion , the reduction of hepatic blood flow due to the production of albumin is decreased , thus lead to the gradual development of hypoproteinemia . in the prognosis , 
 women with chronic and gestational hypertension usually have good outcomes . however , pe represents one - third of cases of severe obstetric morbidity ; the mortality associated with pe and eclampsia was about 0.83 per 100,000 maternities . 
 simultaneously , pe is also strongly related with intrauterine growth restriction , neonatal asphyxia , low birth weight , preterm delivery , and neonatal respiratory distress syndrome . however , a common robust biomarker has not been detected . in this study , we evaluate maternal and perinatal outcomes in women with pe according to the different value of albumin ( 25 g / l and < 25 
 hypoalbuminemia is considered a factor of poor maternal and neonatal outcomes . in this study , 
 shp was a significant risk factor for cs ( or 2.99 , 95% ci 1.137.91 ) , poor maternal outcomes ( or 5.83 , 95% ci 3.3210.24 ) , and poor neonatal outcomes ( or 4.43 , 95% ci 2.577.62 ) in pe women . 
 the systemic small artery of women with pe was in spasm ; protein absorption and utilization in the gastrointestinal tract were affected , often causing the emergence of hypoproteinemia . 
 hypoalbuminemia indicates that the conditions are further exacerbated in pe and further affects the function of various organs , such as retinal damage , white matter lesions , pericardial effusion , ascites , and liver and kidney dysfunction . in our study , the presence of shp has been associated with increased risk of cs , severe hypertension , abnormal liver or renal function , ascites , and abruptio placenta . 
 the percentage of cs in shp was 93.7% , higher than in the mhp group . 
 the incidence of abnormal liver or renal function in shp ( 13.9% ) was significantly higher than in mhp ( 5.5% and 1.8% , respectively ) , and the difference was statistically significant ( p = 0.03 and < 0.01 , respectively ) . 
 placental vascular spasm in pe increases blood flow resistance from mother to the fetus , resulting in chronic fetal hypoxia . with 
 a high number of the mother s protein lost and protein and other nutrient deficiencies , these factors can lead to intrauterine growth restriction and low birth weight . 
 women with shp pe were more likely to deliver preterm infants ( 41 , 51.9% , p < 0.01 ) , lower average birth weight ( 2498.1  866.5 vs. 2940.1  768.0 g , p < 0.01 ) , fgr ( 15 , 19.0% , p < 0.01 ) than those with mhp pe . 
 it seems likely that many unknown risk factors may be present in women with pe , and it is unclear whether this finding occurred as a pathophysiology of latent pe or was responsible for it . 
 however , we show here that shp is a risk factor for women with pe . 
 shp pe is associated with a higher risk of adverse maternal and neonatal outcomes than mhp pe , deserving closer surveillance during pregnancy . 
  
  
 hbc and xtw performed the design and coordinated the study , data collecting , agreed for all aspects of the work , and prepared the manuscript . 
 ft provided assistance in the design , data collecting , analysis , and interpretation of data of the study and participated in manuscript preparation .","background : the aim of this study was to evaluate maternal and perinatal outcomes in preeclampsia ( pe ) , according to the value of albumin.materials and methods : preeclamptic women were retrospectively divided into mild hypoproteinemia ( mhp , n = 220 ) and severe hypoproteinemia ( shp , n = 79 ) pe according to the value of albumin . 
 the maternal and perinatal outcomes were evaluated in both groups.results:two hundred and ninety - nine single pregnancies complicated by pe were included in this study . 
 gestational age at delivery was earlier in shp than mhp ( p < 0.01 ) . 
 severe hypertension , abnormal liver function , abnormal renal function , ascites , and abruption occurred more frequently in shp than in mhp ( p < 0.01 , 0.03 , < 0.01 , 0.01 , and 0.04 , respectively ) . 
 women in shp had a higher rate of cesarean section than those in mhp ( p = 0.04 ) . 
 fetal growth restriction infants were more frequent in shp than in mhp ( p < 0.01 ) . 
 the occupancy rate of the neonatal intensive care unit was higher in shp than in mhp ( p < 0.01).conclusion : shp pe is associated with a higher risk of adverse pregnancy outcome than mhp pe , deserving closer surveillance during pregnancy .",pubmed
"the generation of a humanized monoclonal antibody ( mab ) that inhibits the interaction of vegf - a with its receptors vegfr-1 and vegfr-2 and the development of small - molecule protein tyrosine kinase ( ptk ) inhibitors to the cytoplasmic regions of the vegf receptors have provided avenues to explore the efficacy of targeting the vegf - a signaling pathway in cancer . 
 extensive mouse tumor models developed through the 1990s led to the success of the vegf - a mab known as avastin ( or bevacizumab ) in treating a range of human cancers including colorectal cancer , lung cancer , and renal cell carcinoma . 
 however , the recent withdrawal of avastin for treating breast cancer in the united states has highlighted the limitations of anti - angiogenic treatments , as well as the need for a careful cost - benefit analysis to ensure that scarce healthcare resources are well spent in the treatment of cancer . while avastin is clearly beneficial in a subset of cancer patients , the development of resistance to avastin and 
 the pre - existing refractoriness of some tumors suggest the role of other growth factors in sustaining tumor angiogenesis . 
 alternative ligands ( other than vegf - a ) for the angiogenic receptor vegfr-2 ( i.e. , vegf - c and vegf - d ; see below ) and other angiogenic factors from distinct growth factor families [ e.g. , fibroblast growth factor ( fgf ) ] are logical candidates for promoting resistance to avastin . in addition , 
 other mechanisms , such as vascular mimicry , the involvement of endothelial precursor cells ( epcs ) or altered tumor metabolism leading to reduced oxygen requirement , may also contribute to the development of resistance . 
 pigf is a ligand for vegfr-1 , which has been reported to play a dual role in cancer as a stimulator of angiogenesis and as an autocrine or paracrine factor promoting proliferation of tumor cells expressing vegfr-11 , . 
 it has also been proposed that pigf may modulate the effect of vegf - a by displacing vegf - a from vegfr-1 expressed on endothelial cells . 
 a range of animal tumor models have provided supportive or contradictory data on the role of pigf in driving tumor angiogenesis and tumor growth , . 
 these findings suggest a context - dependent activity for pigf in cancer , either due to the presence of other vegf family members or the diverse expression of vegf receptors on various cell types including endothelial cells , tumor cells , or associated immune cells , . 
 while vegf - b , a ligand for vegfr-1 , has been shown to be expressed in human cancer , its functions in arteriogenesis and in fatty acid uptake by cells have highlighted its role in other pathologies , predominantly cardiovascular disease . 
 recently , vegf - b was shown to regulate endothelial cell uptake and transport of fatty acids in muscle , leading to the hypothesis that vegf - b antagonists could be used as novel pharmacologic agents in the treatment of type 2 diabetes . 
 although inhibitory antibodies against vegf - b have been developed , so far there is little data on the role of this growth factor in tumor biology ; it was reported , however , that overexpression of vegf - b in a mouse model of pancreatic cancer suppressed tumor growth , highlighting the differences in biological functions between the vegf family members . 
 vegf - c and vegf - d form a subfamily within the vegf family , consisting of highly related polypeptides that require post - translational cleavage of the n- and c - terminal propeptides to generate mature forms with enhanced binding affinities for vegfr-2 and vegfr-3,. notably , vegfr-3 is expressed on lymphatic endothelium , where it can promote lymphangiogenesis , and it is also expressed on angiogenic blood vessels . 
 vegf - c is expressed in a range of human cancers , including solid tumors such as nasopharyngeal , liver and gastric cancers , with a preponderance in asian populations . 
 mouse tumor studies have displayed the ability of vegf - c to promote tumor angiogenesis and lymphangiogenesis in vivo and to drive tumor growth and metastatic spread. given that the mature form of vegf - c is a high affinity ligand for vegfr-2 and that vegf - c is expressed in many human cancers , vegf - c is likely to be an alternative ligand to vegf - a for vegfr-2binding , which could in turn promote tumor angiogenesis . 
 therefore , vegf - c , in combination with anti - angiogenic drugs such as avastin , may be a viable target for anti - cancer therapy . 
 the growth factor vegf - d is closely related in structure to vegf - c , and it includes a central vegf homology domain ( vhd ) related to other vegf family members , with n- and c - terminal propeptides that can be proteolytically cleaved by enzymes such as proprotein convertases and plasm in , , . 
 vegf - d is expressed in a range of human cancers , , and has been associated with poor patient outcome in some tumor types , . 
 importantly , animal models of cancer have demonstrated that vegf - d can promote tumor angiogenesis and lymphangiogenesis , solid tumor growth , dilation of collecting lymphatic vessels , and lymphatic and distant organ metastasis. the proteolytic processing of vegf - d is required for promoting tumor growth and spread . 
 opportunities for targeting vegf - d signaling in cancer could involve mabs specific to the vhd of vegf - d that are capable of inhibiting binding to vegfr-2 and vegfr-3 , , . 
 alternatively , ptk inhibitors that block vegfr-2 and vegfr-3 signaling would interfere with vegf - d - mediated signal transduction . 
 further , mabs to vegfr-2 and vegfr-3 , that would prevent the binding of vegf - d , or a soluble form of vegfr-3 that could sequester both vegf - c and vegf - d , could be employed . 
 targeting the vegf - d signaling pathway would likely have the merit of inhibiting both tumor angiogenesis and lymphangiogenesis , which could , in turn , restrict both solid tumor growth and metastatic spread . 
 what have the past 20 years taught us about targeting vegf - a signaling ? clearly , this period has provided biochemists , biologists , and clinicians the time to design and evaluate a variety of agents that modify or inhibit these signaling pathways . 
 the agents that were developed have included a broad range of molecules targeting different components of the pathways , including vegf - a itself , vegf receptors , vegf co - receptors , ptks and signaling intermediates , and transcription factors . during this period , 
 other vegf family members were also identified , enhancing the diversity of signaling induced by the vegf family of ligands . 
 avastin , a humanized mab to vegf - a , has been widely used in a range of prevalent human cancers over the past 8 years , typically in combination with cytotoxic chemotherapy . 
 although this agent has provided significant benefit to cancer patients , there is a need for other drugs that could be combined with avastin to deliver improved clinical outcomes . 
 use of agents targeting other vegf family members , in combination with avastin , may be a potential approach . 
 further , small - molecule ptk inhibitors of vegf receptors ( that are not highly specific ) have been employed although dose - limiting toxicity in combination with cytotoxic chemotherapy has restricted their widespread use . 
 further insights into the structure and function of the ptk domains of vegf receptors may allow development of more specific small - molecule ptk inhibitors . 
 further studies delving into the complex network of signaling cascades that drive angiogenesis , lymphangiogenesis , and resistance to anti - angiogenic drugs such as avastin are clearly required . 
 genome - wide functional approaches such as those using small interfering rna ( sirna ) technology , supported by bioinformatics , could help strategize effective targeting of growth factors , receptors , and ptks . 
 agents specifically targeting vegf family members and their receptors are currently in various stages of development ; mabs to vegf - b , vegf - c , vegf - d , pigf , vegfr-1 , vegfr-2 , and vegfr-3 are being evaluated by the biotechnology and pharmaceutic industries for their efficacy as anti - cancer agents . for these mabs , 
 a key challenge will be identifying specific cancer indications in which clinical benefit can be achieved . 
 given that many cancers are resistant to avastin or develop resistance over the course of treatment , these mabs may be tested in patients whose cancers are avastin - resistant and/or whose cancers express the appropriate target growth factor . 
 hence , assays that quantitate the levels of these growth factors or other relevant biomarkers will be required to identify the appropriate patients , although assessing these biomarkers may not be straightforward . 
 vegf family members are secreted proteins , which may easily allow testing in blood - drawn samples ; however , some family members ( e.g. , vegf - c and vegf - d ) are proteo - lytically processed to generate forms with a different bioactivity . 
 hence , it will be important to fully appreciate which forms are most bioactive and clinically relevant . 
 it is important to note that inhibiting signaling by vegf family members could potentially have pro - tumor effects . 
 recent studies have demonstrated that anti - angiogenic treatments could cause increased tumor metastasis through mechanisms such as the induction of hypoxia ( caused by vegf - a inhibition ) , resulting in increased tumor cell motility , . 
 nonetheless , these issues have been addressed in various tumor models and by comparing the effect of antibodies and small - molecule inhibitors to show that anti 
 clearly , other signaling pathways ( i.e. , not directly involving vegf family members ) such as those driven by members of the platelet - derived growth factor ( pdgf ) , insulin - like growth factor ( igf ) , and fgf growth factor families , exert control on tumor angiogenesis and may be important in promoting tumor growth . other alternative molecular targets , such as c - met , may also be able to affect multiple tumor properties such as metastasis , angiogenesis , tumor growth , and cellular motility upon hypoxia. although not the subject of this review , such pathways may provide valid therapeutic targets for inhibiting angiogenesis and/or lymphangio - genesis in cancer , thereby restricting tumor growth and spread . 
 furthermore , recent studies from our own laboratory have indicated the role of known inflammatory pathways in enhancing the spread of cancer , opening avenues for the use of existing anti - inflammatory drugs in cancer . this possibility is further exemplified by the recent studies published by rothwell et al . 
 , demonstrating the anti - cancer effect of non - steroidal anti- inflam matory drugs ( nsaids ) such as aspirin . while expensive drugs such as avastin allow the treatment of specific cancers in the western world , the capacity to employ relatively inexpensive  off - patent  small molecules ( such as aspirin ) with a defined chemistry and known biological effects could provide a cost - effective and population - based preventative approach to cancer in asian countries .","the vascular endothelial growth factor ( vegf ) family of soluble protein growth factors consists of key mediators of angiogenesis and lymphangiogenesis in the context of tumor biology . 
 the members of the family , vegf - a ( also known as vegf ) , vegf - b , vegf - c , vegf - d , and placenta growth factor ( pigf ) , play important roles in vascular biology in both normal physiology and pathology . 
 the generation of a humanized neutralizing antibody to vegf - a ( bevacizumab , also known as avastin ) and the demonstration of its benefit in numerous human cancers have confirmed the merit of an anti - angiogenesis approach to cancer treatment and have validated the vegf - a signaling pathway as a therapeutic target . 
 other members of the vegf family are now being targeted , and their relevance to human cancer and the development of resistance to anti - vegf - a treatment are being evaluated in the clinic . here , we discuss the potential of targeting vegf family members in the diagnosis and treatment of cancer .",pubmed
"malaria is still one of the main sources of consideration for public health in many countries including iran , particularly in south and southeast regions of the country . 
 deliberate implementation of control measures has resulted in a substantial reduction of malaria incidence in iran in recent years ( 1 ) , therefore malaria cases were reduced from 11,460 cases in 2008 to 3,239 in 2011(iranian center for disease management and control , cdmc , unpublished data ) . in iran , p. vivax is responsible for the majority of malaria cases while plasmodium falciparum accounts for 1015% of the cases ( cdmc , unpublished ) . 
 iran entered the malaria pre - elimination program since 2009 and interventions such as preventing strategies , active case detection and timely appropriate treatment of patients have been performed in all endemic areas of the country . in order to achieve malaria elimination , which is a global concept to reduce the incidence of locally acquired infection into zero , early recognition of all parasite carriers bearing various disease symptoms and parasite rates is crucial . in this situation 
 , active surveillance by precise laboratory methods in endemic areas provides an appropriate tool for detection of all kinds of infections particularly asymptomatic ones containing low parasite densities ( 2 ) . 
 formerly asymptomatic malaria infections were described in hyper - endemic regions following frequent infective bites and repetitive immune system response toward the parasite attenuating parasitemia to undetectable levels in blood smear ( 3 , 4 ) . 
 the prevalence of this kind of malaria infection was determined by means of different laboratory techniques in various endemic countries previously ( 59 ) , in parallel with other counties there are a few studies in iran which assessed asymptomatic malaria prevalence in different low endemic areas of the country ( 1012 ) . based on the controversial results of previous studies which show the fluctuation of asymptomatic infection among the plenty of endemic regions , the importance of regional acquiring of symptomless infection 
 another highly important issue that has to be taking account in asymptomatic malaria investigation is using accurate detection methods to find all carriers , principally who does not have clinical malaria symptoms . 
 this highlights the vitality of evaluating diagnostic method s delicacy for further decisions on malaria elimination interventions . to date 
 , light microscopy of giemsa - stained thick and thin blood smears has been remained as the standard laboratory method of malaria diagnosis due to specificity and ability to quantify the level of parasitemia ( 13 ) . 
 however , the reliance to the results of light microscopy is restricted due to the limitations associated with this method ( 14 ) . 
 although microscopy is the most commonly used method of malaria diagnosis as the gold standard in iran , chromatographic rapid diagnostic tests ( rdts ) are introduced in remote settings , where microscopic diagnosis is either impractical or unfeasible and as a complementary method for confirming the parasite - infected cases . besides the mentioned methods , the usefulness of molecular methods such as nested - polymerase chain reaction ( pcr ) has been emphasized as highly sensitive means of malaria detection , particularly in sub - clinical parasitemia levels ( 1517 ) . 
 the aim of this study was to detect low parasite and asymptomatic malaria infections by means of three malaria diagnostic tests ( microscopy , rdt and nested - pcr ) , in a low transmission region of bagh - e - malek in minab district , hormozgan province , southern iran . the results of this investigation would help further case surveillance strategies in the region and additionally all endemic settings of iran , as an essential approach toward achieving malaria elimination all over the country . 
 the present study was performed as a cross - sectional survey in bagh - e - malek region , which is located in minab district , southeast of hormozgan province . 
 hormozgan is one of the endemic areas in southern iran , containing about 10.8% of all iranian malaria patients in 2011 ( 349 cases reported based on the iranian cdmc unpublished report ) ( fig . 
 the higher rate of transmission has been reported from bagh - e malek area , therefore this region was selected for this project . 
 bagh - e - malek is a lowland area surrounded by mountains , which isolate it from the neighboring regions in minab , which has a population of 1,451 people . 
 the annual average temperature of minab is 28 c ; pluviometer index of 249 mm and an annual relative humidity of 55.5% . 
 iran s map , depicting the study area the total malaria cases in this area were 15 in 2011 ( iranian cdmc ) , while plasmodium vivax is the predominant species in bagh - e malek and whole province , also the chief anopheles vectors in minab are anopheles stephensi , an . 
 . mainly two annual peaks of malaria are seen in this area ; which occurs in may and october . 
 a total of 200 healthy volunteers were participated in this study from bagh - e malek area ( 62 males and 138 females ) . 
 briefly 3 ml fresh blood sample were collected during july 2011 from the participants after getting informed consent , for further microscopic , rdt and molecular analysis . 
 the study was approved by the ethics committee of hormozgan university of medical sciences ( approval no . 
 inclusion criteria such as lack of having any malaria symptom or receiving anti - malarial drug in three weeks prior to the study and having no travel history to other malaria endemic areas in the past three months were considered in sample collection . 
 thin films fixed using methanol then every slide stained with 10% giemsa and examined at 1000 with oil immersion to inspect malaria parasites by a skilled microscopist . 
 further thick and thin blood smear preparation were carried out from all participants , 30 days following the sampling date as well as 60 days afterwards , in order to follow up the samples . 
 all specimens were tested with the first response malaria pldh / hrp2 combo assay ( premier medical corporation ltd . 
 the kit contains a two line strip pre - coated with two monoclonal antibodies , one against the pan - specific lactate dehydrogenase , pldh , of the plasmodium species ( p. falciparum , vivax , malariae , ovale ) and the other antibody was against p. falciparum species histidine - rich protein 2 ( hrp2 ) . 
 briefly , 5 l of whole blood was dispensed onto sample well followed by two drops of assay buffer ( 60 l ) into developer well . 
 the test control line was observed as an indicator , in order to validate the performance of the test . 
 finally , differential diagnosis between p. falciparum and non - falciparum species were interpreted as described previously ( 19 ) . 
 the molecular detection of plasmodium species was performed using nested - pcr amplification of 18 ssrrna of the parasite . to achieve this purpose , 
 dna was extracted from whole blood samples using promega kit ( promega , madison , wi , usa ) and stored at 20c until further analysis with primers and cycling conditions that described previously ( 11,20 ) . in brief , 5 l of extracted dna was used in the first nest , by use of primers targeting an external region specific to the plasmodium genus followed by second nest using 2 l of the first nested - pcr product as template to amplify specific p. vivax and p. falciparum fragments using internal primers . 
 amplification was performed in a total volume of 25 l including , 25 l of templates , 250 nm of primers , 10 mm tris 
 hcl ( ph 8.3 ) , 50 mm kcl , 2 mm mgcl2 , 125 m of each of the four deoxynucleotide triphosphates and 0.4 u of taq polymerase ( invitrogen , carlsbad , ca ) . first and second nests repeated for 25 and 30 cycles , respectively while the annealing temperature was 72 c for both reactions . 
 amplicons from the second nest of pcr were electrophoresed on 2% agarose gel and visualized under uv light after staining with ethidium bromide . 
 a sample was considered positive for p. vivax and p. falciparum if a 120 and 419 base - pair fragment was detected , respectively . 
 the present study was performed as a cross - sectional survey in bagh - e - malek region , which is located in minab district , southeast of hormozgan province . 
 hormozgan is one of the endemic areas in southern iran , containing about 10.8% of all iranian malaria patients in 2011 ( 349 cases reported based on the iranian cdmc unpublished report ) ( fig . 
 the higher rate of transmission has been reported from bagh - e malek area , therefore this region was selected for this project . 
 bagh - e - malek is a lowland area surrounded by mountains , which isolate it from the neighboring regions in minab , which has a population of 1,451 people . 
 the annual average temperature of minab is 28 c ; pluviometer index of 249 mm and an annual relative humidity of 55.5% . 
 iran s map , depicting the study area the total malaria cases in this area were 15 in 2011 ( iranian cdmc ) , while plasmodium vivax is the predominant species in bagh - e malek and whole province , also the chief anopheles vectors in minab are anopheles stephensi , an . 
 . mainly two annual peaks of malaria are seen in this area ; which occurs in may and october . 
 a total of 200 healthy volunteers were participated in this study from bagh - e malek area ( 62 males and 138 females ) . 
 briefly 3 ml fresh blood sample were collected during july 2011 from the participants after getting informed consent , for further microscopic , rdt and molecular analysis . 
 the study was approved by the ethics committee of hormozgan university of medical sciences ( approval no . 
 inclusion criteria such as lack of having any malaria symptom or receiving anti - malarial drug in three weeks prior to the study and having no travel history to other malaria endemic areas in the past three months were considered in sample collection . 
 thin films fixed using methanol then every slide stained with 10% giemsa and examined at 1000 with oil immersion to inspect malaria parasites by a skilled microscopist . 
 further thick and thin blood smear preparation were carried out from all participants , 30 days following the sampling date as well as 60 days afterwards , in order to follow up the samples . 
 all specimens were tested with the first response malaria pldh / hrp2 combo assay ( premier medical corporation ltd . , 
 the kit contains a two line strip pre - coated with two monoclonal antibodies , one against the pan - specific lactate dehydrogenase , pldh , of the plasmodium species ( p. falciparum , vivax , malariae , ovale ) and the other antibody was against p. falciparum species histidine - rich protein 2 ( hrp2 ) . 
 briefly , 5 l of whole blood was dispensed onto sample well followed by two drops of assay buffer ( 60 l ) into developer well . 
 the test control line was observed as an indicator , in order to validate the performance of the test . 
 finally , differential diagnosis between p. falciparum and non - falciparum species were interpreted as described previously ( 19 ) . 
 the molecular detection of plasmodium species was performed using nested - pcr amplification of 18 ssrrna of the parasite . to achieve this purpose , 
 dna was extracted from whole blood samples using promega kit ( promega , madison , wi , usa ) and stored at 20c until further analysis with primers and cycling conditions that described previously ( 11,20 ) . in brief , 5 l of extracted dna was used in the first nest , by use of primers targeting an external region specific to the plasmodium genus followed by second nest using 2 l of the first nested - pcr product as template to amplify specific p. vivax and p. falciparum fragments using internal primers . 
 amplification was performed in a total volume of 25 l including , 25 l of templates , 250 nm of primers , 10 mm tris 
 hcl ( ph 8.3 ) , 50 mm kcl , 2 mm mgcl2 , 125 m of each of the four deoxynucleotide triphosphates and 0.4 u of taq polymerase ( invitrogen , carlsbad , ca ) . first and second nests repeated for 25 and 30 cycles , respectively while the annealing temperature was 72 c for both reactions . 
 amplicons from the second nest of pcr were electrophoresed on 2% agarose gel and visualized under uv light after staining with ethidium bromide . 
 a sample was considered positive for p. vivax and p. falciparum if a 120 and 419 base - pair fragment was detected , respectively . 
 this investigation was carried out in order to assay the concordance between the outcomes of routine malaria diagnostic techniques with nested - pcr detecting method in finding asymptomatic cases in elimination phase of malaria control . 
 hence , blood samples and smears were collected from 200 individuals residing in the endemic region of bagh - e - malek located in minab district , hormozgan province of iran . 
 all of specimens were evaluated using microscopic , rdt and nested - pcr analysis in hormozgan provincial health center laboratory . 
 all of participants were healthy individuals and age range was from 3 to 60 years old ( mean age 27.2 ) ( table 1 ) . 
 details of demographic data and the frequency of asymptomatic infection detected by use of nested - pcr the entire 200 thick and thin giemsa - stained blood smears were inspected to detect plasmodium parasite by well - trained microscopist in hormozgan provincial health center laboratory and no plasmodium parasite were detected in any examined sample . additionally none of samples was diagnosed to have any malaria parasite during the 60-days follow up protocol , while all of samples remained symptomless . 
 rdts were tested as a complementary analysis for all of 200 samples using monoclonal antibody coated sticks . 
 no positive result was discovered for either p. falciparum and/or non - falciparum species in the performed tests . in this cross sectional study , 
 nested - pcr amplification was performed as a sensitive and specific method to find parasite genome in extracted dna from asymptomatic samples . 
 interestingly 3 p. vivax positive samples ( 1.5% ) were discovered by nested - pcr using specific primers , although they were considered uninfected using microscopic and rdt tests ( table 1 ) . 
 these cases were considered as asymptomatic malaria patients since they remained symptomless in follow up evaluations ( i.e. thick blood smear and rdt ) prior to treatment . 
 all positive cases were female and only one of them had a malaria history 20 years prior to the time of this investigation . 
 moreover , no plasmodium falciparum infection was detected by this technique for all examined samples . 
 all infected subjects were treated after diagnosis by standard anti - malarial drugs according cdmc of iran protocol and reevaluated after treatment by means of molecular method . 
 the entire 200 thick and thin giemsa - stained blood smears were inspected to detect plasmodium parasite by well - trained microscopist in hormozgan provincial health center laboratory and no plasmodium parasite were detected in any examined sample . additionally none of samples was diagnosed to have any malaria parasite during the 60-days follow up protocol , while all of samples remained symptomless . 
 rdts were tested as a complementary analysis for all of 200 samples using monoclonal antibody coated sticks . 
 no positive result was discovered for either p. falciparum and/or non - falciparum species in the performed tests . 
 in this cross sectional study , nested - pcr amplification was performed as a sensitive and specific method to find parasite genome in extracted dna from asymptomatic samples . interestingly 3 p. vivax positive samples ( 1.5% ) were discovered by nested - pcr using specific primers , although they were considered uninfected using microscopic and rdt tests ( table 1 ) . 
 these cases were considered as asymptomatic malaria patients since they remained symptomless in follow up evaluations ( i.e. thick blood smear and rdt ) prior to treatment . 
 all positive cases were female and only one of them had a malaria history 20 years prior to the time of this investigation . 
 moreover , no plasmodium falciparum infection was detected by this technique for all examined samples . 
 all infected subjects were treated after diagnosis by standard anti - malarial drugs according cdmc of iran protocol and reevaluated after treatment by means of molecular method . 
 in the elimination phase of malaria control , monitoring all cases particularly hidden parasites is of major importance . 
 this highlights choosing of an acceptable detecting method , which should be , applied in all incoming asymptomatic investigation afterwards in order to detect every single carrier which continue disease transmission in the population ( 21 ) . currently in malaria endemic areas of iran , 
 microscopic and rdt assays are used for active case surveillance , although these methods could detect nearly all of symptomatic infections , in case of asymptomatic and mixed infections a more reliable technique would be helpful especially in elimination program as a public health concern in the country . 
 although , microscopy is still the diagnosis gold standard , limitations such as requiring well skilled microscopists , technical equipment maintenance , discrepancy in microscopist training and insufficient quality control are associated with this method ( 22 ) . 
 although expert microscopy could theoretically detect 10 p/l parasitemia , average microscopy has proven to be unable in detecting lower than 100 p/l parasitemia ( 23 ) . 
 however , it should be mentioned that microscopy is the primary and easiest to establish method in the field situation . 
 alternatively , despite the simplicity and being easy to use without particular training which highlight the role of rdts in malaria detection , they may have some disadvantages ; for instance , the intensity of test band is subject to amount of antigen particularly low parasite densities , and this may lead to reader variation in test results . 
 furthermore , the short shelf life of rdts emphasizes requiring of proficient transportation , storage and distribution systems , additionally , diagnose of asymptomatic malaria infection realized to be unreliable using rdts ( 2325 ) . 
 in addition to the importance of microscopy and rdt , mainly in field settings , in a number of recent investigations molecular methods were nominated as valuable and robust approaches particularly in detecting sub - clinical parasitaemia i.e. asymptomatic carriers , mixed infections and confirmation of plasmodium species in low - transmission areas ( 2629 ) . 
 however , regarding pcr expenses and equipment requirements in contrast to usual diagnosis methods , the performance of nested - pcr in identifying asymptomatic carriers was compared to conventional microscopic and rdts in symptomless population of bagh - e - malek , a malaria endemic region , in southern iran . 
 interestingly nested - pcr found three p. vivax carriers while microscopy and rdt missed the cases , one of the patients was a 12-year - old symptomless girl , who experienced a period of fever in the year prior to the study , and her disease has not been diagnosed since her thick and thin blood smears analyzed negative by light microscopy . 
 the blood films of this patient remained negative even in re - sampling after identifying a positive pcr result . 
 the current results emphasize the importance of using molecular methods in order to interrupt malaria transmission in iran besides microscopy and rdts as routine methods . it should be noted that toward eliminating malaria from the country and prevention of re - introduction , all the three types of malaria detection tools ( microscopy , rdt and molecular methods such as nested - pcr ) should be used to improve sensitivity , specificity and reliability for active case surveillance in endemic settings .","background : the aim of this study was to detect low parasite and asymptomatic malaria infections by means of three malaria diagnostic tests , in a low transmission region of minab district , hormozgan province , southern iran.methods:blood samples of 200 healthy volunteers from bagh - e - malek area were evaluated using microscopic , rapid diagnostic tests ( rdt ) and nested - pcr to inspect malaria parasite.results:the results showed no plasmodium parasite in subjects by means of microscopy and rdt . 
 however , 3 p. vivax positive samples ( 1.5% ) were discovered by nested - pcr while microscopy and rdt missed the cases.conclusion:microscopy as the gold standard method and rdt correctly identified 98.5% of cases , and molecular analysis is sensitive and reliable , especially in the detection of  asymptomatic  infections for active case surveillance . regarding the existence of asymptomatic malaria in endemic area of hormozgan , iran , 
 nested - pcr could be considered as a sensitive tool to interrupt malaria transmission in the country , beside the microscopic and rdt methods .",pubmed
"over the last two decades many new immunotherapy approaches to the treatment of cancer have entered clinical development due to the increased understanding of the mechanisms of antigen presentation , lymphocyte recognition , functions of the innate immune system , and the means of regulation of these responses and the means by which tumors can circumvent these responses . 
 many of these investigations have led to agents approved for standard clinical use , including infusional cytokine therapies for melanoma or renal cell cancer , intravesical bcg therapy for bladder cancer , and most recently an active cellular therapy targeting prostatic acid phosphatase ( pap , sipuleucel - t , provenge , dendreon ) for patients with advanced metastatic prostate cancer . 
 many other agents have demonstrated benefit in large clinical trials , and approval is anticipated in the case of a monoclonal antibody targeting a t - cell checkpoint inhibitor targeting ctla-4 ( ipilimumab , bristol - myers squibb ) for advanced melanoma . 
 ultimately , for these agents to be clinically approved there needs to be a demonstration that these treatments are relatively safe and patient care and outcome are positively affected .   however , there is also an increasing recognition that some of these agents , while likely safe , may best be used in combination with other immune - activating or conventional therapies . 
 this has presented challenges for evaluating these agents using traditional paradigms for clinical development . 
 consequently there is a need to identify markers of biological response , ideally associated with clinical outcome , but permitting an evaluation of biological effect of these agents used in combination .   in the case of antigen - specific vaccines 
 , it has been relatively straightforward to evaluate immune responses to the target antigen as a 
 biomarker  of immunological efficacy .   unfortunately , there are few instances in which target antigen immune response has been associated with clinical benefit . 
 the situation is more difficult for broadly active immune modulating agents such as t - cell checkpoint inhibitors , including antibodies targeting ctla-4 or pd-1 , or tlr agonists , in which appropriate biomarkers of response have been more elusive . 
 studies with anti - ctla-4 monoclonal antibodies , in particular , have sought to identify whether amplification of other t - cell costimulatory molecules , or antibodies to defined antigenic tumor - associated proteins [ 2 , 3 ] , might be useful as biomarkers . 
 for whole cell tumor vaccines where there is not a specific , defined antigen being targeted , surrogate antigens known to be expressed by the tumor vaccine have been used as a means of monitoring immune responses from the vaccine . 
 the use of immunologically recognized surrogate antigens , including her-2/neu , muc1 , and p53 , has been possible in the case of breast cancer where t - cell and igg responses to these antigens have been identified . 
 however it is unknown whether responses to these antigens can be useful to study agents in combination or whether changes in responses to these antigens are associated with clinical outcome .      over the last several years we have used 
 serex- ( serological analysis of recombinant cdna expression libraries ) based studies to identify immunologically recognized proteins expressed by normal and malignant prostate tissue that might serve as targets for anti - tumor vaccines . 
 in particular , we have evaluated the targets of igg responses in patients with chronic prostatitis or autoimmune disorders [ 8 , 9 ] , patients with prostate cancer treated with immune - modulating therapies , and igg responses to cancer - testis antigens in patients with prostate cancer [ 11 , 12 ] .   over the course of these studies we have effectively identified hundreds of immunologically recognized proteins associated with prostate tissue and/or recognized by patients with prostate cancer . 
 while the identification of hundreds of proteins presents challenges in prioritization for the development of antigen - specific vaccines , we previously questioned whether these antigens might also have diagnostic value with igg responses being able to distinguish individuals with prostate cancer ( or other inflammatory conditions of the prostate ) from men without prostate disease . 
 other groups have similarly reported that igg responses to tissue - associated antigens might have diagnostic value in identifying patients with prostate cancer   or nonsmall cell lung cancer . 
 we have previously reported that a subset of 23 of these antigens were recognized in patients with prostate cancer as well as individuals with symptomatic prostatitis , suggesting that such autoantibody signatures might be useful to identify inflammatory conditions of the prostate , and potentially in a premalignant setting .    in the current report 
 , we hypothesized that this same panel of previously identified prostate - associated antigens might be used as a monitoring tool to assess immune responses elicited following immune - modulating therapy . 
 while b - cells or igg production might not be an intended target of a particular therapeutic approach , igg responses are often elicited with concurrent t - cell activation . 
 we reasoned that igg responses are easier to measure compared with antigen - specific t cells , and might be more stable over time in the peripheral blood compared with t - cell frequencies . 
 moreover , the identification of  off - target  igg immune responses might further serve as an indication of  antigen spread  with secondary antigens recognized following immunological targeting and thus be more relevant to developing biomarkers associated with favorable clinical responses .   to detect antibody responses to previously defined antigens , we applied a similar phage immunoblot approach evaluating igg responses to multiple antigens simultaneously . 
 these types of complex biomarker data sets are historically very difficult to work with for two reasons : first is the complexity associated with biological networks ; second is the challenge of infrequent observation of immune biomarkers in a complex system .   as such , the identification of useful biomarkers in data sets such as 
 this study can be very challenging .   in this paper , we sought to evaluate the use of machine - learned bayesian belief networks ( ml - bbns ) as a method for identifying potentially promising biomarkers and potential biomarkers networks [ 16 , 17 ] . 
 we sought to train several ml - bbns to identify promising biomarkers and then use these networks to select a subset of features to train a network of immune biomarkers as they related to observed declines in serum prostate - specific antigen ( psa ) . 
 our objective was to demonstrate the feasibility of this method to identify promising early biomarkers of immune response to vaccine therapies in our data . 
 for the current studies , sera samples were collected prior to treatment and after three months of treatment from three separate trials , one in which patients ( n = 34 ) were treated with androgen deprivation ( adt ) therapy only , a standard therapy known to elicit prostate - associated immune responses [ 1820 ] , a trial in which patients with castrate - resistant prostate cancer ( n = 31 ) were treated with a viral vaccine encoding psa ( psav ) , and one in which patients with early recurrent prostate cancer ( n = 21 ) were treated with a plasmid dna vaccine encoding pap ( papv ) . 
 patients treated with vaccines were subclassified as immunologic or clinical  responders  based on previously reported criteria to distinguish these groups . 
 we report here that igg immune responses could be detected to individual antigens , and as long as one year after therapy the recognition of specific antigens was associated with individual treatments . 
 the evaluation of igg responses to groups of antigens at three months suggests that predictive models might be developed with diagnostic potential . 
 these findings support the concept of using measures of  antigen spread  as biomarkers of immunological efficacy for immune - active therapies , and igg responses to panels of tissue - associated antigens as measures of this antigen spread . 
 sera used for the studies had been previously collected with irb - approved , written consent as part of three separate clinical trials ( figure 1 ) . 
 these samples were all obtained prior to study treatment and after 3 months of treatment in the following settings :   ( 1 ) a trial ( adt ) in which patients ( n = 34 ) with psa - recurrent , or newly metastatic prostate cancer , who had never previously received androgen deprivation , received 22.5  mg leuprolide by intramuscular injection with or without daily oral bicalutamide ; ( 2 ) a trial ( psav ) in which patients ( n = 31 ) with castrate - resistant , metastatic prostate cancer were vaccinated at 2-week intervals with a poxviral vaccine ( prostvac , vaccinia virus encoding psa priming immunization followed by fowlpox virus encoding psa booster immunizations ; ( 3 ) a trial ( papv ) in which patients ( n = 21 ) with psa - recurrent nonmetastatic prostate cancer , not receiving androgen deprivation , were vaccinated at 2-week intervals with a plasmid dna vaccine encoding pap .   from the majority of patients treated with adt ( n = 24 of 34 ) or papv ( n = 19 of 21 ) , serum samples were also available 12 months after the baseline sample .   from the vaccine studies , patients were grouped as clinical 
 responders  or  non - responders  as simply defined by a decrease in serum psa level at the 3-month time point relative to the baseline value . 
 100,000  pfu lambda phage encoding 126 unique antigens were spotted manually in triplicate in a 1624 array onto xl-1 blue e. coli . 
 these individual antigens included 29 cancer - testis antigens , 40 proteins identified in patients with chronic prostatitis , and 57 antigens identified in individual patients , some of whom were treated with androgen deprivation or other immunomodulatory therapies [ 9 , 10 , 20 ] .   a listing of antigens and their genbank accession numbers is included in supplemental table 1 in supplementary material available online at doi:10.1155/2011/454861 . 
 plates were allowed to air - dry after which 10-mm isopropyl  - d - thiogalactopyranoside- ( iptg ) suffused nitrocellulose membranes were overlain , and plates incubated at 37c overnight to allow recombinant protein expression . 
 membranes were then washed , blocked , and probed with sera from patients pre- or post - treatment , diluted 1  :  100 in isotonic buffer . 
 human igg was then detected with an igg - specific secondary antibody conjugated to alkaline phosphatase and immunoreactivity detected by development with 0.3  mg / ml nitro blue tertazolium chloride ( nbt ) ( fisher biotech ) and 0.15  mg / ml 5-bromo 4-chloro 3-indoylphosphate ( bcip ) ( fisher biotech ) .   membranes were scanned and the digital format was assessed visually , with individual plaques scored positive or not by four independent observers , blinded to the treatment , timing of sample acquisition and membrane layout , as previously reported [ 8 , 15 ] . 
 all of the membranes for the entire study were evaluated by the same observers at the same time . 
 triplicate samples were evaluated for each antigen , and immunoreactivity to individual antigens was scored positive if there was concordance among 3 of 4 observers , and if immunoreactivity was scored positive in at least two of the three replicates . 
 heatmap builder software ( version 1.1 , stanford university ) was used to generate heatmaps displaying changes ( gain , loss , or no change ) of antibody immune responses following treatment .    our statistical analysis consisted of using a commercially available machine - learning software package ( fasteranalytics , decisionq corporation , washington , dc ) . 
 machine learning is a field of computer science that uses intelligent algorithms to allow a computer to mimic the process of human learning . 
 machine learning algorithms allow the computer to learn dynamically from the data that resides in the training dataset , detecting associations between features without human supervision . 
 decisionq software generates several networks simultaneously and then continues to generate new hypotheses for each network . 
 the software promotes the network with the best score as determined by goodness of fit relative to compactness . 
 this allows for de novo exploration of associations in complex data sets .    in preprocessing our data , we compared the pre- and post - treatment status of biomarkers and encoded the change in each biomarker as a feature . 
 we then used these encoded features and clinical response ( psa decline ) to train models . 
 the output of our machine - learning algorithms is a bayesian belief network ( bbn ) . 
 a bbn encodes the joint probability distribution of all the variables in the domain by building a hierarchical network of conditional dependence . 
 the graphical nature of the network allows the user to query the structure of conditional dependence to identify those features which provide the most information content in the network .   in order to select a subset of features for inclusion in a final model 
 , we used a stepwise process and trained a series of machine - learned ( ml)bbns for feature selection . 
 we used this stepwise process as a means of identifying nodes with relatively high information content given our statistically challenging biomarker data sets . 
 because these data sets have a very high degree of dimensionality ( features ) relative to evidence ( number of subjects ) , finding those features with the highest information content can be very challenging .   to address this challenge , we trained multiple bbn - ml models and identified those features which recurred across multiple models as evidence of high information content . 
 we modeled each of our study cohorts ( adt , psav , papv ) and then compared to the model structures between individual cohorts to identify shared nodes . 
 we also identified high - content nodes ( greater than 10 associations ) and combined these with the shared nodes to create a selected subset or training a final model to evaluate a network of biomarkers to evaluate clinical response ( psa decline ) . 
 we used our selected markers to then train three additional models : ( i ) a final subset model including clinical response ( psa decline ) on the vaccine cohorts , ( ii ) a model of subjects in the vaccine studies who were immune responders , and ( iii ) a model of subjects who were not immune responders in the vaccine studies . 
 finally , we performed tenfold cross - validation on our clinical response subset model and used receiver operating characteristic ( roc ) curve analysis to calculate an area - under - the - curve ( auc ) metric for the feature psa decline , to determine if the subset model could robustly classify clinical response given immune biomarkers 
 . the frequencies of igg responses to individual antigens were compared between treatment study populations using a chi - square test . 
 we have previously reported that antibody responses to prostate antigens can be detected in patients with prostate cancer or other inflammatory conditions of the prostate . 
 moreover , a subset of these prostate - associated antigens was commonly recognized in patients , relative to men without prostate disease , suggesting that the detection of igg responses to specific prostate - associated antigens might have diagnostic value .   in the current analysis , we wished to determine whether the detection of igg responses to a panel of prostate - associated antigens might have utility in the evaluation of vaccine or other immunomodulatory therapies aimed specifically at eliciting immune responses to the prostate .   for this 
 , we obtained sera from men with prostate cancer prior to and following three months of therapy with standard androgen deprivation therapy ( n = 34 ) , and from men with prostate cancer ( n = 52 ) prior to and following three months of therapy with one of two different antigen - specific vaccines ( figure 1 ) . 
 sera from these individuals were used to screen for igg responses to a panel of 126 antigens by immunoblot , as previously described . 
 responses to all antigens were evaluated in blinded fashion at both time points , and in figure 2 , changes in igg responses ( gain or loss of response ) after 3 months were determined . 
 as demonstrated , androgen deprivation elicited immune responses to multiple antigens , and in particular to antigens previously identified as antigens recognized in patients with chronic prostatitis [ 8 , 15 ] . 
 responses to these prostatitis antigens were uncommon over a similar 3-month period in patients treated with either of the vaccines .   gain or loss of igg responses to some antigens appeared to be shared by these different treatments , while responses to some appeared more specific for individual treatments .   of note , 
 gain or loss of igg responses to psa , while detected in one individual treated with adt , were not detected in patients receiving the psa - tricom vaccine . 
 similarly igg responses to pap were not detected in any of the patients , including those receiving the pap - targeted vaccine , as previously reported . 
 we next wanted to determine whether igg responses observed were generally stable , or increased over time , and also identify more specifically whether responses to some antigens were more generally associated with different therapies .   in the majority of patients treated with adt and papv , 
 evaluation of igg responses gained or lost after 12 months of adt , or 12 months after pap vaccine treatment compared with baseline demonstrated overall an increased number of antigens recognized ( figure 3 ) . 
 interestingly , responses to individual antigens were observed to be highly specific for the treatment . 
 for example , igg responses were elicited to the ribosomal l5 protein in 8/24 patients receiving adt , and 0/19 patients after receiving the papv ( p = .005 , chi - square test ) . 
 similarly , igg responses elicited to the neuronal pas domain protein 2 ( npas2 ) antigen were observed more frequently in patients receiving the pap vaccine ( 5/19 ) compared with patients receiving androgen deprivation ( 0/24 , p = .0075 , chi - square test ) .   even after one year 
 , responses gained or lost to antigens previously identified as prostatitis antigens were not detected in patients treated with the vaccine .   while we did not have access to a control population of sera from untreated men , given that these represented populations of subjects with nearly identical stage of disease , collected at the same institution , the differences in igg response patterns to individual antigens appears most related to the difference in treatment . 
 moreover , these findings suggest that igg responses are elicited to  off - target  antigens by means of prostate - directed therapies , and the patterns of igg responses differ with respect to therapy . 
 the results above demonstrated that , indeed , igg immune responses are elicited as a result of prostate - directed immune - active therapies . 
 moreover , antigen - specific igg immune responses were highly associated with specific treatments , suggesting that the generation of immune responses to these  off - target  antigens might be associated with other measures of immune response or clinical outcome .   these responses , however , were most detectable at 12 months after therapy initiation , a time relatively late to be useful in most circumstances as a predictive biomarker . 
 responses detectable at three months would be more useful .   however , the sample sizes for each individual trial were small , and multiple comparisons made by evaluating igg responses to multiple antigens present difficulties in statistically assessing the importance of an individual marker .    consequently , we evaluated igg responses to multiple antigens by training a ml - bbn model to determine whether we could identify groups of igg responses that are associated with clinical response , using data obtained at three months .   because the vaccine trials were conducted in different patient populations where different definitions of clinical response were used , we defined it simply for this purpose as a serum psa value at the 3-month time point lower than the baseline time point ( n = 1 for the papv trial , and n = 4 for the psav trial subjects ) . 
 we identified nine ( 9 ) biomarkers that were shared between one or more model structures , as described in table 1 , as well as two high - content nodes in the all - cohort model . 
 this resulted in a final subset of biomarkers to include in the final ml - bbn model : chromosome 20 gene contig change , rp11 - 738b7 dna on chromosome 7 change , chromosome 1 gene contig change1 , prolactin - induced protein change , acetyl - coenzyme a acyltransferase 1 change , bac rp11 - 321g3 change , cutaneous t - cell lymphoma tumor antigen sel-1 change , neuronal pas domain protein 2 change , o - fucosyltansferase change , page 1 change , and recombination signal binding protein ( rbpjk ) change . 
 this indicates that there are two first - degree associates of psa decline , igg responses to chromosome 1 gene contig 1 and bac rp11 - 321g3 , and three immune biomarkers features which can be used to estimate psa decline : igg responses to chromosome 1 gene contig 1 , bac rp11 - 321g3 , and rp11 - 738b7 dna on chromosome 7 . 
 further , these biomarkers are associated with igg responses to chromosome 20 gene contig , o - fucosyltansferase , page 1 , and cutaneous t - cell lymphoma tumor antigen sel-1 
 .   to evaluate the robustness of this model , we performed tenfold cross - validation and calculated an auc for clinical response ( psa decline ) of 0.357 . 
 this indicates that our first model is not a robust classifier , but is rather an exploratory model . 
 in the current report , we sought to determine whether serum antibody responses to a panel of prostate tissue- and prostate cancer - associated antigens might be developed as a diagnostic tool to evaluate immune responses elicited following immune - active therapies , and further to determine whether this might be developed in the future as a biomarker of clinical response . 
 using sera obtained from patients treated with three different therapies , we found that antigen - specific igg responses could be detected , likely elicited as a result of therapy . 
 the patterns of response differed with respect to the individual therapy , and recognition of specific antigens was most evident at a later ( 12 months following treatment ) than at an earlier time point ( 3 months following treatment ) .   using a ml - bbn model to evaluate groups of igg responses detected three months after treatment , we prioritized a cohort of antigens , immune responses to which were most associated with psa decline . 
 these findings suggest that , with data from larger populations of subjects , models could be developed to assist in the detection of potentially therapeutic immune responses resulting from immune - based therapies . 
 our results demonstrate that immune - active therapies , including androgen deprivation , elicit igg responses to individual prostate - associated antigens .   this has already been demonstrated in previous studies [ 18 , 20 ] ; however , the igg responses from androgen deprivation therapy were most obvious many months after treatment , where responses to individual antigens were common and predominantly induced rather than lost .   it is conceivable that some immune responses wax and wane over time , and in fact the detection of frequent gains and losses of immune responses to individual antigens , common across treatment groups detected earlier at three months , suggests that this can happen with some antigens . 
 ideally , to control for this , we would have preferred to have sera samples from men without prostate cancer and/or not undergoing active treatment over the same periods of time .   in the absence of this , however , we did have cohorts of subjects treated with different therapies . 
 given that different individual and sets of antigens were specifically recognized following these different therapies suggests that the responses observed were not purely by chance or due to the waxing and waning of responses to individual antigens . 
 the antigens recognized following androgen deprivation , in particular , were ones previously demonstrated to be commonly recognized by igg in patients with prostate cancer or inflammatory conditions of the prostate [ 8 , 15 ] . 
 we did not observe igg responses to pap in patients receiving the pap - targeted vaccine , nor igg responses to psa in patients receiving the psa - targeted vaccine . 
 this was actually not unexpected , as we have previously reported that these vaccines , while able to elicit antigen - specific t - cell responses , do not elicit robust antigen - specific igg in patients as detected by more sensitive elisa methods [ 6 , 22 ] . 
 the observation of igg responses elicited with these treatments to other antigens suggests that they may be presented by cross - presentation following immune - mediated tumor cell targeting .   of interest was the observation that the antigens recognized following androgen deprivation were different from those recognized following vaccine treatment .   theoretically , the recognition of other nonvaccine target antigens represents antigen spread induced by immune targeting and presentation of other tissue - associated antigens . 
 the recognition of different antigens suggests different mechanisms of antigen spread , or potentially recognition of other tissue - derived antigens , since most of the antigens in this panel are not prostate specific in expression .   at present 
 it is unclear whether the generation of such responses is favorable or not ; at least one report has suggested that the generation of igg responses to non - target antigens might be associated with a worse outcome . 
 future studies will explore whether the antigens recognized are shared among different vaccine approaches , suggesting common mechanisms of antigen spread , or whether different vaccine therapies elicit specific responses to different  off - target  antigens .   with larger group sizes 
 , we also hope to address whether responses to these antigens are associated with measures of t - cell immune responses to the target antigen , further implicating antigen spread as the mechanism of their recognition . 
 in addition , with larger group sizes we hope to answer whether these are clearly associated with improved clinical benefit or not , or whether this is dependent on treatment context and the specific antigen(s ) recognized , as we expect .    given the small sample size and the multitude of igg response data points , we sought to identify if the use of ml - bbn modeling was feasible to identify biomarker cohorts in our study data . 
 we were able to use a stepwise process and bbn model structures to identify those biomarkers which had high information content for use in a selected subset for ml - bbn modeling . 
 we were subsequently able to use this subset to train an ml - bbn including clinical response , however on cross - validation , our auc for clinical response was poor . 
 this is likely due to the fact that of the 52 vaccine subjects we only had 5  responders  as defined . 
 this resulted in a very small set of training outcomes , making models very sensitive to record deletion , as in the case of cross - validation . 
 psa response has itself not been validated as a surrogate clinical endpoint , and adt itself elicits initial psa responses in the vast majority of patients . 
 in addition , as further data are collected from additional subjects treated by vaccines , we expect this will produce a more robust predictive model .    in any case , the use of ml - bbn modeling appears to provide a promising method for identifying biomarkers in complex data sets that can then be selected for further analysis , as the same subset of biomarkers appeared to produce high information content in models across different populations . 
 further , once we have sufficient subjects to produce a robust model , tables of posterior estimates for clinical response given combinations of igg response biomarkers can be developed . 
 an example inference table is provided in table 2 , where those biomarkers that are predictive of clinical response can provide a posterior estimate of response . 
 this type of inference could support the translation of this research into a clinical application for determining whether an individual patient has  responded  from a particular vaccine therapy or potentially whether ongoing immunization should be performed . 
 future modeling might further permit the selection of patients who would be appropriate to receive vaccine therapy based on pre - existing immunological response parameters .","we previously demonstrated that igg responses to a panel of 126 prostate tissue - associated antigens are common in patients with prostate cancer .   in the current report we questioned whether changes in igg responses to this panel might be used as a measure of immune response , and potentially antigen spread , following prostate cancer - directed immune - active therapies . 
 sera were obtained from prostate cancer patients prior to and three months following treatment with androgen deprivation therapy ( n = 34 ) , a poxviral vaccine ( n = 31 ) , and a dna vaccine ( n = 21 ) 
 .   changes in igg responses to individual antigens were identified by phage immunoblot . 
 patterns of igg recognition following three months of treatment were evaluated using a machine - learned bayesian belief network ( ml - bbn ) . 
 we found that different antigens were recognized following androgen deprivation compared with vaccine therapies .   while the number of clinical responders was low in the vaccine - treated populations , 
 we demonstrate that ml - bbn can be used to develop potentially predictive models .",pubmed
"it is known that bone mass density and architecture are intimately dependent upon mechanical factors . 
 several in vitro studies suggest that cell mechanical stimulation promotes proliferation and differentiation of osteogenic lineage [ 1 , 2 ] , in agreement with the results from several in vivo studies [ 35 ] . 
 stress shielding causes bone loss through remodeling due to decreased loading , particularly in noncemented prostheses [ 6 , 7 ] . 
 several solutions have been proposed for these two mechanical problems , namely , design variations and development of controlled - stiffness implants [ 8 , 9 ] . a new approach to this problem , with potential application to other orthopedic problems and other medical fields , would be the development of smart implants integrating systems for bone mechanical stimulation . 
 the present in vivo study explores the use of the converse piezoelectric effect to mechanically stimulate bone with resource to polyvinylidene fluoride films ( pvdf ) ( measurement specialties inc company , usa ) . 
 these films were used , after dip - coating to improve cell adhesion and assure electrical insulation , as a substrate for mc3t3 cells to explore the concept of using piezoelectric materials and converse piezoelectric effect to produce effective osteoblast mechanical stimulation . 
 the coating was performed by dip - coating at constant velocity of 0,238  mm / s . 
 the chosen material for coating was an acrylic , poly(methyl methacrylate ) ( pmma ) , ( perfex , international dental products , usa ) , used alone in the first three layers and a in forth layer along with 4% of bonelike ( 250500  m ) particles added ( kindly offered by inescporto ) . 
 the displacement was higher where the cells were seeded , in the central area of the coated devices , in the order of 700  nm along the z - axis , in a semisinusoidal fashion . 
 on the substrates submitted to dynamic conditions , stimulation was done with an alternating sinusoidal current ( ac ) , of 5  v , at 1  hz and 3  hz for 15 minutes at each frequency . in this exploratory in vivo study , 
 the same coating procedures and daily stimulation regimens were applied . the biological response around the osteotomies 
 was assessed through histology and histomorphometry in undecalcified sections , and through histochemistry and immunohistochemistry in decalcified sections , namely , through masson 's trichrome , and labeling of osteopontin ( opn ) , proliferating cell nuclear antigen ( pcna ) , and tartrate - resistant acid phosphatase ( trap ) . 
 the actuator device was composed of a microboard containing an ultra - low power 16-bit microcontroller ( ez430-rf2500 , texas instruments , usa ) , powered by lithium battery and encapsulated in pmma and a set of six actuators composed of polyvinylidene fluoride ( pvdf ) and silver electrodes , electrically insulated by dip - coating as described elsewhere . 
 each actuator measured 15  mm width and 40  mm length , with an active area of 12  31  mm . 
 the actuator device was programmed to apply a 5  v square wave of 1  hz and 3  hz for 15 minutes at each frequency , once every 24 hours . 
 finite numerical method studies and electronic speckle pattern interferometry process ( espi ) studies have shown the lower deformation levels occurred in the clamped region of the films , for a potential differential of 5  v , and the higher deformation occurred in the opposite extremity of the film . a control device , similar but static was also developed . a sterilization method was developed and validated based on iso 11737 - 1 and iso 14937 , using a series of decontaminating solutions ( 10% sodium hypochlorite for 20 minutes , without agitation , washing in sterile water , and immersion in 10% hydrogen peroxide for 30 minutes , without agitation , followed by rinse in sterile water and drying under laminar flux ) , before 2 hours of exposure to uv ( in two different positions ) . 
 the actuator device was implanted in the left hind limb , and a control device was implanted in the right hind limb , of a 4-year - old merino ewe , with 45  kg body - weight . 
 the animal was premedicated with atropine ( 0.7  mg / kg ) , xylazine ( 0.1  mg / kg ) and butorphanol ( 0.01  mg / kg ) , and anesthesia induced with thiopental sodium 5% ( 5  mg / kg ) by intravenous injection . the anesthesia was maintained through isoflurane with spontaneous ventilation . 
 the stifle joint of both legs was prepared with a solution of povidone - iodine and ethanol 70% , after being shorn and shaved . 
 two longitudinal incisions were made on the craniomedial border of the femur and at tibial crest level . 
 the tibial bone was exposed by reflecting deep medial fascia , the vastus medialis and cranial part of the sartorius muscles and the periosteum , and two osteotomies were made using an especially metal designed guide to make two regular and well - orientated osteotomies using an oscillating saw . 
 the bone was continuously irrigated with a sterile saline solution during the process of low - speed drilling and cutting . 
 the same procedure was followed with a different design guide for the distal femoral physis , where four osteotomies were done . 
 a stab wound was made in the fascia lata , and the extremities of the piezoelectric films passed through it , and then , the active area of each piezoelectric film was placed inside the slot osteotomy cavity . the actuator position inside the tibial bone can be clearly viewed in figure 1(a ) , obtained post - mortem . 
 the portion of the device containing the microprocessor and the power supply was left in the nearby subcutaneous space . 
 the actuator device was implanted in the left hind limb , and control on the right hind limb . after placing the implants , 
 incisions were closed routinely with a continuous - pattern suture in the retinaculum and fascia , using absorbable surgicryl 2/0 ( smi ag , belgium ) and in the skin with silk 0 ( smi ag ) . 
 postoperative x - ray images were obtained thirty days after implantation , before sacrifice , to ensure the proper evolution of the bone cuts and to detect signs of infection / inflammation ( figure 2 ) . 
 one week after implantation calcein ( 15  mg / kg ; sigma , usa ) was injected subcutaneously and one week prior to sacrifice the same procedure was done with alizarin complexone ( 25  mg / kg ; sigma , usa ) . 
 the present study was authorized by competent national authorities and conducted accordingly to felasa 's guidelines for animal care . 
 both hind limbs were dissected , the implanted materials and surrounding tissue removed and fixed in 4% paraformaldehyde for two weeks . 
 bone samples were cut transversally to the long axis of the bone , each including a piezoelectric film and the surrounding bone ( figure 1(a ) ) . 
 soft tissues ( local lymph nodes and samples of the fibrous capsule surrounding the implants ) were routinely processed and embedded in paraffin . 
 undecalcified bone samples of each of the implants were included in resin ( technovit 9100 , heraeus kulzer , germany ) according to the manufacturer 's instructions and 80  m thick sections cut with a saw microtome ( leica 1600 , germany ) parallel to the piezoelectric film long axis 
 sections were then appropriately processed for routine staining ( giemsa eosin ) , mounted for fluorescence microscopy . 
 for histomorphometric studies , the interface between the bone and implant was divided in four distinct areas : z1 , z2 , z3 , and z4 , from cortical towards bone surrounding the free extremity of the piezoelectric film ( figure 1(b ) ) . 
 pictures were taken from the bone surrounding both sides of the film in areas z1 to z3 and z4 , using a 40x magnification ( figure 3 ) . 
 for immunohistochemistry and histochemistry , bone sections were decalcified in formic acid 5% for 3 weeks , dehydrated in ethanol , cleared in xylene , and embedded in paraffin wax and 3  m sections cut . 
 after deparaffinization and rehydration , immunohistochemistry sections were treated with 3% hydrogen peroxide for 10 minutes . 
 primary antibodies for tartrate - resistant acid phosphatase ( trap ) ( thermo scientific , usa , mouse monoclonal antibody , ab-1 , clone 26e5 , cat . 
 no.ms-1768 ) , proliferating cell nuclear antigen ( pcna ) ( neomarkers , usa , mouse monoclonal antibody , ab-1 , clone pc10 , cat . 
 no.rb-9097 ) were diluted to 1  :  20 , 1  :  200 , and 1  :  50 , respectively . 
 prior to immunostaining , the sections were pretreated for antigen retrieval at 100c in 10  mm citrate buffer , ph 6 , for 20 minutes in microwave oven , followed by cooling for 30 minutes at room temperature . for double staining of pcna and osteopontin , 
 immunohistochemistry was done using a double staining kit picture ( zymed laboratories inc , usa ) , according with the manufacturer 's instructions . 
 trap immunohystochemistry was performed with resource to kit picture - max polymer ( invitrogen , usa ) . 
 slides were counterstained with mayer 's hematoxilin , dehydrated , mounted with entellan ( merck , germany ) , and coverslipped . for all sections , 
 as negative controls adjacent sections were incubated : ( a ) without primary antibody and ( b ) with rabbit / rat normal serum ( similar concentration as that of primary ) . 
 masson trichrome was done using a kit ( bio - optica , italy ) , followed by dehydration and mounting . 
 observation and pictures were done using optic ( olympus bx41 ) and fluorescence microscope equipped with wb fluorescence cube ( olympus u - lh100hg ) . 
 total bone area , immature bone area , and mature bone area were measured using sigma scanpro 5 ( systat software inc . 
 the results , in percentage of total bone area , mature bone , and immature bone , were determined for each piezoelectric film in the pre - determined areas z1 to z4 . 
 normal distribution of the results was verified using the kolmogorov - smirnov test , homogeneity of variance assessed through the levene test , and differences between groups tested using one - way anova ( at a 0.05 level ) . 
 the statistical analysis was done using software originpro 8 ( originlab corporation , usa ) . 
 total bone area around the actuators was significantly higher , when comparing to static controls ( 39.91  14.08% versus 27.20  11.98% ) ( figure 4 ) . 
 the increment of bone occupied area was due to new bone formation , and in actuators the area occupied by woven bone and osteoid was 64.89  19.32% of the total bone area versus 31.72  14.54% in static devices ( figure 5 ) . 
 the chosen staining technique , giemsa eosin , stains older bone in a darker shade of pink , whilst more recently deposed bone is of a lighter pink color and frequently adjacent to much less prominent osteoid ( dark blue ) areas ( figures 5(a ) and 5(b ) ) . 
 taking into account the close relation in between osteoid and woven , recently formed bone , the authors chose not to make separated measurements that could be misleading . 
 the significant differences arise from the bone areas surrounding the two distal thirds of the devices ; around the devices ' clamped area , no differences were found between actuated and static devices ( figure 6 ) . 
 when assessing the mineral apposition rate in the distal third of the films , with the aid of the fluorochrome labeling , the mineral apposition rate was significantly higher around actuated devices ( 4.44  1.67  m / day ) than around static devices ( 2.70  0.95  m / day ) ( figure 5 ) . 
 a fibrous capsule was obvious both in actuators and static devices , with no statistical significant differences in capsule thickness between the two groups ( 289.59  131.20  m in actuated films versus 293.93  84.79  m ) . 
 immunohistochemistry failed to detect differences in trap expression between the two groups , but there is a marked elevation in opn detection around actuators , in z3 and z4 ( figure 7 ) . 
 opn is also strongly labeled in the fibroblast layers closer to the pvdf films , both in actuators and static controls , and this is the area where positive pcna labeling was found more often . 
 pcna labeling was also positive in some of the preosteoblastic cells in layers apposed to opn - positive columnar osteoblasts . 
 the existence of micro - movements at the interface , depending on the magnitude , may enhance osteogenesis or impair it and promote the formation of fibrous tissue [ 11 , 12 ] . considering the deformation levels of the actuator devices , under the experimental conditions , the generated strains were low , but the results suggest they are enough to elicit response . 
 mineral apposition rate and total bone mass are significantly higher around actuated films when compared to static controls , and these differences arise from around the areas of the films known to have higher displacement values . 
 the increased opn expression we found around actuators when comparing to static controls is most likely associated with mechanical stress , and it was not accompanied by a proportional increase in trap . 
 opn is a multifunctional noncollagenous glycoprotein present in bone matrix , expressed in various degrees by proliferating preosteoblasts , osteoblasts , and osteocytes but also by fibroblasts , osteoclasts , and macrophages [ 1315 ] . 
 opn production is known to be increased in association with mechanical loading [ 15 , 16 ] , and its deficiency significantly decreases bone fracture toughness , leading to heterogeneous mineral distribution , since opn is known to bind strongly to hydroxyapatite , and cell surface as well [ 17 , 18 ] . 
 we observed that all the devices were separated from neighboring bone by a fibrous capsule . 
 this is probably due to the material itself , since there were no differences between actuators and static controls . 
 it would be mandatory to develop and test a material with improved biocompatibility to evaluate accurately the bone material interface . 
 one other issue arises from reflecting upon the material and the coating properties and behavior . 
 the in vitro study previously developed shows evidence of adequate electric insulation for 48 hours in the experimental conditions . 
 however , the in vivo environment is not as controlled and , in addition to a longer period of time , the implantation procedure itself may induce damage on the coating . if coating damage occurs , one should consider the possible effects of the material ( pvdf and silver nitrate particles from the electrode ) and electrical current . 
 previous studies have shown cytotoxic silver nitrate activity , dependent on the concentration [ 19 , 20 ] . 
 the clinical advantages of applying electrical and electromagnetic fields in the treatment of fractures and mal - unions have been long known although the mechanisms leading to the clinical response are not well understood [ 2123 ] . 
 electric fields as low as 0.1  mv / cm promote osteoblast proliferation , with cell metabolic activity being influenced by the intensity of the electric field , and cell signaling based on the voltage - dependent calcium channels [ 24 , 25 ] . 
 electric and electromagnetic fields also exert effects on fibroblasts , enhancing proliferation , protein synthesis and influencing cell alignment , by calcium - independent mechanisms [ 2629 ] . 
 the fact that we found no significant differences in fibrous capsule thickness surrounding the devices suggests it is likely that the electric insulation remained satisfactory , but further studies would be needed to prove this . 
 however , if an electric field was being created locally , increased bone formation along the whole actuator could also be expected . 
 instead , new bone formation follows a pattern in accordance with what is known to be the areas of increased motion and deformation . 
 this study was performed in one animal only but using an adequate number of devices ( total of twelve ) . 
 the sheep was chosen as an animal model considering the bone biology similarities with the human species and the size of the devices . 
 the results are very clear in evidencing qualitative and quantitative statistically significant differences when comparing static and actuated films , but it would be necessary to enlarge the animal study and to study response with longer implantation times . 
 however , from an ethical point of view , it would be desirable to do so after developing a material other than pvdf , more biocompatible . 
 in conclusion , the present in vivo study suggests that in fact piezoelectric materials and the converse piezoelectric effect may be used to effectively stimulate bone growth at the bone implant interface , eliciting an early response . 
 piezoelectric materials have the advantages of enabling a wide range of deformation / displacement , frequencies , and regimens . 
 therefore , they are excellent candidate components of actuators for clinical purposes . however , new materials should be developed , aiming better biocompatibility .","this in vivo study presents the preliminary results of the use of a novel piezoelectric actuator for orthopedic application . 
 the innovative use of the converse piezoelectric effect to mechanically stimulate bone was achieved with polyvinylidene fluoride actuators implanted in osteotomy cuts in sheep femur and tibia . 
 the biological response around the osteotomies was assessed through histology and histomorphometry in nondecalcified sections and histochemistry and immunohistochemistry in decalcified sections , namely , through masson 's trichrome , and labeling of osteopontin , proliferating cell nuclear antigen , and tartrate - resistant acid phosphatase . 
 after one - month implantation , total bone area and new bone area were significantly higher around actuators when compared to static controls . 
 bone deposition rate was also significantly higher in the mechanically stimulated areas . in these areas , osteopontin increased expression was observed . 
 the present in vivo study suggests that piezoelectric materials and the converse piezoelectric effect may be used to effectively stimulate bone growth .",pubmed
"the study was conducted in the department of oral medicine and radiology and a cancer centre . 
 the total sample consisting of 90 individuals was divided into a study group of 60 individuals and a control group of 30 individuals . 
 the study group had 60 oscc patients ( both male and female ) between the ages of 25 and 75 years with histologically proven oral cancer but before onset of any form of treatment . 
 individuals with other malignancies , liver disease , tuberculosis , diabetes mellitus , cardiovascular diseases , or any other chronic systemic diseases were excluded from the study . 
 a group of 30 healthy volunteers aged 2575 years without any history of debilitating systemic illness and no history of tobacco or alcohol consumption formed the control . 
 details of the study are explained to the volunteers and written informed consent is obtained . 
 serum l - fucose level was estimated using clinical chemistry autoanalyzer based on winzler method . 
 chicago : spss inc .. to compare the mean values between two groups ( between genders ) , independent samples t - test was used . to compare the mean values between three or more groups ( stages of cancer ) , one - way anova was used . 
 chicago : spss inc .. to compare the mean values between two groups ( between genders ) , independent samples t - test was used . to compare the mean values between three or more groups ( stages of cancer ) , one - way anova was used . 
 a comparison of the overall serum l - fucose levels between the oscc patients and healthy controls showed the mean value of serum l - fucose levels of oscc patients to be 10.85 mg / dl and the healthy controls had 3.47 mg / dl , thereby showing a difference of 7.38 mg / dl . 
 statistical analysis revealed a significant p value ( p < 0.001 ) [ graph 1 ] . 
 serum level of levo - fucose between oral cancer and healthy individuals among the 60 oscc , 2 belong to stage i with a mean serum l - fucose level of 8.13 mg / dl . 
 the serum l - fucose level progressively increased with its level being 9.18 mg / dl in stage ii , 10.53 mg / dl in stage iii , and 11.59 mg / dl in stage iv . 
 stage iv showed the highest level with a maximum value of 12.54 mg / dl and minimum of 10.55 mg / dl . 
 statistical analysis shows a significant p value ( p < 0.001 ) [ table 1 ] . 
 serum level of l - fucose in various stages of oral cancer an attempt to correlate the serum l - fucose levels with histopathological grading between genders and ages yielded no significant results thereby inferring that serum l - fucose levels are directly indicative of the degree of tissue destruction . 
 group of neoplasms affecting any region of the oral cavity is termed as oral cancer , among which oscc represents > 90% of all the neoplasm . 
 recently , the knowledge about cancer biomarkers has increased , and numerous studies have been carried out regarding these . 
 this has paved the way for improving the management of cancer patients by enhancing the accuracy of detection and efficacy of treatment . 
 understanding the relevance of biomarkers before using them is very important for diagnosis , treatment , and proper follow - up . 
 tumor markers are produced either by the tumor itself , as a tumor by product , by the body in response to the presence of cancer or certain benign ( noncancerous ) conditions . 
 these can aid in the diagnosis of cancer and in the assessment of tumor burden . 
 l - fucose is a monosaccharide , which is a common component of many n- and o - linked glycans and glycolipids produced by mammalian cells . 
 lack of a hydroxyl group on the carbon at the 6-position ( c-6 ) and the l - configuration is the two structural features which help to distinguish fucose from other six - carbon sugars present in mammals . 
 it is the most essential sugars that the body demands for normal cell - to - cell communication . 
 physiologically , the concentrations in serum are less but are increased in cancer and other diseases . 
 it is important in many of the signaling pathways which turns a healthy cell into a dysplastic cell . 
 the protein diversity is achieved by varying sequence and structure of sugar moieties or glycan attachment . 
 mammalian cells either express or mediate many of their properties through the cell surface . change in glycosylation of cell surface proteins 
 is essential in tumor progression , especially the terminal epitopes of glycoproteins , which have been proposed to play an essential role in cell  cell interactions , cell adhesion , malignant transformation , and metastasis . 
 fucosylation of glycoproteins ( the addition of l - fucose at the terminal end of the oligosaccharide chain ) is one of the most important features that mediate several specific biologic functions . 
 tumor cells alter their surface by increasing fucosylation levels to escape recognition ; this contributes to numerous abnormal characteristics of tumor cells such as decreased adhesion and uncontrolled tumor growth . 
 several studies have suggested that estimating the serum / tissue fucose levels could be a promising approach for the early detection , diagnosis , and prognosis of various cancer types . 
 group of enzymes catalyze incorporation of fucose from activated nucleotide donor guanosine diphosphate - fucose to the reducing end of complex glycans in a linkage - specific manner . 
 these enzymes are expressed in many tissues and are elevated in serum and tumors of cancer patients . 
 it has been mentioned in the article that increased fucosylation is associated with elevated fuct activity . 
 cancer cells which are shed or released into circulation from the primary tumor often overexpress fucosylated glycans on their surface . 
 the expression of fucosylated glycoproteins ( i.e. , fucoproteins ) has been detected by means of specific lectins . 
 profound fucosylation of the serum microenvironment may be a reason that interrupts adhesion and induces the formation of metastases ( for example ) , several fucose - containing  natural ligands 
 increased expression of fucosylated cell surface antigens , such as lewis x / y ( lex / y ) or sialyl lex/ , and the upregulation of 1 , 3/4-fuct have been associated with malignant transformation and increased metastatic potential of tumors , which leads to poor prognosis of patients with cancer . 
 -l - fucosidase is a lysosomal enzyme that catalyzes the hydrolytic cleavage of terminal fucose residue that is involved in maintaining the homeostasis of fucose metabolism . 
 extensive studies on the nature of metastasis have shown that only a small subpopulation of cells in tumors possess the characteristics necessary for their release from the primary tumor and transport to and establishment of tumor foci in distant organs . 
 carbohydrate moieties on cell surface glycoconjugates play an important role in this metastatic spread since it could be demonstrated that they are involved in adhesion processes . 
 several studies have mentioned that estimation of serum l - fucose levels could be a reassuring approach for the early detection , diagnosis , and prognosis of various types of cancer including oral carcinoma . 
 thus , the present study was undertaken to estimate the serum level of l - fucose ( tumor marker ) among various tnm stages in oral cancer patients and compare them with healthy individuals . 
 present study suggested that serum level of l - fucose can not be correlated with age and gender . 
 this was similar to other studies which were published in the literatures . in our study , out of 60 cancer patients , 
 31 were well differentiated , 27 - moderately differentiated , and 2 poorly differentiated and anaplastic - nil . 
 however , there was a negative correlation between serum fucose levels and histopathological tumor differentiation in our study as well as in the literatures . in our study 
 , the mean serum value of l - fucose in cancer group ( 10.85 mg / dl ) and control group ( 3.47 mg / dl ) shows a wide margin of difference ( 7.38 mg / dl ) . 
 moreover , all the 60 patients in the cancer group were categorized according to tnm staging . 
 comparison of all the four tnm stages in cancer group showed increase in fucose levels as the stages progressed . 
 they assessed the levels of glycoprotein - associated carbohydrates such as hexose ; hexosamine , fucose , and sialic acid . 
 they found a significant rise in the level in cases when compared to control subjects . 
 furthermore , there was a progressive rise in these markers as the stages of oral cancer advances . in cancer patients , elevated levels 
 have been observed more so in advanced stages and in cases with metastasis than in subjects without metastasis . 
 some studies have concluded that it is the most effective of the essential sugars when it comes to slowing the growth of cancer cells . 
 fall in serum fucose levels after treatment has been reported in many studies although it is emphasized that the follow - up period has to be long enough for significant serological alteration . 
 unfortunately , rise in serum fucose level is not specific for cancers , as elevated serum fucose levels have also been reported in other pathological states such as cirrhosis liver , meningitis , rickets , osteomalacia , tuberculosis , cardiovascular disorders as well as in depressive disorders . 
 furthermore , it has been observed that the serum fucose level is raised in different groups of malignancies such as breast cancer , ovarian cancer , colorectal adenocarcinomas , and leukemia as well as brain tumors . 
 thus , it becomes important to exclude other degenerative and proliferative diseases , while estimating the serum fucose levels in oral cancer . 
 literature is replete with studies on fucose levels in various malignancies including a few on oral cancer . 
 based on the analysis of the results of our study , we can conclude that there was a positive correlation between the serum l - fucose levels and tnm stages of oscc . 
 serum l - fucose levels remained constant among healthy individuals and was raised proportionately in oral cancer patients , commensurate with the stage of cancer . in conjunction with clinic - diagnostic procedures , 
 serum l - fucose levels can be used as an easy , noninvasive , cost - effective , biochemical indicator of cancer detection , staging , therapeutic success , prognosis , and as a posttreatment evaluation tool . 
 further investigation on a large scale would in all probability prove serum l - fucose as an effective tumor marker in the diagnosis and management of oral cancer . 
  
 ","background : oral cancer is a result of disordered cellular behavior initiated by various stimuli which is characterized by the alteration of serum glycoproteins consisting of different monosaccharides . 
 one of these is levo - fucose ( l - fucose ) , a methyl pentose . 
 elevated levels of protein - bound fucose have been reported in various malignancies.aim:the present study attempted to correlate levels of serum l - fucose as a biomarker with the various tumor node metastasis ( tnm ) stages of oral cancer.methodology:the study was carried out on 90 subjects consisting of 30 healthy controls and 60 histopathologically proven oral squamous cell carcinoma ( oscc ) cases . 
 the serum fucose level estimation was done based on the method adopted by winzler . 
 statistical analysis included independent sample 's t - test , one - way anova test , karl  pearson correlation test , and tukey 's hsd post hoc test to evaluate the significance and variability of values between groups.results:significant elevation in serum fucose levels was noticed among oscc patients when compared with the controls and a progressive ascent of l - fucose levels were noted as the stage of severity increased . 
 serum fucose levels were independent of histopathological grading , age , and sex.conclusion:serum l - fucose levels were increased in oscc patients , and a positive correlation was observed between serum l - fucose levels and tnm staging of oscc . 
 thus , serum l - fucose can be used as an effective diagnostic and prognostic biomarker in oscc patients .",pubmed
"alan aderem ( institute for systems biology , seattle , washington ) , which were originally generated by dr . 
 shizuo akira ( research institute for microbial diseases , osaka university , osaka , japan ) . 
 lcn2 mice were generated by gene targeting in embryonic stem ( es ) cells from mouse strain 129 , and targeted es cells were injected into c57bl/6 blastocysts as described previously ( 12 ) . 
 lcn2-null mice were backcrossed onto the b6 background for 10 generations before mice were used for the experiments . 
 heterozygous mating scheme was used to generate wild - type and lcn2 mice for the experiments . 
 aminals were housed in a specific pathegon - free facility at the university of minnesota . 
 animal handling followed the national institutes of health guidelines , and experimental procedures were approved by the university of minnesota animal care and use committee . 
 age - matched male wild - type and lcn2 mice were allocated into groups ( 34 mice / cage ) and fed an hfd ( fat calories : 60% ) obtained from bio - serv ( f3282 ; new brunswick , nj ) or a regular chow diet ( rcd ) , with free access to water for all studies . in the adaptive thermogenesis study , 
 age - matched wild - type and lcn2 mice on an rcd were exposed to 4c , with free acess to diet and water . 
 rectal temperature of the mice was measured at the indicated time points using a microtherma thermometer with rectal probe for mice ( braintree scientific , braintree , ma ) . during the experiemental period of hfd feeding , 
 glucose and insulin tolerance tests were conducted by the intraperitoneal injection of glucose and insulin . 
 mice were fasted overnight ( 12 h ) for the glucose tolerance test ( gtt ) and for 6 h for insulin tolerance tests ( itts ) . 
 gtts and itts were conducted by intraperitoneal injection of glucose ( 1 mg / g body wt ) or insulin ( 0.75 units / kg body wt ) with blood collection via the tail vein at 0 , 15 , 30 , 60 , 90 , and 120 min . 
 briefly , frozen liver tissue ( 100 mg ) was homogenized in 1 ml water . 
 an aliquot of the organic phase was collected and dried with nitrogen and then dissolved in isopropanol alcohol containing 1% triton . 
 preparation of isolated adipose cells from wild - type and lcn2 mice was performed as described previously ( 14,15 ) . 
 after mincing , epididymal fat pads were digested with collagenase ( 2 mg / ml solution ) in digestion vials containing krebs - ringer bicarbonate hepes buffer , ph 7.4 ; 200 nmol / l adenosine ; and 3.5% bsa . after a 2-h digestion , adipose cells were separated by centrifugation at 1,200 rpm for 10 min and washed twice with krebs - ringer bicarbonate hepes buffer . 
 adipose tissue was obtained from the epididymal and inguinal fat pad of wild - type and lcn2 mice fed an rcd . 
 tissue samples ( 2030 mg ) were immediately fixed in osmium tetroxide and incubated in a water bath at 37c for 48 h , as described previously ( 16 ) , and then adipose cell size was determined by a beckman coulter multisizer iii with a 400-m aperture . 
 the range of cell sizes that can effectively be measured using this aperture is 20240 m . 
 after collection of pulse sizes , the data were expressed as particle diameters and displayed as histograms of counts against diameter using linear bins and a linear scale for the x - axis . 
 the figures were generated using graphpad prism 5.01 for windows ( graphpad software , la jolla , ca ) . 
 differences in parameters between wild - type and lcn2 mice were evaluated using a two - group t test with a 0.05 two - sided significance level . 
 during the experiemental period of hfd feeding , glucose and insulin tolerance tests were conducted by the intraperitoneal injection of glucose and insulin . 
 mice were fasted overnight ( 12 h ) for the glucose tolerance test ( gtt ) and for 6 h for insulin tolerance tests ( itts ) . 
 gtts and itts were conducted by intraperitoneal injection of glucose ( 1 mg / g body wt ) or insulin ( 0.75 units / kg body wt ) with blood collection via the tail vein at 0 , 15 , 30 , 60 , 90 , and 120 min . 
 briefly , frozen liver tissue ( 100 mg ) was homogenized in 1 ml water . 
 an aliquot of the organic phase was collected and dried with nitrogen and then dissolved in isopropanol alcohol containing 1% triton . 
 preparation of isolated adipose cells from wild - type and lcn2 mice was performed as described previously ( 14,15 ) . after mincing , 
 epididymal fat pads were digested with collagenase ( 2 mg / ml solution ) in digestion vials containing krebs - ringer bicarbonate hepes buffer , ph 7.4 ; 200 nmol / l adenosine ; and 3.5% bsa . after a 2-h digestion , adipose cells were separated by centrifugation at 1,200 rpm for 10 min and washed twice with krebs - ringer bicarbonate hepes buffer . after the final wash , adipose cells were collected for rna extraction . 
 adipose tissue was obtained from the epididymal and inguinal fat pad of wild - type and lcn2 mice fed an rcd . 
 tissue samples ( 2030 mg ) were immediately fixed in osmium tetroxide and incubated in a water bath at 37c for 48 h , as described previously ( 16 ) , and then adipose cell size was determined by a beckman coulter multisizer iii with a 400-m aperture . 
 the range of cell sizes that can effectively be measured using this aperture is 20240 m . 
 after collection of pulse sizes , the data were expressed as particle diameters and displayed as histograms of counts against diameter using linear bins and a linear scale for the x - axis . 
 the figures were generated using graphpad prism 5.01 for windows ( graphpad software , la jolla , ca ) . 
 differences in parameters between wild - type and lcn2 mice were evaluated using a two - group t test with a 0.05 two - sided significance level . 
 at 3 weeks of age , male wild - type and lcn2 mice were fed either an hfd for 12 weeks or rcd . throughout the experimental period , 
 body weight was significantly different between wild - type and lcn2 mice fed an hfd ( fig . 
 lcn2 mice were significantly heavier than wild - type mice on either an hfd or rcd . 
 the weight of white and brown fat pads was significantly higher in hfd - fed ( fig . 
 1d ) mice than that in wild - type mice . both hfd - fed and aged lcn2 mice showed a significantly higher weight of liver , heart , kidney , and spleen ( fig . 
 similar metabolic phenotypes were also observed in female lcn2 mice ( data not shown ) . 
 however , the overall growth did not appear to be significantly affected , as the body length and bone length ( trabecular ) were not significantly different between the genotypes ( data not shown ) . 
 figure 1 g and h demonstrated the fat - cell size distributions of wild - type and lcn2 mice on an rcd at 15 weeks of age , as measured by multisizer analysis using cells isolated from osmium - fixed epididymal and inguinal adipose tissue . 
 the average peak diameter of the large - cell population was significantly larger for lcn2 mice compared with wild - type mice , indicating that lcn2 adipose tissue contains larger adipose cells . 
 growth curve , weight of white and brown fat pads and organs in lcn2 mice . 
 growth curve of lcn2 mice on an hfd ( n = 1014 ) ( a ) and rcd ( n = 13 ) ( b ) . 
 fat pad weight of lcn2 mice on an hfd at 16 weeks of age ( c ) and on rcd at 30 weeks of age ( d ) . 
 organ weight of lcn2 mice on hfd at 16 weeks of age ( e ) and on rcd at 30 weeks of age ( f ) . 
 * p < 0.05 ; * * p < 0.01 . fat - cell size distribution of mice on rcd diet at 15 weeks of age ( g and h ) . 
 epi : epididymal ; ing : inguinal . because lcn2-deficient mice gained more body weight and increased adiposity , particularly when mice were fed an hfd or aged , we examined food intake and energy expenditure in lcn2 mice . 
 an analysis of indirect calorimetry measurements showed that food intake , ambulatory activity , vo2 , and rq were not significantly different between rcd - fed wild - type and lcn2 mice at 18 weeks of age ( fig . 
 we then assessed thermogenic activity of brown adipose tissue ( bat ) under thermoneutral and thermal stress conditions . 
 body temperature was measured in an ambient temperature of 28c and 22c as well as during acute exposure to 4c . 
 wild - type and lcn2 mice at 12 weeks of age exhibited a similar body temperature in an ambient temperature of 28c ( wild type , 38.34  0.34 ; lcn2 , 38.2  0.30 ) . 
 however , female lcn2 mice had a significantly lower rectal temperature than wild - type mice ( fig . 
 2a ) , while male lcn2 mice had a trend toward decrease in rectal temperature when kept in an ambient temperature of 22c . 
 more strikingly , lcn2 mice displayed cold sensitivity and could not survive when exposed to 4c for > 10 h. lcn2 mice ( seven of seven ) died after being exposed to 4c for 12 h , while all of the wild - type mice ( n = 7 ) in the experiment survived . 
 when acutely exposed to 4c , rectal temperature of lcn2 mice dropped significantly within 3 h compared with wild - type mice ( fig . 
 . five - hour cold exposure caused a 10-fold increase in lcn2 gene expression in adipose tissue in wild - type mice ( fig . 
 a : basal rectal body temperature of 8-week - old male and female wild - type and lcn2 mice on an rcd ( n = 710 ) measured during the daytime in an ambient temperature of 22c . 
 b : body temperature curve of lcn2 mice ( n = 8) and wild - type mice ( n = 7 ) exposed to 4c . 
 c : gene expression of lcn2 in wat of wild - type mice exposed to 4c for 5 h. d : gene expression of ucp-1 and pgc-1 in bat and ppar , cpt-1 , tfam , and nrf1 in skeletal muscle of hfd - fed mice ( n = 6 ) . 
 g : gene expression of hsl in bat and wat of mice exposed to 4c for 5 h. h : gene expression of mitochondrial genes in bat of lcn2 mice under the hfd condition . 
 * p < 0.05 ; * * p < 0.01 . to explore the possible mechanisms for cold intolerance in lcn2 mice , uncoupling protein ( ucp)-1mediated thermogenic activity and capacity of bat 
 we observed that upon hfd feeding , lcn2 mice expressed significantly lower levels of ucp-1 and peroxisome proliferator  activated receptor ( ppar )  coactivator ( pgc-1) genes in bat than wild - type mice mice ( fig . 
 however , ucp-1 and pgc-1 expression in bat were markedly stimulated at an even higher level in lcn2 mice , as wild - type mice after exposure to 4c for 5 h ( fig . 
 figure 2f showed that the protein expression levels of adipose triglyceride lipase ( atgl ) in white adipose tissue ( wat ) were slightly reduced in lcn2 mice under the hfd and cold condition compared with wild - type mice . however , cold - induced hsl gene expression in bat and wat was completely diminished in lcn2 mice ( fig . 
 lastly , we investigated the mitochondrial oxidative capacity of bat , as this function of bat is critical for providing the energy for thermogenesis . 
 interestingly , we observed that there was a significant reduction in the expression levels of mitochondrial genes involved in mitochondrial biogenesis and oxidative function , such as nuclear respiratory factor ( nrf)1 , transcription factor a , mitochondrial ( tfam ) , atp synthase 5 ( atp5 ) , cytochrome c oxidase ( cox ) 4 , and carnitine palmitoyltransferase ( cpt)-1 in bat of lcn2 mice fed an hfd ( fig . 
 in addition , the expression of transcription factors ppar , nrf1 and tfam , and cpt-1 gene in skeletal muscle was also significantly downregulated in lcn2 mice fed an hfd ( fig . 
 2d ) . to assess the effects of lcn2 deficiency on systemic insulin sensitivity , gtts and itts were performed . 
 glucose clearance curves in response to glucose administration were significantly increased in hfd - fed ( fig . 
 3b ) , indicating that lcn2 mice are more glucose intolerant upon hfd feeding . in the itt , 
 increased insulin - stimulated glucose disposal curve was observed in lcn2 mice , indicating that lcn2 mice are more insulin resistant than wild - type controls when fed an hfd ( fig . 
 3d ) . analysis of serum metabolic parameters , as shown in table 1 , demonstrated that levels of fasting serum glucose and insulin were significantly elevated , while adiponectin levels were reduced in hfd - fed lcn2 mice when compared with wild - type controls . 
 serum levels of leptin and plasminogen activator inhibitor-1 were not significantly changed in lcn2 mice on an rcd , although serum leptin levels had a trend toward a decrease in hfd - fed lcn2 mice . 
 after adjustment to body weight , the serum levels of glucose , insulin , and adiponectin were consistently different at the statistically significant level , indicating that the changes in glucose tolerance and insulin sensitivity after hfd in lcn2 mice is not simply due to body weight effects . to further confirm that lcn2 mice have decreased insulin sensitivity , in vivo insulin - stimulated akt phosphorylation was evaluated . 
 wild - type and lcn2 mice on rcd were injected intraperitoneally with insulin , killed after 10 min , and tissues were collected for evaluation of akt phosohorylation . 
 consistently , insulin - stimulated akt phosphorylation in liver , muscle , and adipose cells was significantly reduced in lcn2 mice as compared with wild - type mice ( fig . 
 the examination of lcn2 effect on upstream signaling components of insulin signaling pathway demonstrated that the expression levels of insulin receptor substrate-1 protein were markedly reduced in wat of lcn2 mice on an hfd compared with wild - type mice ( fig . 
 this data further support that lcn2 mice developed more insulin resistance and lcn2 regulates insulin signaling activity likely at the upstream level . 
 gtts ( a ) and itts ( c ) conducted in lcn2 mice on an hfd ( n = 1012 , age 1415 weeks ) . 
 gtts ( b ) and itts ( d ) conducted in lcn2 mice on an rcd ( n = 1012 , age 2829 weeks ) . 
 serum metabolic parameters in wild - type and lcn2 mice data are means  se . 
 measurements were performed on 6-h  fasted mice fed an rcd and hfd ( n = 911 ) . 
 * p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . in vivo insulin - stimulated akt phosphorylation in lcn2 mice . 
 representative western blot analysis of serine - phosphorylated akt in muscle , liver , and isolated primary adipose cells from lcn2 ( n = 4 per group ) and wild - type mice ( n = 4 per group ) under basal and in vivo insulin - stimulated conditions . to test the role of lcn2 in inflammatory response , expression of lcn2 and inflammatory molecules was examined in adipose tissue and liver of hfd - induced wild - type and lcn2 mice . 
 the gene expression of proinflammatory cytokine tnf and monocyte chemoattractant protein ( mcp)-1 were upregulated , while expression of arginase 1 , an anti - inflammatory marker , was downregulated in adipose tissue of hfd - fed lcn2 mice compared with wild - type control mice ( fig . 
 downregulation of arginase 1 and interleukin ( il)-10 was also observed in the liver of hfd - fed lcn2 mice ( fig . 
 a : protein expression levels of lcn2 in adipose tissues of wild - type mice fed an rcd and hfd . 
 b : protein expression levels of lcn2 in bone marrow macrophages ( bm - mac ) isolated from wild - type mice fed an rcd and hfd . 
 c : gene expression of inflammatory molecules in adipose tissue and liver of wild - type and lcn2 mice fed on hfd . 
 * p < 0.05 . to investigate whether increased liver weight in lcn2 mice is associated with the development of fatty liver and dyslipidemia , liver triglyceride content and blood lipid profiles were measured . in comparison with wild - type controls , hfd - fed and 
 aged lcn2 mice demonstrated a significant increase in lipid accumulation as detected by oil - red o staining of the liver section ( fig . 
 the development of more severe fatty liver disease , together with elevated fasting blood glucose levels , suggests hepatic insulin resistance in lcn2 mice . 
 to elucidate hepatic glucose production in wild - type and lcn2-deficient mice fed an hfd , mice were fasted for 18 h and gene expression of two key gluconeogenic enzymes pepck and glucose-6-phosphatase ( g6pase ) were detected by quantitative pcr . 
 6c , expression levels of pepck1 , pepck2 , and g6pase genes were significantly higher in the liver of lcn2 mice than that in wild - type controls , suggesting that increased hepatic glucose production is attributed to hyperglycemia in lcn2 mice . 
 we next examined the hepatic capabilities for fatty acid synthesis and oxidation to explore the possible mechanism for the development of fatty liver in lcn2 mice . 
 6d , the lipogenic genes sterol regulatory element - binding protein-1c ( srebp-1c ) , acetyl - coa carboxylase 1 ( acc1 ) , spot 14 ( s14 ) , and scd-1 were expressed at a significantly higher level in hfd - fed lcn2 mice compared with wild - type controls , while genes involved in fatty acid oxidation such as ppar , cpt-1 , and acetyl - coa carboxylase 2 were similarly expressed between genotypes ( data not shown ) . therefore , the development of fatty liver results primarily from the increased capability for fatty acid synthesis in the liver in lcn2 mice . 
 b : liver triacyglyceride levels in lcn2 mice on an rcd ( n = 68 , age 30 weeks ) and an hfd ( n = 11 , age 1516 weeks ) . 
 c : the mrna expression of gluocneogenic genes in liver ( n = 6 ) . 
 d : the mrna expression of lipogenic genes in liver ( n = 6 ) . 
 the data are represented as means  se . * p < 0.05 ; * * p < 0.01 . 
 ( a high - quality digital color representation of this figure is available in the online issue . ) 
 compared with wild - type mice , lcn2 mice on an rcd had significantly higher serum levels of triglycerides , cholesterol , and free fatty acids . 
 the hfd feeding did not induce a further increase in serum levels of triglyceride and cholesterol but significantly augmented the levels of ldl and -hydroxybutyrate in lcn2 mice compared with wild - type controls . 
 the above data have suggested that lcn2-deficient mice became more insulin resistant upon hfd feeding and age . 
 since adipose tissue is a key site for insulin - regulated glucose / lipid metabolism and glucose disposal , we examined the expression of genes involved in lipid metabolism in primary adipose cells isolated from lcn2 mice . 
 compared with wild - type adipose cells , expression levels of genes involved lipid metabolism and adipokines , including ppar , lipoprotein lipase ( lpl ) , glut4 , adiponectin , and leptin were markedly reduced in adipose tissue of hfd - fed lcn2 mice ( online appendix fig . 
 plasma lipid profile in wild - type and lcn2-null mice data are means  se . 
 measurements were performed on 6-h  fasted mice fed an rcd and hfd ( n = 911 ) . * 
 p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . 
 at 3 weeks of age , male wild - type and lcn2 mice were fed either an hfd for 12 weeks or rcd . throughout the experimental period , 
 body weight was significantly different between wild - type and lcn2 mice fed an hfd ( fig . 
 lcn2 mice were significantly heavier than wild - type mice on either an hfd or rcd . 
 the weight of white and brown fat pads was significantly higher in hfd - fed ( fig . 
 1d ) mice than that in wild - type mice . both hfd - fed and aged lcn2 mice showed a significantly higher weight of liver , heart , kidney , and spleen ( fig . 
 similar metabolic phenotypes were also observed in female lcn2 mice ( data not shown ) . 
 however , the overall growth did not appear to be significantly affected , as the body length and bone length ( trabecular ) were not significantly different between the genotypes ( data not shown ) . 
 figure 1 g and h demonstrated the fat - cell size distributions of wild - type and lcn2 mice on an rcd at 15 weeks of age , as measured by multisizer analysis using cells isolated from osmium - fixed epididymal and inguinal adipose tissue . 
 the average peak diameter of the large - cell population was significantly larger for lcn2 mice compared with wild - type mice , indicating that lcn2 adipose tissue contains larger adipose cells . 
 growth curve , weight of white and brown fat pads and organs in lcn2 mice . 
 growth curve of lcn2 mice on an hfd ( n = 1014 ) ( a ) and rcd ( n = 13 ) ( b ) . 
 fat pad weight of lcn2 mice on an hfd at 16 weeks of age ( c ) and on rcd at 30 weeks of age ( d ) . 
 organ weight of lcn2 mice on hfd at 16 weeks of age ( e ) and on rcd at 30 weeks of age ( f ) . 
 * p < 0.05 ; * * p < 0.01 . fat - cell size distribution of mice on rcd diet at 15 weeks of age ( g and h ) . 
 because lcn2-deficient mice gained more body weight and increased adiposity , particularly when mice were fed an hfd or aged , we examined food intake and energy expenditure in lcn2 mice . 
 an analysis of indirect calorimetry measurements showed that food intake , ambulatory activity , vo2 , and rq were not significantly different between rcd - fed wild - type and lcn2 mice at 18 weeks of age ( fig . 
 we then assessed thermogenic activity of brown adipose tissue ( bat ) under thermoneutral and thermal stress conditions . 
 body temperature was measured in an ambient temperature of 28c and 22c as well as during acute exposure to 4c . 
 wild - type and lcn2 mice at 12 weeks of age exhibited a similar body temperature in an ambient temperature of 28c ( wild type , 38.34  0.34 ; lcn2 , 38.2  0.30 ) . 
 however , female lcn2 mice had a significantly lower rectal temperature than wild - type mice ( fig . 
 2a ) , while male lcn2 mice had a trend toward decrease in rectal temperature when kept in an ambient temperature of 22c . 
 more strikingly , lcn2 mice displayed cold sensitivity and could not survive when exposed to 4c for > 10 h. lcn2 mice ( seven of seven ) died after being exposed to 4c for 12 h , while all of the wild - type mice ( n = 7 ) in the experiment survived . 
 when acutely exposed to 4c , rectal temperature of lcn2 mice dropped significantly within 3 h compared with wild - type mice ( fig . 
 five - hour cold exposure caused a 10-fold increase in lcn2 gene expression in adipose tissue in wild - type mice ( fig . 
 a : basal rectal body temperature of 8-week - old male and female wild - type and lcn2 mice on an rcd ( n = 710 ) measured during the daytime in an ambient temperature of 22c . 
 b : body temperature curve of lcn2 mice ( n = 8) and wild - type mice ( n = 7 ) exposed to 4c . 
 c : gene expression of lcn2 in wat of wild - type mice exposed to 4c for 5 h. d : gene expression of ucp-1 and pgc-1 in bat and ppar , cpt-1 , tfam , and nrf1 in skeletal muscle of hfd - fed mice ( n = 6 ) . 
 g : gene expression of hsl in bat and wat of mice exposed to 4c for 5 h. h : gene expression of mitochondrial genes in bat of lcn2 mice under the hfd condition . 
 * p < 0.05 ; * * p < 0.01 . to explore the possible mechanisms for cold intolerance in lcn2 mice , uncoupling protein ( ucp)-1mediated thermogenic activity and capacity of bat 
 we observed that upon hfd feeding , lcn2 mice expressed significantly lower levels of ucp-1 and peroxisome proliferator  activated receptor ( ppar )  coactivator ( pgc-1) genes in bat than wild - type mice mice ( fig . 
 however , ucp-1 and pgc-1 expression in bat were markedly stimulated at an even higher level in lcn2 mice , as wild - type mice after exposure to 4c for 5 h ( fig . 
 figure 2f showed that the protein expression levels of adipose triglyceride lipase ( atgl ) in white adipose tissue ( wat ) were slightly reduced in lcn2 mice under the hfd and cold condition compared with wild - type mice . 
 however , cold - induced hsl gene expression in bat and wat was completely diminished in lcn2 mice ( fig . 
 lastly , we investigated the mitochondrial oxidative capacity of bat , as this function of bat is critical for providing the energy for thermogenesis . interestingly 
 , we observed that there was a significant reduction in the expression levels of mitochondrial genes involved in mitochondrial biogenesis and oxidative function , such as nuclear respiratory factor ( nrf)1 , transcription factor a , mitochondrial ( tfam ) , atp synthase 5 ( atp5 ) , cytochrome c oxidase ( cox ) 4 , and carnitine palmitoyltransferase ( cpt)-1 in bat of lcn2 mice fed an hfd ( fig . 
 in addition , the expression of transcription factors ppar , nrf1 and tfam , and cpt-1 gene in skeletal muscle was also significantly downregulated in lcn2 mice fed an hfd ( fig . 
 to assess the effects of lcn2 deficiency on systemic insulin sensitivity , gtts and itts were performed . 
 glucose clearance curves in response to glucose administration were significantly increased in hfd - fed ( fig . 
 3b ) , indicating that lcn2 mice are more glucose intolerant upon hfd feeding . in the itt , 
 increased insulin - stimulated glucose disposal curve was observed in lcn2 mice , indicating that lcn2 mice are more insulin resistant than wild - type controls when fed an hfd ( fig . 
 3d ) . analysis of serum metabolic parameters , as shown in table 1 , demonstrated that levels of fasting serum glucose and insulin were significantly elevated , while adiponectin levels were reduced in hfd - fed lcn2 mice when compared with wild - type controls . 
 serum levels of leptin and plasminogen activator inhibitor-1 were not significantly changed in lcn2 mice on an rcd , although serum leptin levels had a trend toward a decrease in hfd - fed lcn2 mice . 
 after adjustment to body weight , the serum levels of glucose , insulin , and adiponectin were consistently different at the statistically significant level , indicating that the changes in glucose tolerance and insulin sensitivity after hfd in lcn2 mice is not simply due to body weight effects . to further confirm that lcn2 mice have decreased insulin sensitivity , in vivo insulin - stimulated akt phosphorylation was evaluated . 
 wild - type and lcn2 mice on rcd were injected intraperitoneally with insulin , killed after 10 min , and tissues were collected for evaluation of akt phosohorylation . 
 consistently , insulin - stimulated akt phosphorylation in liver , muscle , and adipose cells was significantly reduced in lcn2 mice as compared with wild - type mice ( fig . 
 the examination of lcn2 effect on upstream signaling components of insulin signaling pathway demonstrated that the expression levels of insulin receptor substrate-1 protein were markedly reduced in wat of lcn2 mice on an hfd compared with wild - type mice ( fig . 
 this data further support that lcn2 mice developed more insulin resistance and lcn2 regulates insulin signaling activity likely at the upstream level . 
 gtts ( a ) and itts ( c ) conducted in lcn2 mice on an hfd ( n = 1012 , age 1415 weeks ) . 
 gtts ( b ) and itts ( d ) conducted in lcn2 mice on an rcd ( n = 1012 , age 2829 weeks ) . 
 serum metabolic parameters in wild - type and lcn2 mice data are means  se . 
 measurements were performed on 6-h  fasted mice fed an rcd and hfd ( n = 911 ) . * 
 p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . in vivo insulin - stimulated akt phosphorylation in lcn2 mice . 
 representative western blot analysis of serine - phosphorylated akt in muscle , liver , and isolated primary adipose cells from lcn2 ( n = 4 per group ) and wild - type mice ( n = 4 per group ) under basal and in vivo insulin - stimulated conditions . to test the role of lcn2 in inflammatory response , expression of lcn2 and inflammatory molecules 
 was examined in adipose tissue and liver of hfd - induced wild - type and lcn2 mice . 
 5b ) . furthermore , the gene expression of proinflammatory cytokine tnf and monocyte chemoattractant protein ( mcp)-1 were upregulated , while expression of arginase 1 , an anti - inflammatory marker , was downregulated in adipose tissue of hfd - fed lcn2 mice compared with wild - type control mice ( fig . 
 downregulation of arginase 1 and interleukin ( il)-10 was also observed in the liver of hfd - fed lcn2 mice ( fig . 
 a : protein expression levels of lcn2 in adipose tissues of wild - type mice fed an rcd and hfd . 
 b : protein expression levels of lcn2 in bone marrow macrophages ( bm - mac ) isolated from wild - type mice fed an rcd and hfd . 
 c : gene expression of inflammatory molecules in adipose tissue and liver of wild - type and lcn2 mice fed on hfd . 
 to investigate whether increased liver weight in lcn2 mice is associated with the development of fatty liver and dyslipidemia , liver triglyceride content and blood lipid profiles were measured . in comparison with wild - type controls , hfd - fed and 
 aged lcn2 mice demonstrated a significant increase in lipid accumulation as detected by oil - red o staining of the liver section ( fig . 
 the development of more severe fatty liver disease , together with elevated fasting blood glucose levels , suggests hepatic insulin resistance in lcn2 mice . to elucidate hepatic glucose production in wild - type and 
 lcn2-deficient mice fed an hfd , mice were fasted for 18 h and gene expression of two key gluconeogenic enzymes pepck and glucose-6-phosphatase ( g6pase ) were detected by quantitative pcr . as illustrated in fig . 
 6c , expression levels of pepck1 , pepck2 , and g6pase genes were significantly higher in the liver of lcn2 mice than that in wild - type controls , suggesting that increased hepatic glucose production is attributed to hyperglycemia in lcn2 mice . 
 we next examined the hepatic capabilities for fatty acid synthesis and oxidation to explore the possible mechanism for the development of fatty liver in lcn2 mice . 
 6d , the lipogenic genes sterol regulatory element - binding protein-1c ( srebp-1c ) , acetyl - coa carboxylase 1 ( acc1 ) , spot 14 ( s14 ) , and scd-1 were expressed at a significantly higher level in hfd - fed lcn2 mice compared with wild - type controls , while genes involved in fatty acid oxidation such as ppar , cpt-1 , and acetyl - coa carboxylase 2 were similarly expressed between genotypes ( data not shown ) . 
 therefore , the development of fatty liver results primarily from the increased capability for fatty acid synthesis in the liver in lcn2 mice . 
 b : liver triacyglyceride levels in lcn2 mice on an rcd ( n = 68 , age 30 weeks ) and an hfd ( n = 11 , age 1516 weeks ) . 
 c : the mrna expression of gluocneogenic genes in liver ( n = 6 ) . 
 d : the mrna expression of lipogenic genes in liver ( n = 6 ) . 
 the data are represented as means  se . * p < 0.05 ; * * p < 0.01 . 
 ( a high - quality digital color representation of this figure is available in the online issue . ) 
 compared with wild - type mice , lcn2 mice on an rcd had significantly higher serum levels of triglycerides , cholesterol , and free fatty acids . 
 the hfd feeding did not induce a further increase in serum levels of triglyceride and cholesterol but significantly augmented the levels of ldl and -hydroxybutyrate in lcn2 mice compared with wild - type controls . 
 the above data have suggested that lcn2-deficient mice became more insulin resistant upon hfd feeding and age . since adipose tissue is a key site for insulin - regulated glucose / lipid metabolism and glucose disposal , we examined the expression of genes involved in lipid metabolism in primary adipose cells isolated from lcn2 mice . compared with wild - type adipose cells , expression levels of genes involved lipid metabolism and adipokines , including ppar , lipoprotein lipase ( lpl ) , glut4 , adiponectin , and leptin were markedly reduced in adipose tissue of hfd - fed lcn2 mice ( online appendix fig 
 plasma lipid profile in wild - type and lcn2-null mice data are means  se . 
 measurements were performed on 6-h  fasted mice fed an rcd and hfd ( n = 911 ) . * 
 p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . 
 we show here that lcn2 plays a critical role in lipid metabolism , adaptive thermoregulation , and diet - induced obesity and insulin resistance in vivo . 
 lack of lcn2 in mice potentiates hfd - induced obesity , dyslipidemia , fatty liver , glucose intolerance , and insulin resistance . 
 we also discovered that lcn2 is a critical regulator of adaptive thermogenesis and mitochondrial oxidative function in bat . 
 lcn2 deficiency impairs adaptive thermogenesis and increases the susceptibility of lcn2 mice to hfd - induced obesity . 
 our data demonstrated that lcn2 mice gained more body weight when they were fed hfd or aged . increased body fat mass together with enlarged multiple organs 
 in particular , increased organ weights may largely account for increased body weight in lcn2 mice under the rcd condition . 
 in general , wild - type and lcn2 mice at a young age did not demonstrate significantly different metabolic phenotypes when they were fed rcd . however , lcn2 mice developed more severe systemic insulin resistance , hyperglycemia , hyperinsulinemia , and hypoadiponectinemia under the hfd condition 
 . one of the most profound phenotypic features of lcn2 mice is cold sensitivity and lower body temperature under thermal stress conditions . 
 it has been known for a long time that nonshivering thermogenesis is used to maintain constant body temperature and energy balance , controlling body weight , especially in small mammals ( 1719 ) . 
 both neural and hormonal signals , including norepinephrine , epinephrine , thyroid hormones , glucagon , insulin , glucocorticoid , and leptin , are known to regulate thermogenesis ( 17,20 ) through different thermogenic mechanisms involving stimulating atp utilization , increasing ucp-1mediated proton leak and heat production and affecting substrate availability and utilization for energy production . in rodents , 
 bat is the main tissue that is responsible for sympathetic nervous system  regulated nonshivering themogenesis with the involvement of norepinephrine and epinephrine ( 17,20,21 ) . ucp-1 and pgc-1 are the key genes that mediate sympathetic thermogenic responses in bat . 
 for example , ucp-1 gene expression in bat of ob / ob mice was reduced ( 23 ) . however 
 , bat ucp-1 expression could be normally stimulated in response to cold environment in young ob / ob mice , albeit ob / ob mice show decreased capability of utilizing fat and cold intolerance ( 23 ) . in this study 
 , we found that 5-h cold exposure leads to a significant increase in lcn2 expression in wat , which is in agreement with the result in a recent study ( 24 ) . in the absence of lcn2 , mice were cold intolerant and failed to maintain the body temperature when acutely exposed to cold temperature . interestingly , cold exposure could still significantly stimulate gene expression of ucp-1 and pgc-1 in bat in lcn2 mice , suggesting that cold - induced sympathetic thermogenesis remained intact in the absence of lcn2 . 
 the role of leptin in thermogenesis has been well documented , and its thermogenic effect is considered to be primarily central ( 25 ) . 
 for example , leptin administration has been reported to rescue cold - induced hypothermia in the absence of ucp-1 in leptin and ucp-1 double knockout mice ( 26,27 ) , while the opposite results were perceived in the other two independent studies ( 28,29 ) . in lcn2 mice on rcd , 
 serum leptin levels were not different from that in wild - type mice , albeit serum leptin levels had a trend toward decreased in lcn2 mice in response to hfd feeding . 
 additionally , wild - type and lcn2 mice on an rcd did not exhibit a difference in body temperature in thermoneutral conditions . 
 therefore , it seems rather unlikely that leptin could be the major contributor to cold intolerance in lcn2 mice ; leptin may be only partially involved in lcn2-regulated thermogenesis . 
 in addition to hormonal signals , defects in key metabolic regulators of substrate availability for energy production , atp synthesis and mitochondrial biogenesis and oxidative function could all possibly affect thermogenic function . 
 indeed , studies in mice with the genetic disruption of key metabolic regulators have proved this assumption ; mice without expressing atgl could similarly result in cold intolerance ( 30 ) . in the present study 
 , the disruption of lcn2 completely diminished cold - induced hsl gene expression in bat and wat , whereas atgl expression in wat was only slightly affected in lcn2 mice under both the hfd and cold conditions . 
 since hsl mice are not cold sensitive ( 31 ) , hsl is not an important regulator for adaptive thermogenesis . 
 therefore , the effect of changes in atgl levels on the supply of substrates for energy production for thermogenesis is minimal in lcn2 mice . on the other hand 
 , the production of the energy necessary for thermogenesis requires normal mitochondrial oxidative function , and mitochondrial dysfunction could have a significant impact on adaptive thermogenesis . 
 this hypothesis has been supported by a previous study in estrogen - related receptor  ( eer ) knockout mice . in eer-deficient mice , mitochondrial biogenesis and oxidative function in bat 
 was disturbed , and mice failed to maintain body temperature when exposed to cold ( 32 ) . to this end 
 interestingly , we found that the expression levels of mitochondrial genes involved in mitochondrial biogenesis and oxidative function such as nrf1 , tfam , atp5b , cox 4 , and cpt-1 were significantly reduced in bat of lcn2 mice fed an hfd . in the skeletal muscle , the expression of -oxidation genes ppar and cpt-1 , as well as mitochondrial genes nrf1 and tfam , were also downregulated in hfd - fed lcn2 mice . 
 all the above data together suggest that mitochondrial metabolism is dysfunctional in bat and muscle of lcn2 mice , which mainly contributes to decreased thermogenic capacity , decreased energy expenditure , and the high susceptibility to diet - induced obesity and insulin resistance during the thermal stress conditions ( mice have been living at 22c ) . in acute cold , 
 muscle - shivering thermogenesis is considered to be the primary mechanism for a body temperature defense ( 21,33,34 ) . 
 bat nonshivering thermogenesis participates in combating the cold temperature only when the extra capacity is already recruited for using during the acute cold exposure ( 35 ) . therefore , cold intolerance ( acute exposure to 4c for 5 h ) in lcn2 mice may largely result from decreased oxidative capacity in skeletal muscle and bat as well as decreased atgl expression in wat . 
 our results clearly demonstrated that insulin responsiveness in muscle , adipose tissue , and liver was reduced , indicating systemic insulin resistance in lcn2 mice . 
 furthermore , fasting for 18 h induces significantly higher levels of expression for pepck and g6pase in the liver of hfd - fed lcn2 mice , supporting that the increased hepatic glucose production is an important contributor to fasting hyperglycemia in lcn2 mice . 
 this phenomenon is likely due to the impaired regulation of lipid metabolism in adipose tissue and liver in lcn2 mice . 
 we showed that lcn2 mice on an rcd had higher serum levels of triglycerides but lower serum triglyceride levels when fed an hfd as compared with wild - type mice . 
 lower serum triglycerides in hfd - fed lcn2 mice could be due to the increased triglycerides synthesis and decreased vldl ( or triglyceride ) secretion into the circulation resulting from the development of fatty liver in lcn2 mice . 
 this explanation is strengthened by a previous study ( 36 ) in lirko ( insulin receptor knockout ) mice with hepatic insulin resistance and 50% decrease in serum triglyceride levels . as reported in our previous in vitro study ( 6 ) , lcn2 is capable of suppressing inflammatory response of macrophages to lps stimulation . 
 we speculated that lcn2 possesses anti - inflammatory role in metabolic regulation . in this in vivo study , we show that lcn2 expression is downregulated in adipose tissue and bone marrow macrophages ( bm - mac ) in hfd - induced obese mice . in the absence of lcn2 , 
 the expression of proinflammatory cytokines such as mcp-1 and tnf are upregulated in adipose tissue , while expression levels of anti - inflammatory markers arginase 1 and il-10 are decreased in adipose tissue and liver , suggesting an increased inflammatory state in lcn2 mice . 
 these results further suggest that lcn2 mice are more susceptible to hfd - induced proinflammatory response than wild - type mice . in summary , herein we discovered a novel role for lcn2 in energy metabolism , adaptive thermogenesis , and insulin resistance . 
 mice lacking lcn2 display impaired adaptive thermogenesis and deteriorated diet - induced obesity and insulin resistance . 
 decreased mitochondrial oxidative capacity in bat and skeletal muscle may be the primary mechanisms for the development of diet - induced insulin resistance and cold sensitive in lcn2 mice . 
 however , the mechanisms for how lcn2 regulates mitochondrial function and lipid metabolism remain to be further identified . 
 it is likely that the binding of lcn2 to small hydrophobic molecules such as retinoic acids , fatty acids , and other unknown ligands or to the cell surface receptor such as megalin or ldl - related protein 2 ( 37 ) could potentially involve or mediate lcn2 effects in metabolism . 
 our findings provide new insight into the role of lcn2 in lipid metabolism and metabolic homeostasis . in this manner 
 , lcn2 may be a potential therapeutic target for controlling obesity - associated type 2 diabetes and metabolic complications . 
 ","objectivelipocalin ( lcn ) 2 belongs to the lipocalin subfamily of low  molecular mass  secreted proteins that bind small hydrophobic molecules . 
 lcn2 has been recently characterized as an adipose - derived cytokine , and its expression is upregulated in adipose tissue in genetically obese rodents . 
 the objective of this study was to investigate the role of lcn2 in diet - induced insulin resistance and metabolic homeostasis in vivo.research design and methodssystemic insulin sensitivity , adaptive thermogenesis , and serum metabolic and lipid profile were assessed in lcn2-deficient mice fed a high - fat diet ( hfd ) or regular chow diet.resultsthe molecular disruption of lcn2 in mice resulted in significantly potentiated diet - induced obesity , dyslipidemia , fatty liver disease , and insulin resistance . 
 lcn2/ mice exhibit impaired adaptive thermogenesis and cold intolerance . 
 gene expression patterns in white and brown adipose tissue , liver , and muscle indicate that lcn2/ mice have increased hepatic gluconeogenesis , decreased mitochondrial oxidative capacity , impaired lipid metabolism , and increased inflammatory state under the hfd condition.conclusionslcn2 has a novel role in adaptive thermoregulation and diet - induced insulin resistance .",pubmed
"male wistar rats ( 10 weeks old ) were obtained from sankyo labo service , co. ltd . 
 ( sapporo , japan ) . each animal was housed in an individual cage in a temperature- and humidity - controlled room with unlimited food and water and a 12-h light / dark cycle . all animals ( n = 22 ) were given 0.3-mg / kg lps ( from escherichia coli serotype 055 : b5 ; sigma , st . 
 louis , mo , usa ) intravenously on days 1 and 2 . on days 3 , 4 and 5 , 
 the animals were given 20-mg / kg methylprednisolone intramuscularly to promote development of non - traumatic onfh as described previously ( okazaki et al . 
 all the injections were administered at 7:00 p.m. hindlimb unloading was accomplished as described by wronski and morey - holton ( morey - holton & globus 1998 ) with some modifications ( okazaki et al . , unpublished observation ) . 
 the tail was then varnished with instant adhesive ( aron alpha ; toagosei co. , ltd . , 
 tokyo , japan ) , and a strip of traction tape ( skin - trac , 3  40 in . ; zimmer , warsaw , in , usa ) was then attached to the tail beginning at the base of the tail above the hairline . to facilitate free movement about the cage 
 , the cast was attached to a swivel anchored to the cage that allowed a 360 range of movement . 
 the animals were then suspended in a head - down tilted position . over time , the suspension angle was readjusted as the body size increased to prevent any weight coming to bear on the hindlimbs . in nwb rats ( n = 11 ) , the tail was suspended from day 1 to sacrifice . 
 the femur and humerus were harvested from each rat and fixed with 10% formalin-0.1 m phosphate buffer , ph 7.4 . 
 the tissues were processed for routine haematoxylin and eosin staining to assess the general architecture and osteonecrosis of the femoral and humeral heads . 
 we defined histopathological osteonecrosis as the diffuse presence of empty lacunae or pyknotic osteocyte nuclei in the bone trabeculae , accompanied by surrounding bone marrow cell necrosis ( figure 1 ) ( ichiseki et al . 
 histopathological appearance of proximal femoral epiphysis of weight - bearing ( wb ) rat with haematoxylin and eosin staining . 
 data were compared between wb and nwb groups using the chi - square test . a p - value of < 0.05 was considered to be significant . 
 male wistar rats ( 10 weeks old ) were obtained from sankyo labo service , co. ltd . 
 ( sapporo , japan ) . each animal was housed in an individual cage in a temperature- and humidity - controlled room with unlimited food and water and a 12-h light / dark cycle . all animals ( n = 22 ) were given 0.3-mg / kg lps ( from escherichia coli serotype 055 : b5 ; sigma , st . 
 louis , mo , usa ) intravenously on days 1 and 2 . on days 3 , 4 and 5 , 
 the animals were given 20-mg / kg methylprednisolone intramuscularly to promote development of non - traumatic onfh as described previously ( okazaki et al . 
 all the injections were administered at 7:00 p.m. hindlimb unloading was accomplished as described by wronski and morey - holton ( morey - holton & globus 1998 ) with some modifications ( okazaki et al . , unpublished observation ) . 
 the tail was then varnished with instant adhesive ( aron alpha ; toagosei co. , ltd . , 
 tokyo , japan ) , and a strip of traction tape ( skin - trac , 3  40 in . ; zimmer , warsaw , in , usa ) was then attached to the tail beginning at the base of the tail above the hairline . to facilitate free movement about the cage 
 , the cast was attached to a swivel anchored to the cage that allowed a 360 range of movement . 
 the animals were then suspended in a head - down tilted position . over time , the suspension angle was readjusted as the body size increased to prevent any weight coming to bear on the hindlimbs . in nwb rats ( n = 11 ) , the tail was suspended from day 1 to sacrifice . 
 the femur and humerus were harvested from each rat and fixed with 10% formalin-0.1 m phosphate buffer , ph 7.4 . 
 bone samples were decalcified with kalkitox ( wako pure chemical industries , ltd . , osaka , japan ) and then neutralized with sodium sulphate buffer . 
 the tissues were processed for routine haematoxylin and eosin staining to assess the general architecture and osteonecrosis of the femoral and humeral heads . 
 we defined histopathological osteonecrosis as the diffuse presence of empty lacunae or pyknotic osteocyte nuclei in the bone trabeculae , accompanied by surrounding bone marrow cell necrosis ( figure 1 ) ( ichiseki et al . 
 histopathological appearance of proximal femoral epiphysis of weight - bearing ( wb ) rat with haematoxylin and eosin staining . 
  
 all experiments adhered to the guidelines of the ministry of sports , culture , science and technology of japan and followed protocols approved by the animal ethics committee of the sapporo medical university ( # 09 - 008 ) . 
 femoral head osteonecrosis was observed in seven of 11 ( 63.6% ) wb rats and six of 11 ( 54.5% ) nwb rats , with no significant intergroup difference observed ( p = 0.66 ) . 
 figure 2 shows the histopathological appearance of the femoral head after haematoxylin and eosin staining in typical specimens from the wb ( a , b ) and nwb ( c , d ) groups . in the wb group , 
 empty lacunae were seen at the necrotic bone trabeculae , and the number of hematopoietic cells was decreased in the medullary space at most areas of the femoral head ( figure 2a , b ) . in the nwb group , empty lacunae 
 in addition , the number of haematopoietic cells was again decreased , and necrotic cell debris was accumulated in the medullary space at most areas of the femoral head ( figure 2c , d ) , as observed in the wb group . on the other hand , no humeral head osteonecrosis was observed in any of the wb or nwb rats . 
 typical specimens of weight - bearing ( wb ) ( a , b ) and non - weight - bearing ( nwb ) ( c , d ) rats . both wb and nwb group shows diffuse presence of empty lacunae in the bone trabeculae , accompanied by surrounding bone marrow cell necrosis at most areas of femoral head . 
 figure 3 shows the histopathological appearance of the humeral head in typical specimens from the wb ( a , b ) and nwb ( c , d ) groups . in the wb group , 
 normal trabeculae , as well as haematopoietic and fat cells , were observed ( figure 3a , b ) . 
 normal trabeculae were also observed in the nwb group rats . in comparison with the wb group 
 , a large number of haematopoietic cells , but few fat cells were observed in the nwb group rats ( figure 3c , d ) . 
 typical specimens of weight - bearing ( wb ) ( a , b ) and non - weight - bearing ( nwb ) ( c , d ) rats . 
 osteonecrosis of the femoral head often develops after corticosteroid therapy for inflammatory diseases , such as autoimmune diseases ( fukushima et al . 
 , it has been reported that mega - dose corticosteroid therapy for trauma , such as spinal cord injury , does not induce onfh ( wing et al . 
 therefore , we developed a non - traumatic onfh animal model in which onfh was encouraged by the administration of lps ( a ligand of toll - like receptor 4 , and implicated in inducing inflammatory status ) and methylpredonisolone treatment in the rats . in this animal model 
 , however , rats were allowed to walk and move independently in individual cages , with the hip joints supporting the loading from the lower body . 
 we could not exclude the possible influence of weight bearing on the hip joints on the induction of onfh . 
 therefore , we employed a hindlimb unloading procedure , in which the rat tail was fitted on to a suspension system with the rats in a head - down tilted position to prevent weight bearing on the hindlimbs . in this system , 
 we observed that nwb group rats developed onfh at a comparable rate as did wb group rats . 
 taken together , the results of the present study confirm that weight bearing does not contribute to the development of non - traumatic onfh in rats . to clarify the pathogenesis of onfh , 
 several quadruped animal models of osteonecrosis , including both traumatic and non - traumatic forms , have been reported ( yamamoto et al . 1995 ; norman et al . 
 however , the quadruped animals in these models fail to progress to end stage mechanical collapse similar to that in bipedal animals , such as humans ( conzemius et al . 
 xu et al . ( 2010 ) reported that this is caused by a difference in weight bearing through the hip joint between quadruped and bipedal animals . 
 however , our results showed that weight bearing did not contribute to the initial development of onfh in nwb rats . 
 therefore , it is speculated that the rat model may help us to elucidate the pathogenesis of onfh and to devise a prevention strategy for the condition , but may not be useful for the observation of the mechanism of femoral head collapse . in conclusion , the present study revealed that weight bearing is not related to the development of non - traumatic onfh in the rat model , although the aetiology , pathogenesis and prognosis of non - traumatic onfh remain unclear . 
 ","the hip joint is one of the major structures in the human body and the resultant force acting through the hip joint is 300% of body weight . 
 therefore , weight bearing , as a cause of ischaemia , may contribute to the development of non - traumatic osteonecrosis of the femoral head ( onfh ) . 
 however , it remains unclear whether weight bearing is related to the development of non - traumatic onfh . 
 therefore the aim of this study was to clarify the role of weight bearing in the development of non - traumatic onfh . 
 non - weight - bearing ( nwb ) rats were tail suspended to prevent any weight coming to bear on the hindlimbs from day 1 to the time of sacrifice . 
 the weight - bearing ( wb ) group rats were also housed individually , although without tail suspension . 
 all rats were injected with lipopolysaccharide and methylprednisolone to promote the development of non - traumatic onfh . 
 all animals were sacrificed three weeks after the final methylprednisolone injection . 
 histopathological analysis was performed . 
 osteonecrosis of the femoral head was observed not only in the nwb but also in the wb rats ; however , no osteonecrosis of the humeral head was observed in either group . 
 we confirmed that non - traumatic onfh developed in nwb rats , suggesting that weight bearing does not contribute to the development of non - traumatic onfh in rats .",pubmed
"with the global debates around health policy reforms , there have been increasing calls for nurses to be actively engaged in national policy processes [ 13 ] . 
 first , nurses comprise the majority of the global health care workforce , working closely with patients and their families in a variety of settings . 
 thus , nurses ' experiences and insights may help guide improvements in the quality of health service delivery and inform health systems strengthening . 
 second , many health sector policies have an impact on nurses ' work environments and consequently their professional practice . 
 thus , nurses ' input on health sector policies could help ensure that supportive work environments for clinical practice are taken into account when policies are reformed . 
 given nurses ' pivotal role in health care delivery , their engagement in policy changes in the system has been described as both a moral and professional obligation as well as a responsibility to the people they serve [ 2 , 4 ] . 
 the engagement of nurses in national policy processes is especially important in lower income settings where nurses comprise an even larger proportion of the health sector workforce and where the largest burden of illness exists . 
 historically , nurses have had limited involvement in national policy and political decisions that affect health service delivery [ 58 ] . 
 for instance , a quantitative study of national nurse leaders and hospital based nurses in thailand revealed that majority of nurses were not involved in policy formulation or modification stage . 
 literature further reveals several reasons for nurses underengagement in policy including lack of awareness of policy related issues and processes , inadequate knowledege and skills on how to contribute to policies , and lack of opportunities to contribute to policy decisions [ 911 ] . 
 nurses ' limited participation in policy processes has also been attributed to gender differences in the health care system [ 12 , 13 ] . 
 nursing is predominately viewed as a woman 's occupation involving caring for others , while men are typically seen in positions of power and decision - making [ 1416 ] . 
 thus , men are more likely than women to be involved in public policy and to influence policy decisions . in kenya , 
 nurses ' underengament has also been attributed , in part , to an inadequate policy focus in undergraduate and graduate nursing programs . 
 studies provide several strategies that can be applied to enhance nurses ' engagement in policy processes . 
 several studies have emphasized that nurses can engage in policy processes directly by assuming formal leadership positions in the health care system , and indirectly through lobbying and advocacy with policy - makers via professional organizations [ 12 , 17 ] . 
 the nurse leaders can identify issues strategically , work with decision - makers , and understand who holds the power in the health care organizations and who controls the resources for health care in order to influence them . 
 another strategy is to build nurses ' understanding of and competencies in policy and politics by integrating pertinent courses in nursing training and fellowship programs [ 1720 ] . 
 examples of programs developed to enhance nurses ' competency in leadership and policy influence exist mainly in developed countries [ 21 , 22 ] . 
 for instance in the usa a program was developed to train nurse leaders who would influence policy change . 
 the training helped the nurse leaders to understand how policy reforms affect health care , the policy formulation process , and the challenges affecting health care including access , quality , and care as well as the role of media . 
 several authors have emphasized the need for nurses to utilize research as credible evidence to inform policy decisions . 
 however , the studies have found that nurses have not been adequately supported to generate evidence to inform health policies [ 2326 ] . 
 furthermore , nurses from low- and middle - income countries ( lmics ) have inadequate training and mentoring in research as well as scarce resources for conducting research . 
 thus , there is a need to strengthen lmic nurses ' research abilities to enhance their ability to understand , generate , and utilize research knowledge that is useful for policy change . 
 very few emperical studies have examined extent of nurses ' involment policy developemnt process in the african context . 
 a survey of nurse leaders involvement in health policy process in east africa found that nurse leaders ' participation in all stages of policy development was limited and inconsistent . 
 another study looked at nurses involvement in hiv / aids policy in four lmics including kenya and uganda . 
 the study revealed nurses ' lack of involvement in policy processes , poor communication of policies from the national level , and a lack of resources for nurses to implement the policies . 
 published studies are mainly descriptive reviews and have not used a critical theory lens to analyze nurses ' contributions to policy processes . 
 furthermore , studies have rarely compared the perspectives of nurses and other decision - makers regarding nurses ' engagement in policy development and implementation processes . 
 this study applied a critical theory perspective to examine how nurses had been involved in national health policy processes in kenya , eliciting the perspectives of nurses and decision - makers working at various levels of the health care system . 
 feminist theories are related to critical theory but focus on gender domination and discrimination within patriarchal societies [ 28 , 29 ] . from feminist perspectives , 
 analysis examines formal power structures that create or maintain gender inequality , thereby systematically elevating men above women [ 31 , 32 ] . in this study , 
 critical theory helped interrogate historical hierarchies and structures as well as gender - related issues affecting nurses ' participation in policy processes . 
 the overall study applied a two - phased mixed method ( qualitative / quantitative ) design to examine the influence of policies on nurses ' work as well as how nurses were involved in policy processes in the kenyan health care system . 
 this paper focuses on the qualitative results , describing how nurses were involved in national policy processes in the kenyan public health sector . 
 the perspectives of nonnursing decision - makers , national level nurse leaders , and frontline nurses and managers were sought . 
 the specific objectives were ( 1 ) to analyze the extent of nurses ' involvement in the national policy development processes in the health sector ; ( 2 ) to analyse the reasons for nurses ' inadequate involvement in the national policy development in the health sector ; and ( 3 ) to describe mechanisms though which nurses ' involvement in policy development processes can be improved . 
 the study was carried out in the kenyan public health care system , since this sector has primary responsibility for policy development and provides about 55% of health care services . 
 the health delivery system is organized in a pyramidal structure with six levels of care . 
 the community is the first level , followed by dispensaries and medical clinics , health centers and nursing homes , district level hospitals , and provincial level and national level hospitals . 
 decision - making structures exist at national , provincial , and district levels , all of which include nurse representation . 
 although these nurses work at all levels of the health care system , the majority are employed at the district levels . 
 the majority of nurses entered the health care service with diploma or certificate level preparation until the early 1990s when the country started training degree level nurses . 
 kenya 's nursing data base for 2011 indicates that among those employed in the public sector , the majority were certificate nurses ( 53% ) , followed by diploma nurses ( 46% ) , with a small minority of degree level nurses ( 1% ) . 
 decision - makers at national , provincial , and district levels were purposively sampled based on their positions involving policy development or implementation . 
 national level nonnursing and nursing decision - makers employed by the ministry of health were eligible for selection . 
 two provinces ( nyanza and central ) were purposively selected because of their varied geographical locations and differences in health indicators . 
 nyanza province had poor health indicators and few health service resources , while the central province had better health indicators and more health service resources . 
 one district was then purposefully selected from each of these provinces and the two main district public hospitals were the sites for sampling district - level nurses and managers . 
 nurses and nurse managers in these district hospitals were selected based on a sufficiently long work history in the system and 2 years or more in the facility which gave them potential familiarity with many of the policy changes affecting nursing and health system reforms in kenya . 
 open - ended interview guides tailored to participant groups were used with all key informant interviewees . 
 the groups included national level nursing and nonnursing decision - makers , decision - makers at provincial and district levels , and frontline nurses and managers at the hospital level . 
 participants were asked questions about national level health policy - making processes ; extent to which nurses engaged in policy - making and implementation processes ; and ways to improve nurses ' involvement in policy processes . 
 data collection was stopped when saturation was reached . additionally , the researcher kept field notes during the visits . 
 audio - taped data were transcribed verbatim and they were analyzed simultaneously with data collection . 
 transcripts from each participant were read repeatedly before and after coding to gain a sense of the whole interview . 
 interview data were clustered in three groups including nonnursing decision - makers , nurse leaders and frontline nurses , and nurse mangers ; then all the data sets and field notes were triangulated as the analysis proceeded . 
 data was initially coded and divided into contents including nurses ' engagement in policy , reasons for under engagement , and ways to improve nurse ' engagement . 
 matrices were then used to compare categorized responses of national level nonnursing decision - makers , national nurse leaders , and frontline nurses and managers [ 36 , 37 ] . 
 further analysis and interpretation of findings was then done using critical and feminist lens to identify hierarchies and gender related factors that influenced nurses ' engagement in policy development . 
 credibility was enhanced throughout the study process by maintaining accuracy in data collection , analyzing , and reporting . 
 critical reflexivity involving cyclical and continuous comparisons of data collected were used throughout data collection and analysis . 
 these included journaling and writing reflections on processes and decisions [ 36 , 37 ] . 
 transferability was enhanced through the purposive recruitment of participants at different health care system levels . 
 triangulation of data from different types of respondents was done making it possible to compare and confirm similar perceptions across different groups . 
 ethical approval was obtained from university of ottawa health sciences and science research ethics board in canada and great lakes university of kenya ethics review committee . 
 administrative approval was also obtained from the ministry of health in kenya , and appropriate authorities in provincial health offices , district offices , and the two district hospitals where study participants were recruited . 
 a total of 32 interviews were conducted ( table 1 ) . at the national level , 
 five interviews were conducted with nonnursing decision - makers at the ministry of health , one interview with the world health organization country representative and three interviews with national nurse leaders . 
 each of the three national nurse leaders had risen up the ranks from the district level to the national level and had worked for more than 20 years in public service . at the provincial level , 
 two nonnursing decision - makers and two nurse leaders were interviewed in nyanza and one nonnursing decision - maker and two nurse leaders interviewed in central province . at the district level , one nurse manager in each district office and 
 most respondents were of the opinion that nurses were not adequately involved in the health sector policy development processes at the national level . 
 they mentioned that only three nurse leaders from the offices of the chief nurse , the nursing council and the , national nurses association had been invited to participate in national policy committees as part of their jobs . 
 the nurse leaders ' input on these committees was described as revolving around representing nurses ' issues at selected policy tables , communicating policies to nurses , and regulating nursing education and practice . 
 the chief nurse was charged with the highest responsibility of representing all kenyan nurses at policy meetings . 
 two of the nonnursing decision - makers felt that kenyan nurses were adequately involved in national policy - making processes through the nurse leaders ' representation at the national level . 
 in contrast with this view , all nursing participants felt that this representation was inadequate given the large number and diversity of nurses in the country.generally nurses have not been fully involved in policy formulation . 
 many a time nurses are involved after policies have been formulated , and maybe just a few nurses [ national nurses leaders are involved in the policy formulation stage ] ; the province is just informed later that this is happening , but really there has been no good input from nurses . 
 ( national nurse leader ) generally nurses have not been fully involved in policy formulation . 
 many a time nurses are involved after policies have been formulated , and maybe just a few nurses [ national nurses leaders are involved in the policy formulation stage ] ; the province is just informed later that this is happening , but really there has been no good input from nurses . 
 ( national nurse leader ) furthermore , the contributions of these three nursing leaders to the national policy formulation processes were considered inadequate by both the nurse leaders and nonnursing decision - makers but for different reasons . 
 while nonnursing decision - makers emphasized the limitation of nurse leaders ' singular focus on nursing issues , the nurse leaders described the problem of inadequate representation of nurses on a wider set of national policy committees . 
 they described representation on these committees as being primarily heads of departments , the majority of whom were doctors . 
 the public health act , which allows doctors to be the heads of ministerial departments , as well as provincial and district health offices , affords doctors this role.normally the heads of the various departments sit down ; many of them are headed by doctors because of the public health act , and others would be the technical people . 
 ( national nurse leader ) normally the heads of the various departments sit down ; many of them are headed by doctors because of the public health act , and others would be the technical people . 
 ( national nurse leader ) the findings also revealed nurse leaders ' uneven participation in policy meetings . according to the nurse leaders 
 , nurses only attended those policy meetings requiring reports on nurse situations , while doctors were active in every policy meeting regardless of the policy content . 
 furthermore , nurse leaders noted that many of the final policy decisions had been made without nurse leaders being present . 
 frontline nurses and nurse managers at district levels described their primary policy role as being implementers of national policies . even though policy - making was considered a national level activity , nurses at the frontline were concerned that they were not involved . 
 they added that their contribution could be important since nurses better understood the care environment.nurses should be consulted before policies are started because we are the majority , but they decide for us (  ) . 
 nurses have everything that they need ,  education , brains , but they lack chance to air views . 
 ( provincial nurse leader ) nurses should be consulted before policies are started because we are the majority , but they decide for us (  ) . 
 nurses have everything that they need ,  education , brains , but they lack chance to air views . 
 ( provincial nurse leader ) a common view , among frontline nurses , was that they were excluded from policy decisions even in the workplace.we are not involved , yet we are the ones who really are on the ground , we are the backbone of the hospitals . 
 we are like a mother in the kitchen , we are the ones who know how much we need , so we should be really involved in every step , because you can not just tell me  now cook  and even i was not there in the shopping but you want to see what i am cooking ; we should be involved from the word go . 
 ( frontline nurse ) we are not involved , yet we are the ones who really are on the ground , we are the backbone of the hospitals . the other people come to write papers and go away (  ) . 
 we are like a mother in the kitchen , we are the ones who know how much we need , so we should be really involved in every step , because you can not just tell me  now cook  and even i was not there in the shopping but you want to see what i am cooking ; we should be involved from the word go . 
 ( frontline nurse ) due to their limited involvement , frontline nurses and managers felt that their priority work - related issues such as staff shortages , motivation , and resource inadequacies were not being addressed . yet 
 they felt that policies were just pushed on them , so they sat back and watched or did only what they could , as one nurse manager commented : lack of nurses ' involvement in formulation is the cause of poor policy implementation . 
 they are not positive towards it , but since they have to comply , they do it , but not in the right way . 
 when you are involved from the beginning , you understand and own it , and during implementation , you have inward drive to accomplish what you planned (  ) . 
 we feel like we are pushed at the corner , so we remain there and watch things as they are done , or do things because they must be done , not that we feel motivated to do them . 
 they are not positive towards it , but since they have to comply , they do it , but not in the right way . 
 when you are involved from the beginning , you understand and own it , and during implementation , you have inward drive to accomplish what you planned (  ) . 
 we feel like we are pushed at the corner , so we remain there and watch things as they are done , or do things because they must be done , not that we feel motivated to do them . 
 the participants gave several reasons as to why there was inadequate nurses ' participation in national health policy processes . 
 one reason was that policy - making primarily followed a top down approach , emanating from the national level . 
 respondents felt that this approach denied nurses opportunities for direct involvement in national policy decisions because most nurses are employed at lower levels of the health care system . 
 as one nurse manager said  for me policy - making is a thing for those at the top , whether they are doing the right thing or not , to them it does not matter . 
 another frontline nurse commented that  policies are made from above , they are not made according to how you want things to be done , they are just brought and you are supposed to follow them . 
 other reasons for nurses ' lack of engagement in policy formulation mentioned by nonnursing decision - makers were their lack of knowledge about the policy - making process and lack of skills to engage in this process . 
 nurse leaders concurred that nurses did not generally understand the broader issues outside nursing that were necessary to influence policies . 
 they largely blamed this on the omission of any curriculum related to policy issues in basic nursing training . 
 others felt that nurses ' low participation was due to the general design of the health care system , which excluded nurses from policy - making structures.it is by design of health system that nurses are not brought at the policy tables  [ the ] majority of nurses lack knowledge and skills (  ) to support that policy . 
 therefore nurses should be made to add value when an opportunity comes for them to sit at the policy - making tables , not to be a passenger but to be a participant . 
 ( national nurse leader ) it is by design of health system that nurses are not brought at the policy tables  [ the ] majority of nurses lack knowledge and skills (  ) to support that policy . 
 therefore nurses should be made to add value when an opportunity comes for them to sit at the policy - making tables , not to be a passenger but to be a participant . 
 ( national nurse leader ) nurses were also perceived as being isolated from other professionals . 
 nonnursing decision - makers blamed the nursing profession for its tradition of being a  closed  professional group , concerning themselves only with professional issues and not broader issues affecting the health care system . 
 a few of the decision - makers felt that nurses had failed to market themselves as important providers in the health care system . 
 in addition , they felt that nursing had a hierarchical system that hindered junior nurses from participation . 
 one national nonnursing decision - maker was particularly strident in his / her remarks : nurses are allergic to change . 
 nursing profession discourages nurses from thinking because it 's the matron who decides , (  ) . 
 they have put a self - imposed mind block by relying on another person to make decisions (  ) . 
 they go through a very hierarchical system unlike other professions . while a nurse will still report to a nursing officer 
 , she can not make decisions on her own , it is the biggest weakness , there is no freedom , and they maintain the status quo . 
 nursing profession discourages nurses from thinking because it 's the matron who decides , (  ) . 
 they have put a self - imposed mind block by relying on another person to make decisions (  ) . 
 they go through a very hierarchical system unlike other professions . while a nurse will still report to a nursing officer 
 , she can not make decisions on her own , it is the biggest weakness , there is no freedom , and they maintain the status quo . 
 ( nonnursing national decision - maker ) another reason expressed for nurses ' nonengagement was the inability of nurses to bring forward research evidence to inform policy formulation . 
 nurse leaders commented that nurses had inadequate skills for generating and utilizing research evidence to influence policy decisions at national levels . 
 frontline nurses complained that a further impediment was the failure of nurses to take united action to influence policy . 
 they had different professional associations ( the kenya registered nurses ' association and kenya professional nurses association ) , which served different nursing groups and tended to divide nurses and their leaders . 
 participants gave several suggestions regarding how nurses ' participation in the policy process could be enhanced . 
 they mentioned actions to be taken by decision - makers to enhance nurses ' participation in policy - making . 
 these included creating more opportunities for nurses to contribute to policy processes , increasing nurses ' representation on policy - related structures , and reviewing the public health law to allow nurses to compete for leadership positions in the system . 
 in addition , frontline nurses felt that national policy - makers should consult frontline nurses when developing national policies . 
 it was their view that this would enhance bottom - up input on policy - making . 
 as one frontline nurse said : i believe there is a way of channeling suggestions until they reach the national level , maybe through the nurses in charge of hospitals to the provincial then to the national level through writing and meetings . 
 i believe there is a way of channeling suggestions until they reach the national level , maybe through the nurses in charge of hospitals to the provincial then to the national level through writing and meetings . as their representatives 
 , the frontline nurses felt that nurse leaders should come to the ground more often and get views from frontline nurses to include in policies : nurse leaders are at the top , when there are issues to be discussed , they can come down , involve us in discussions and gather information , so that when they make these policies they know it is something that has been discussed , it has been understood and it is needed , because occasionally policies have been made [ and ] when they come down here they do not even work . 
 ( frontline nurse manager ) nurse leaders are at the top , when there are issues to be discussed , they can come down , involve us in discussions and gather information , so that when they make these policies they know it is something that has been discussed , it has been understood and it is needed , because occasionally policies have been made [ and ] when they come down here they do not even work . 
 ( frontline nurse manager ) some nursing participants mentioned that the opinion of frontline nurses could be sought through research and regular meetings and then brought to policy - making processes . 
 in particular , they identified the need to empower nurses through training to enable them to better understand and participate in policy formulation . while the experiences of nurses were thought to be important assets for this input , a broader knowledge of the health care system , of policy - making , and of decision - making processes , were described as essential prerequisites for constructive contributions.the bottom - line 
 is that you must be knowledgeable , you may be a real nursing guru , (  ) your contribution should not only be based on care , of course care is very critical , but you must have general knowledge like how the government operates (  ) . 
 majority of nurses do not know anything apart from nursing , you are supposed to have general knowledge , like international monetary funds policies , how does it affect us ? 
 ( nurse leader 2 ) the bottom - line is that you must be knowledgeable , you may be a real nursing guru , (  ) your contribution should not only be based on care , of course care is very critical , but you must have general knowledge like how the government operates (  ) . 
 majority of nurses do not know anything apart from nursing , you are supposed to have general knowledge , like international monetary funds policies , how does it affect us ? 
 ( nurse leader 2 ) other areas identified for skill - building included political skills needed to engage the government to address policy gaps that affected nurses ' work as one nurse leader said that  nurses in this country lack political skills , they should have political skills in order to dialogue with politicians and other leaders in this country .  in addition the participants mentioned that nurses need data management and research skills to bring evidence to policy . 
 finally , they mentioned actions to be taken by nurses themselves to enhance their ability to participate in policy - making . 
 nonnursing decision - makers strongly suggested that nurses should take the lead to change their situation . 
 they mentioned that nurses should market themselves , progress in their careers , diversify , venture outside the nursing profession , and broaden their curriculum in order to understand broader policy related issues.nurses build empires around themselves (  ) . 
 nurses must be free to venture into other areas and be competent in those areas and not tied to nursing . 
 more often , you find doctors in areas , which are not related to medicine and they fit well . 
 they do not have to link strongly to their profession , they are more independent . 
 ( national nonnurse decision - maker ) nurses build empires around themselves (  ) . 
 nurses must be free to venture into other areas and be competent in those areas and not tied to nursing . 
 more often , you find doctors in areas , which are not related to medicine and they fit well . 
 they do not have to link strongly to their profession , they are more independent . 
 ( national nonnurse decision - maker ) congruent with this decision - maker 's opinion , some of the nurse leaders interviewed added that more nurses should position themselves to take up leadership roles in the system . 
 they indicated that nurses should diversify their knowledge , be united to build a strong voice , collaborate and network within and beyond the country , and be assertive about what they wanted . 
 they added that the nursing council and the professional association should be strengthened to enhance their respective roles in influencing national policies that affect nurses ' work . 
 frontline nurses added that nurse leaders should be more proactive in policy activities so as to influence policy - makers . 
 some frontline nurses felt that this type of advocacy would be an incentive for front line nurses to participate in decision - making in the health care sector . 
 from a critical perspective , two major issues influencing nurses ' involvement in policy processes emerged . 
 these issues were hierarchies and structures influencing decision - making in the health sector and nurse and nursing professional issues . 
 three intersecting hierarchies hindered nurses ' active engagement in policy processes in kenya : health systems hierarchies , interprofessional hierarchies , and intraprofessional hierarchies . 
 the kenyan health systems decision - making structure is organized following political administrative levels including central headquarters and provincial and district levels . 
 the political structures mandate the central administration as the policy - making and service planning level . 
 the kenyan health policy framework of 19942010 emphasized that policy formulation was the role of the central office . this top - down policy - making process limited nurses ' opportunities to engage directly in policy formulation in the health care system . 
 this policy framework delineates a policy implementation rather than a policy formulation role for nurses . 
 given the top - down nature of policy - making processes in the health sector , particularly with the many stakeholders and foreign donors involved , the findings speak to the importance of finding ways to give nurses some voice in decision - making at all levels . 
 the interprofessional hierarchy described within the health sector identified medical doctors as being the dominant policy decision - makers at various levels of the health care system . 
 social constructs of racialized status and gender imbue health care hierarchies and are grounded in the legacy of kenya 's colonial period that ended in 1963 . 
 white colonialists developed health care institutions that favored a male- and white - dominated medical profession over a female- and african - dominated nursing profession . 
 the education system prepared physicians with first degrees while nurses acquired certificates and later diplomas . 
 although baccalaureate degree nursing education in kenya began in the late 1990s , only one percent of nurses in the public health care sector have baccalaureate degrees . 
 resultantly , medical doctors have higher status and are granted more decision - making authority in the system as opposed to nurses whose education and status were , and are considered low , further precluding nurses from higher decision - making positions . 
 the interprofessional hierarchy is further sustained by the legal system , specifically the public health act , which led to the systematic exclusion of nurses from senior leadership positions at various levels of the health care system . according to the act , 
 physicians were to serve as the heads of various departments , administrative directors of districts and hospitals , and managers of various programs , while senior nurses could only be placed in positions to represent nurses , thus limiting their participation in policy and other health care decisions . 
 this legislation thus legitimized the gendered professional hierarchy and further limited the types of issues that nurse leaders could bring to the policy table . 
 nurse leaders ' participation at policy tables was considered nursing centric by both nursing and nonnursing leaders . 
 while the majority of decision - makers blamed nurse leaders for nursing centric participation , this kind of participation was also shaped by the system , which mandated nurse leaders to report only on nursing issues . 
 this is consistent with a global study   of nurse leaders focusing on chief nursing officers , which revealed that , even though these nursing leaders had varied responsibilities associated with their positions , they were only asked to provide nursing - related advice . 
 the study revealed how an intraprofessional hierarchy within the nursing profession played out in the policy arena . 
 nurse leaders at the national level were recognized as the sole professional representatives of other nurses in the country who would provide input on national policy decisions . 
 the intraprofessional hierarchy influenced channels of policy communication to frontline nurses who received new policy directives from their supervisors at every level of the system . 
 this hierarchical pattern of decision - making within nursing may have been exacerbated by the fact that there are only a few nurses in key national level positions , limiting the scope of their policy influence at the national level and precluding them from engaging nurses more proactively at the district level or on the front lines in policy - oriented discussion . 
 in addition , nurses at lower levels felt oppressed by senior nurses who made most of the decisions . from the findings , the nonnursing decision - makers had more to say about nurses ' oppression than nurses themselves . 
 nonnursing decision - makers indicated that nursing was a profession where the oppression of lower level nurses was a problem . 
 however , they were generally pessimistic , indicating that nurses were not yet ready to make a change . 
 the oppression of lower cadre nurses by higher cadre nurses in decision - making has been documented in other studies [ 4042 ] . in the absence of feedback from the grassroots on policy implementation , as identified by participants in this study , the voices of nurses at lower levels might be suppressed further . given the fact that kenya has a decentralized health care system , more participatory approaches could be introduced to enhance nurses ' potential engagement in policy - making , thus improving bottom up planning and decision - making . 
 this would increase the way in which those who have less power in a hierarchy get voice . 
 both nursing and nonnursing participants attributed nurses ' underengagement in policy to issues related to the nurses and the nursing profession . 
 respondents consistently indicated that nurses had inadequate knowledge and skills required to contribute to policy processes . 
 they described this deficiency in nurses ' knowledge as being attributable to a failure of the nursing profession to incorporate policy issues and policy process into nurses ' training curricula , thus maintaining the status quo for nurses ' role in decision - making . 
 studies in other contexts have revealed how lack of policy content in nursing training contributes to nurses ' lack of understanding of policy . 
 given the need to strengthen nursing leadership to influence policies , other programs have emerged to train nurse leaders in ways to influence policy . in the african context , many nurse leaders have been enrolled and trained in a leadership training program offered by international council of nurses . 
 this participation may help strengthen nurses ' competency in engaging in national health care decision - making . 
 study findings also revealed that nurses had inadequate advocacy , research , and political skills required to influence policies . 
 these issues are similar to what has been identified in studies from higher income countries [ 12 , 17 , 23 ] . in these studies , 
 the profession is seen to have been slow in incorporating these issues in nursing education . 
 nonnursing decision - makers in this kenyan study felt that the profession had reinforced its own weaker professional status by failing to enhance nurses ' abilities and competencies to influence decisions at strategic levels in the system . 
 their comments reflected an openness to incorporate nurses ' views if nurses were competent in national policy processes and more knowledgeable about general health care system issues . 
 the findings also revealed that nurses have worked in isolation from other professionals in the system . with the emphasis on professional transformation towards equality and diversity [ 44 , 45 ] 
 this would require nursing champions to take up leadership in working with other health cadres to influence policy change . 
 while leadership competencies have been recognized as key attributes to shaping and influencing policy [ 46 , 47 ] , in this study nursing leadership was viewed as internal to the nursing profession , with concerns about the focus on nurses ' work issues and not general health care issues . 
 nursing champions should not focus on the profession alone ; they must be educated in the broader health systems issues and become patient advocates for health care as they engage with other professionals in leading policy change . 
 kenya has many district level hospitals but data from frontline nurses was mainly collected from two districts given the limitation in funding . 
 in conclusion nurses in kenya have not been adequately involved in national policy development . applying critical theory perspective in the analysis 
 has revealed important health system structures and hierarchy as well as nursing professional factors hindering nurses ' participation in policies making processes . 
 these factors must be addressed if nurses ' input in policy - making is going to be significantly altered . to enhance nurses ' participation in policy processes 
 , the policy - makers should address hierarchies , domination , and gender issues inherent in the health care system and establish mechanisms through which the views of nurses could get to national level decision - makers when policies are being formulated . 
 this can be achieved by enacting legislation in kenya that would allow nurses with competencies in leadership and policy to hold senior positions in the health care system just like doctors . with governance opportunities created through decentralization of power 
 , there is a need for all health professionals to have equal opportunities to be in leadership and management positions that are embedded within the decision - making structures in the health care system . to enhance bottom - up policy - making , national level decision - makers 
 should solicit the views of nurses at the frontline with regard to policies that might affect their work and quality of care . 
 there are well - described processes for this purpose [ 49 , 50 ] but they can not be implemented in isolation of process changes at the district and national levels , which build mechanisms for continuous quality improvement . 
 leadership and advocacy skills should be developed within nursing to enhance nurses ' abilities to carry out policy advocacy at all levels . 
 icn has developed programs aimed at building the capacity for such leadership in societies around the world . 
 these programs include the leadership in negotiation program , the global nursing leadership institute , and the leadership for change program . 
 nurses in this region can benefit from these programs . to enhance their role in advocacy 
 , nurses should unite to build a strong voice to advocate for improvements in national policies that affect their work and quality of care . working in collaboration with other health professionals , nurses can influence decisions to strengthen the health care system , resource allocation , and primary health care as well as patient care . 
 in addition , nursing regulators and educators should be clear that policy involvement is part of nurses ' scope of practice . 
 nurses , particularly at the frontline , should be given orientation on existing national policies in the sector and how these policies are affecting their work environment and patient outcomes . 
 this would be an important step towards better communication of policy impact , creating a much needed feedback chain between those implementing policy and those formulating policy . 
 as members of nursing associations , frontline nurses should encourage and support their nurse leaders to speak for nurses at the national policy tables . 
 in their workplaces , decision - makers should allow other nurses to actively engage in organizational policy decisions during policy implementation 
 . the nursing profession should consider and implement strategies to strengthen nurses ' capacity to contribute to policy decisions that would enhance service delivery . in order to effectively do this 
 , nurses in kenya will need to understand that the policy cycle is complex and influencing policy decisions depends on having a sufficient understanding of the broader issues in the health care system , as well as knowledge and competencies in policy formulation , implementation , and evaluation . to enhance nurses ' awareness , literacy , and competency in policy 
 , nursing educators should introduce a course on policy and policy processes , as well as policy influencing in the nursing training curriculum . 
 finally , the nursing profession should carry forward the notion of evidence - based policy - making . 
 they should develop skills in research and conduct both health systems and clinical research to inform policy - making decisions . 
 research courses should be linked to policy courses and training curricula should include current and locally relevant examples of how evidence can be used to shape policies . 
 this would include strategies and tools for communicating research to policy - makers , like writing policy briefs and position statements based on research evidence . 
 sharing of policy relevant research would contribute to policy change and resource allocation to improve nurses ' work . 
 this is one of the first studies examining the perspectives of both nurses and nonnurses at different system levels regarding nurses ' involvement in national policy - making processes . with the continuing health care policy reforms , further examination of issues of hierarchy , gender , and power inherent in the health care system 
 the question is how do we unravel the deeply embedded hierarchies and power processes that sustain nurses ' status quo in policy change process ? 
 of particular importance is how the hierarchies affect the role and performance of nurses at lower levels and how can nurses influence national leaders to enhance participation in health policy change processes . 
 furthermore , this study did not carry out an in - depth examination of the competencies held by nurse leaders that would be useful for policy change ; thus future research should examine these aspects .","the aim of this study was to critically examine how nurses have been involved in national policy processes in the kenyan health sector . 
 the paper reports qualitative results from a larger mixed method study . 
 national nonnursing decision - makers and nurse leaders , and provincial managers as well as frontline nurse managers from two kenyan districts were purposefully selected for interviews . 
 interviews dealt with nurses ' involvement in national policy processes , factors hindering nurses ' engagement in policy processes , and ways to enhance nurses ' involvement in policy processes . 
 critical theory and feminist perspectives guided the study process . 
 content analysis of data was conducted . 
 findings revealed that nurses ' involvement in policy processes in kenya was limited . 
 only a few nurse leaders were involved in national policy committees as a result of their positions in the sector . 
 critical analysis of the findings revealed that hierarchies and structural factors as well as nursing professional issues were the primary barriers constraining nurses ' involvement in policy processes . 
 thus , there is need to address these factors both by nurses themselves and by nonnursing decision makers , in order to enhance nurses engagement in policy making and further the contribution to quality of services to the communities .",pubmed
"uti is a common problem in infants and young children ( 1 , 2 ) ; however , its frequency , symptoms and causative organisms varies in accordance with sex , age , and gender ( 3 ) . unlike other age groups neonates present with non - specific signs and symptoms ( 4 ) warranting a complete sepsis workup followed by a 7 to 14 day - course of antibiotic therapy ( 2 ) , typically parenteral ampicillin plus an aminoglycoside ( 5 ) or a third - generation cephalosporin ( 6 , 7 ) . 
 recent reports on emerging patterns of resistance toward empirical antibiotics ( 812 ) , especially ampicillin ( 1317 ) , have made the early choice of antibiotics a more challenging decision ( 8) . the criticalness of issue lies in the fact that those diagnosed earlier and treated accordingly , are less likely to bear long - term consequences imposing an increased burden on health care systems ( 18 ) . 
 clinical manifestations of uti in neonates are mostly non - specific and of a systemic nature ( 15 ) like fever , irritability , lethargy , vomiting , growth failure , abnormal urination- namely oliguria , polyuria or malodorous urine , and jaundice ( 14 , 1921 ) . 
 the non - specific presentations often make uti hard to diagnosis ; however , its reported prevalence ranges from 4 - 25% ( 22 ) in in very low weight infants ( birth weight  1000 g ) to 0.1 - 1% in term infants ( 23 ) . 
 additionally , uti in newborns could lead to urosepsis or neonatal sepsis that is why it is often managed as a case of sepsis ( 24 ) . 
 most utis in infants are caused by gram - negative bacilli ( 23 , 2527 ) . 
 e. coli has been the most prevalent organism causing uti in all ages including neonatal period ( 1 , 21 , 2830 ) accounting for as many as 80% of isolates in many studies ( 25 ) . 
 other enterobacteriaceae causing uti in neonates include klebsiella , enterobacter , citrobacter , proteus , providencia , morganella , serratia , and salmonella ( 6 , 26 , 27 ) . 
 gram - positive bacteria like staphylococcus and enterococcus have less been retrieved from neonates with uti ( 2 , 26 ) . 
 this is not possible unless a continuous surveillance is done and let therapeutic choices to be updated accordantly . 
 these motivated researchers to retrospectively review all uti cases managed in an inpatient setting of neonatal division and aim to this end . 
 the primary intention of this study was to see the frequency of uti causative organisms in neonates admitted to neonatal division of bahrami children s hospital over an eight  year period . 
 the study accomplished at the neonatal division of bahrami children s hospital - tehran , iran , the largest tertiary pediatric center in the eastern end of tehran . 
 all cases over an eight - year period ( june 2004 to june 2012 ) constituted the population of current study the source of extracted data were the hospital records of the patients regarding age ( day ) , sex , date and method of urine sample collection , urine culture results , and antibiogram reports . 
 the latter routinely was based on the report of laboratory unit of bahrami children s hospital which reports antibiogram by semi - quantitative approachbased on diffusion technique , and the available laboratory standards . 
 current study designed as a case series using simple non - random sampling to recruit all cases with diagnosis of uti admitted to neonatal division of bahrami children hospital tehran , iran in 2004-to-2012 period . in this study , uti was defined ( 6 ) as : i ) a pure growth of single bacteria from a urine sample obtained by suprapubic aspirate , ii ) 10 cfu / ml from a urine sample obtained by catheterization . 
 cases with more than one episode of uti were separately analyzed if they occurred more than 14 days apart . 
 only the result of the first positive urine culture was extracted . in the event of two separate initial positive cultures , result of suprapubic aspirate 
 cases diagnosed on the basis of a urine bag sample were not included in the study . 
 those with incomplete records , including absence of two confirmatory progress notes regarding urine sample collection method were excluded from the study . to increase accountability of results , 
 only those cases included which had a physician progress note and nursing staff note on the way urine is collected . 
 mixed growth urine cultures denoting growth of two or more organisms in one sample were excluded , as they are likely to represent contaminated samples . 
 17 ) software spreadsheets . ultimately , quantitative data displayed as minimum , maximum , means , standard deviation , and qualitative data as the relative frequency . 
 all research ethics and regulations adopted by the iranian medical commission of scientific research council were considered in every steps of this study and researchers will adhere to it . academic honesty and trustworthiness , impartiality and avoiding certain trends 
 have been respected ; however , the study was not posed with any other ethical prohibition and was approved by the research ethics committee of the tehran university of medical sciences . 
 the study accomplished at the neonatal division of bahrami children s hospital - tehran , iran , the largest tertiary pediatric center in the eastern end of tehran . 
 all cases over an eight - year period ( june 2004 to june 2012 ) constituted the population of current study the source of extracted data were the hospital records of the patients regarding age ( day ) , sex , date and method of urine sample collection , urine culture results , and antibiogram reports . 
 the latter routinely was based on the report of laboratory unit of bahrami children s hospital which reports antibiogram by semi - quantitative approachbased on diffusion technique , and the available laboratory standards . 
 current study designed as a case series using simple non - random sampling to recruit all cases with diagnosis of uti admitted to neonatal division of bahrami children hospital tehran , iran in 2004-to-2012 period . in this study , uti was defined ( 6 ) as : i ) a pure growth of single bacteria from a urine sample obtained by suprapubic aspirate , ii ) 10 cfu / ml from a urine sample obtained by catheterization . 
 cases with more than one episode of uti were separately analyzed if they occurred more than 14 days apart . 
 only the result of the first positive urine culture was extracted . in the event of two separate initial positive cultures , result of suprapubic aspirate 
 cases diagnosed on the basis of a urine bag sample were not included in the study . 
 those with incomplete records , including absence of two confirmatory progress notes regarding urine sample collection method were excluded from the study . to increase accountability of results , 
 only those cases included which had a physician progress note and nursing staff note on the way urine is collected . 
 mixed growth urine cultures denoting growth of two or more organisms in one sample were excluded , as they are likely to represent contaminated samples . 
 17 ) software spreadsheets . ultimately , quantitative data displayed as minimum , maximum , means , standard deviation , and qualitative data as the relative frequency . 
 all research ethics and regulations adopted by the iranian medical commission of scientific research council were considered in every steps of this study and researchers will adhere to it . academic honesty and trustworthiness , impartiality and avoiding certain trends 
 have been respected ; however , the study was not posed with any other ethical prohibition and was approved by the research ethics committee of the tehran university of medical sciences . 
 a total of 73 cases ( mean age , 14.14  7.68 days ; mean birth weight of 3055.85  623.00 grams ; and mean admission weight of 3180.55  610.25 grams ) fulfilled the inclusion criteria and recruited in the study . 
 boys constituted 69.9% ( 51 neonates ) of the study population , bringing a boys - to - girls ratio of 2.32 . 
 four different bacteria were isolated from retrospectively assessed cases , namely e. coli , enterobacter , klebsiella , and s. epidermidis no cases of proteus , pseudomonas , serratia , s. aureus , enterococcus were found over the study period . 
 e. coli was the most prevalent organism of the study with a relative frequency of 64.4% ( 47 cases ) followed by enterobacter with a relative frequency of 19.2% ( 14 case ) . 
 however , frequency of isolated organisms as well as their relative frequency in study population is represented in table 1 distribution of organisms isolated from the urine in the study in this study , e. coli found to be mostly resistant toward ampicillin ( 93.6% ( 44 cases ) ) , cefixime 85.7% and cephalexin 77.3% respectively . 
 the most sensitivity of e. coli toward the antibiotics commonly used as first line neonatal empirical therapy were toward cefotaxime ( 63.6% ) ; however , its most susceptibility was to norfloxacin ( 100% ) and ciprofloxacin ( 100% ) respectively in 14 and 12 patients . 
 the general overview of e. coli susceptibility pattern toward all applied discs is summarized in table 2 . 
 distribution of study escherichia coli antibiotic susceptibility pattern enterobacter susceptibility pattern was representative of a high resistance to amikacin ( 100% ) and ampicillin ( 92.8 ) respectively in 4 and 13 cases , and high sensitivity to ceftazidime ( 100% ) and norfloxacin ( 100% ) respectively in 5 and 6 cases ; nonetheless , its most susceptibility toward commonly used neonatal empirical therapy antibiotics was in face of ceftizoxime ( 71.4% ) . 
 notably less frequent isolated uropathogens of this study , klebsiella and s. epidermidis , were found to be completely ( 100% ) resistant to ampicillin . 
 distribution of study enterobacter antibiotic susceptibility pattern distribution of study s. epidermdiis antibiotic susceptibility pattern distribution of study klebsiella antibiotic susceptibility pattern 
 current study showed e. coli to be the most prevalent ( 64.4% ) organism causinguti in neonates admitted to neonatal division of bahrami children s hospital over an eight  year . besides gram negative bacteria comprised the leading cause ( 95.8% ) of uti in the study population . 
 this finding is in agreement with most of past studies ( e.g. , 1 , 21 , 28 - 30 ) over the subject . 
 it is already known that uti incidence in boys exceeds that of girls in first few months of life ; however , this sex - related dominancy would change toward girls by end of first year and thereafter ( 2 , 6 ) . based on epidemiological data on neonatal uti , 
 this boys - to - girls incidence ratio estimated as 2 to 1 ( 6 ) . 
 current study 69.9% frequency of uti among boys comes to a boys - to - girls ratio of 2.32 , which is compatible with foreseeable higher incidence of in boys . on an overview of previous national studies , 
 e. coli has been reported as the leading uropathogen in neonates with uti with various relative frequencies : 80.5% in qazvin ( 14 ) , 76.5% in tehran ( 20 ) , 76.3% in tehran ( 33 ) , 50% in kashan ( 34 ) , and 50% in uromia ( 35 ) . 
 uunlike previous national based studies , proteus ( 14 ) , pseudomonas ( 14 , 35 ) , serratia ( 36 ) , s. aureus ( 20 ) were not retrieved in this study . in compare to other age groups , continuous assessment of uropathogen epidemiology in neonatal period is of profound significance , as uti is hard to diagnose at this age group and could potentially lead to systemic infection leaving potential life threatening and long term sequel . moreover 
 this would enable clinicians to choose an empirical therapy regimen that is most clinically and epidemiologically ideal till further microbiologic results are available . 
 furthermore it is an accepted practice that neonatal divisions take into account the prevailing antibiotic sensitivities of local bacterial isolates before opting an empirical regimen ( 32 ) . 
 having said all these , to our knowledge , this is an area which has least been underattention of neonatal divisions across the country . 
 most of previous studies on neonatal uti were focused on the relationship of neonatal uti and some non - specific signs and symptoms such as neonatal jaundice . 
 recent observed resistance patterns towards antibiotics commonly being used in empirical therapy of neonates with uti and or sepsis , namely ampicillin along with aminoglycoside or a third - generation cephalosporin ( 1317 ) , have raised concern over the efficacy of current treatment protocols and even the need for changes in present regimens ( 912 ) . 
 resistant toward ampicillin has been reported by many studies ( 1317 ) . however , there are few studies assessing susceptibility pattern of neonatal uti organisms toward a wide spectrum of antibiotics ( 8 , 31 , 32 ) . on the view point of susceptibility to neonatal empirical therapy antibiotics at this study , e. coli was resistant to ampicillin in 93.6% , ceftizoxime in 78.9% gentamycin in 59.5% and amikacin in 56.7% cases . 
 less frequently isolated organisms also showed different but consistent susceptibility patterns : enterobacter resistance was : 92.9% to ampicillin , 69.2 - 100% to aminoglycosides ( gentamycin and amikacin ) , and 0 - 28.6% to third - generation cephalosporins ; klebsiella and s. epidermidis were found to be sensitive to gentamycin 50% and 100% respectively . 
 comparing our results with few past national - based studies is representative of some shift in sensitivity patterns of uropathogens isolated from neonates with uti . 
 movahedian et al . ( 34 ) reported e. coli resistant ratio to be 78.9% , 64.7% , 5.25% , and 5.25% toward cephalexin , ampicillin , amikacin , and gentamycin respectively in 2007 . 
 our study found a dramatic almost ten - fold resistance to amikacin and gentamycin and 144% increase -in resistance rate to ampicillin ; however , the resistance to cephalexin has a little decreased . 
 it is not clear whether this changein susceptibility pattern of e. coli toward ampicillin , amikacin , and gentamycin is secondary to overusage of these antibiotics , as one of accepted empirical regimen , or it is due to an original different in susceptibility patterns in kashan and tehran cities , where the studies accomplished . on the same comparative view to movahedian et al study , 
 klebsiella susceptibility toward ampicillin , cephalexin , and gentamicin has not changed but klebsiella exhibits an almost ten - fold resistance rate to amikacin . 
 the strengths of our study include : i ) the large sample size relative to low prevalence of urinary tract infection in term neonates , 0.1% to 1% ( 23 ) . 
 it needs to be mentioned that in bahrami children hospital , pre - term neonates with diagnosis of sepsis most often would be admitted to nicu than neonatal division where our study accomplished . 
 ii ) the long period of the study which enabled researcher to recruit larger number of cases . 
 iii ) the accountability of the urine samples included in the study ( suprapubic aspirate or catheterization ) . 
 all included cases were diagnosed upon a suprapubic aspirate or catheterizations which are the most accepted means ( 6 , 37 ) of collecting urine sample in acute settings in need of instituting an empirical antibiotic . besides , all cases with incomplete double - notes on the method of urine collection ( physician note and nursing staff note ) were excluded from the study population to : i ) adhere to study inclusion criteria , ii ) decreasing chance of the recruiting contaminated samples in the study 
 i ) inevitably miss some cases due to their incomplete records , ii ) report the susceptibility results on the basis of number of individual antibiotic discs applied over total cases , iii ) not having the chance of assessing isolated uropathogens toward a wide spectrum of antibiotics . 
 nonetheless , ampicillin was the sole antibiotic which had been applied to almost all studied cases . 
 the strengths of our study include : i ) the large sample size relative to low prevalence of urinary tract infection in term neonates , 0.1% to 1% ( 23 ) . 
 it needs to be mentioned that in bahrami children hospital , pre - term neonates with diagnosis of sepsis most often would be admitted to nicu than neonatal division where our study accomplished . 
 ii ) the long period of the study which enabled researcher to recruit larger number of cases . 
 iii ) the accountability of the urine samples included in the study ( suprapubic aspirate or catheterization ) . 
 all included cases were diagnosed upon a suprapubic aspirate or catheterizations which are the most accepted means ( 6 , 37 ) of collecting urine sample in acute settings in need of instituting an empirical antibiotic . besides , all cases with incomplete double - notes on the method of urine collection ( physician note and nursing staff note ) were excluded from the study population to : i ) adhere to study inclusion criteria , ii ) decreasing chance of the recruiting contaminated samples in the study . 
 limitations of our study include its retrospective nature which made us to : i ) inevitably miss some cases due to their incomplete records , ii ) report the susceptibility results on the basis of number of individual antibiotic discs applied over total cases , iii ) not having the chance of assessing isolated uropathogens toward a wide spectrum of antibiotics . 
 nonetheless , ampicillin was the sole antibiotic which had been applied to almost all studied cases . 
 according to previous studies ( 31 , 32 ) , the guiding key to choose the empirical antibiotic with the most efficacy , and the least side effect is understanding of causative organisms susceptibility pattern . otherwise , it is less likely to manage as efficacious as possible . 
 the susceptibility patterns not only are different over various centres but also vary over the time . 
 the most prevalent organisms implicated in this study were significantly resistant to ampicillin - a frequent combining to empirical antibiotic regimen . 
 having reviewed neonatal uropathogens over the past 8 years , authors believes a third generation cephalosporins plus an aminoglycoside would be a better choice to ampicillin plus an aminoglycoside as the first line of empiric therapy of neonatal uti . 
 lack of extensive and multi - central studies over neonatal uti in iran warrants prospective future studies on this subject , and authors believes the results of this study could stress the necessity to conduct such projects and motivate further researcher on assessing the subject . 
 ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .","abstractbackgroundsusceptibility pattern of organisms causing urinary tract infection ( uti ) in neonate would potentially improve the clinical management by enabling clinicians to choose most reasonable first line empirical antibiotics . 
 this study aimed to this end by studying isolated organisms from neonates with uti in an inpatient setting.methodscurrent retrospective study has recruited all cases of neonatal uti diagnosed through a suprapubic / catheterized sample , admitted to neonatal division of bahrami children s hospital , tehran , iran , from june 2004 to june 2012.resultsescherichia coli was the dominant ( 64.4% ) bacteria among a total of 73 cases ( 69.9% boys and 30.1% girls ; aged 14.14  7.68 days ; birth weight of 3055.85  623.00 g ) and enterobacter ( 19.2% ) , klebsiella ( 12.3% ) , and staphylococcus epidermdisis ( 4.1% ) were less frequent isolated bacteria . 
 e. coli was mostly resistant to ampicillin ( 93.6% ) , cefixime ( 85.7% ) and cephalexin ( 77.3% ) , and sensitive to cefotaxime ( 63.6% ) . 
 enterobacter found to be most resistant to amikacin ( 100% ) , ampicillin ( 92.85% ) , and most sensitive to ceftizoxime ( 71.4%).conclusiona high ratio ( > 92.85% ) of resistance toward ampicillin was observed among common neonatal uti bacterial agents . 
 having this finding along with previous reports of emerging resistance of neonatal uropathogensto ampicillin could be a notion that a combination of a third generation cephalosporin and an aminoglycoside would be a more reasonable first choice than ampicillin plus an aminoglycoside .",pubmed
"congenital adrenal hyperplasia ( cah ) describes a group of inherited autosomal recessive disorders that cause a deficiency in an adrenal enzyme resulting in altered cortisol and aldosterone secretion . 
 the most frequent cah variant , accounting for 95% of all affected patients , is 21-hydroxylase deficiency ( 21-ohd ) and is caused by inactivating mutations in the 21-hydroxylase gene ( cyp21a2 ) . 
 the deficiency of 21-hydroxylase causes a decrease in negative feedback to the hypothalamic - pituitary - adrenal ( hpa ) axis , and subsequent hyperplasia of the adrenal gland . 
 cortisol and aldosterone precursors are subsequently diverted to androgens , causing increased virilization and a chronic hyperandrogenic state . 
 there are different clinical forms of cah associated with 21-ohd : classical cah , the most severe form comprises both salt - wasting ( sw ) and simple virilizing ( sv ) forms , and the non - classical ( nc ) form which may be asymptomatic or associated with symptoms of postnatal or even adult onset hyperandrogenism . 
 several studies have suggested high concordance rates between genotype and phenotype in patients with the classic form of cah . 
 treatment of cah consists of replacement of glucocorticoids and , when necessary , mineralocorticoids to prevent adrenal crises and to suppress the abnormal secretion of androgens and steroid precursors from the adrenal cortex . 
 conventional glucocorticoid replacement can not , however , mimic the natural circadian rhythms of cortisol release . even in adequately treated patients , 
 there are reports of clinical evidence of both over - suppression with glucocorticoid including iatrogenic cushing 's syndrome , obesity , and decreasing growth velocity and under - suppression including virilization , menstrual disorders and infertility , growth suppression , and development of adrenal tumors . 
 cah constitutes an interesting natural model for studying interactions between hormones and the brain . until now , only a few studies described brain magnetic resonance imaging ( mri ) abnormalities in cah patients . 
 hormonal imbalances during early development and exposure to excess exogenous glucocorticoids are the most probable explanations for these mri findings.[711 ] in the present study , we investigated the frequency of white matter changes and temporal lobe structures dysgenesis in patients with cah due to 21-ohd . 
 we studied 26 patients with cah due to 21-ohd ( 11 males and 15 females , mean age  sd = 27.4  8.2 years , range = 16.5 - 48 years ) , who were followed at the department of endocrinology in mahdia and sfax , in tunisia . 
 this sample includes all patients with cah , aged more than 16 years and regularly followed at these two clinics since 1982 . 
 the diagnosis of cah was based on clinical and biochemical criteria ( i.e. , elevated levels of 17-hydroxyprogesterone ( 17-ohp ) and androstenedione , acth stimulation test ) . 
 ten patients ( 6 m , 4 f ) had the sw form of cah . they had been diagnosed in their first year of life and had been treated , from the time of diagnosis , with glucocorticoids and mineralocorticoids . 
 eight patients ( 5 m , 3 f , age at diagnosis : between birth and 6 years ) were diagnosed as classical sv patients . in the other eight patients ( women only in this group ) , the nc form was diagnosed between 15 and 44 years of age . 
 twenty - one patients were started on a regimen of hydrocortisone ( hc ) , given 2 or 3 times daily , whereas the remaining five patients ( three with the nc form and two with the classic sv form ) were treated with dexamethasone ( once daily ) . 
 salt wasters were treated with 27.9  9.6 mg / m per day of hc the first 2 years of life , and the doses were decreased to 17.6  6.6 mg / m per day during childhood . 
 daily doses of hc in patients with classical and nc forms were respectively , 17.3  4.6 mg / m and 16.04  3.4 mg / m during adulthood . for dexamethasone , 
 clinical and brain magnetic resonance imaging findings in 26 patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency the adequacy of therapy was monitored periodically on the basis of clinical and laboratory data , in accordance with current guidelines . 
 patients were classified as under adequate hormonal control if 50% or more of the total serum androgen levels were within normal limits for age or if 50% or more of the baseline serum 17-ohp concentrations were 2.0 - 10 ng / ml ( 6 - 30 
 the mean duration of the follow - up period was 18.5  9.3 ( range 3 - 41.5 ) years . 
 seven patients ( five with the sw form and two with the sv form ) had experienced a sw adrenal crisis in the neonatal period . one female patient ( patient 
 she was started on l - thyroxine 10 g / kg per day at the age of 6 months , but she was irregular in follow - up and was not compliant with treatment . 
 4 ) with the sw form developed hypertension at 8 years of age and responded well to nifedipine . 
 detailed investigations ( renal doppler ultrasound , adrenal ct scan , urinary metanephrine , 11-deoxycortisol , and aldosterone concentrations ) failed to detect any cause for secondary hypertension , and a diagnosis of essential hypertension was made . 
 all our patients or their parents gave their informed consent to participate in the study and underwent neurological examination and brain mri . 
 the imaging protocol included axial and sagittal spin - echo ( se ) t1-weighted [ repetition time ( tr ) 550 ms , echo time ( te ) 15 ms , flip angle 90 ] , axial and coronal enhanced t1-weighted ( tr 550 ms , te 15 ms , flip angle 90 , axial fast se ( fse ) t2-weighted ( tr 5000 ms , te 94 ms , flip angle 90 ) , axial proton - density ( pd ) t2-weighted ( tr 2500 ms , te 18 ms , flip angle 90 ) , and coronal fluid - attenuated inversion recovery [ flair ; tr 7500 ms , te 94 ms , inversion time ( ti ) 2200 , flip angle 90 ] sections . 
 white matter changes were defined as areas of increased signal intensity on intermediate and t2-weighted sequences . 
 thin coronal mri slices , perpendicular to the axis of the hippocampus were used for determining hippocampal dysplasia ( hippocampus with distorted anatomy , small volume , disturbed internal architecture , and increased signal on t2 and flair images ) . 
 amygdala was regarded as dysplastic if it was smaller than the contralateral side , if increased signal on t2 and flair sequences was observed , or both . 
 further investigations , including viral serologies , amino acid profile , organic acid profile , lactate , ceruloplasmin , lumbar puncture and arylsulfatase a activity , were performed in a16.5-year - old patient ( patient no . 
 4 ) with the classical sw form of cah and who presented with hypertension , obesity , mental decline , tremor , cerebellar syndrome , white matter hyperintensities , and agenesis of the corpus callosum . 
 we studied 26 patients with cah due to 21-ohd ( 11 males and 15 females , mean age  sd = 27.4  8.2 years , range = 16.5 - 48 years ) , who were followed at the department of endocrinology in mahdia and sfax , in tunisia . 
 this sample includes all patients with cah , aged more than 16 years and regularly followed at these two clinics since 1982 . 
 the diagnosis of cah was based on clinical and biochemical criteria ( i.e. , elevated levels of 17-hydroxyprogesterone ( 17-ohp ) and androstenedione , acth stimulation test ) . 
 ten patients ( 6 m , 4 f ) had the sw form of cah . they had been diagnosed in their first year of life and had been treated , from the time of diagnosis , with glucocorticoids and mineralocorticoids . 
 eight patients ( 5 m , 3 f , age at diagnosis : between birth and 6 years ) were diagnosed as classical sv patients . in the other eight patients ( women only in this group ) , the nc form was diagnosed between 15 and 44 years of age . 
 twenty - one patients were started on a regimen of hydrocortisone ( hc ) , given 2 or 3 times daily , whereas the remaining five patients ( three with the nc form and two with the classic sv form ) were treated with dexamethasone ( once daily ) . 
 salt wasters were treated with 27.9  9.6 mg / m per day of hc the first 2 years of life , and the doses were decreased to 17.6  6.6 mg / m per day during childhood . 
 daily doses of hc in patients with classical and nc forms were respectively , 17.3  4.6 mg / m and 16.04  3.4 mg / m during adulthood . for dexamethasone , 
 clinical and brain magnetic resonance imaging findings in 26 patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency the adequacy of therapy was monitored periodically on the basis of clinical and laboratory data , in accordance with current guidelines . 
 patients were classified as under adequate hormonal control if 50% or more of the total serum androgen levels were within normal limits for age or if 50% or more of the baseline serum 17-ohp concentrations were 2.0 - 10 ng / ml ( 6 - 30 
 the mean duration of the follow - up period was 18.5  9.3 ( range 3 - 41.5 ) years . 
 seven patients ( five with the sw form and two with the sv form ) had experienced a sw adrenal crisis in the neonatal period . one female patient ( patient 
 she was started on l - thyroxine 10 g / kg per day at the age of 6 months , but she was irregular in follow - up and was not compliant with treatment . 
 4 ) with the sw form developed hypertension at 8 years of age and responded well to nifedipine . 
 detailed investigations ( renal doppler ultrasound , adrenal ct scan , urinary metanephrine , 11-deoxycortisol , and aldosterone concentrations ) failed to detect any cause for secondary hypertension , and a diagnosis of essential hypertension was made . 
 all our patients or their parents gave their informed consent to participate in the study and underwent neurological examination and brain mri . 
 all patients underwent neurological and mri examinations . the mri examination was performed with a 1 t. siemens magnetom impact system . 
 the imaging protocol included axial and sagittal spin - echo ( se ) t1-weighted [ repetition time ( tr ) 550 ms , echo time ( te ) 15 ms , flip angle 90 ] , axial and coronal enhanced t1-weighted ( tr 550 ms , te 15 ms , flip angle 90 , axial fast se ( fse ) t2-weighted ( tr 5000 ms , te 94 ms , flip angle 90 ) , axial proton - density ( pd ) t2-weighted ( tr 2500 ms , te 18 ms , flip angle 90 ) , and coronal fluid - attenuated inversion recovery [ flair ; tr 7500 ms , te 94 ms , inversion time ( ti ) 2200 , flip angle 90 ] sections . 
 white matter changes were defined as areas of increased signal intensity on intermediate and t2-weighted sequences . 
 thin coronal mri slices , perpendicular to the axis of the hippocampus were used for determining hippocampal dysplasia ( hippocampus with distorted anatomy , small volume , disturbed internal architecture , and increased signal on t2 and flair images ) . 
 amygdala was regarded as dysplastic if it was smaller than the contralateral side , if increased signal on t2 and flair sequences was observed , or both . 
 further investigations , including viral serologies , amino acid profile , organic acid profile , lactate , ceruloplasmin , lumbar puncture and arylsulfatase a activity , were performed in a16.5-year - old patient ( patient no . 
 4 ) with the classical sw form of cah and who presented with hypertension , obesity , mental decline , tremor , cerebellar syndrome , white matter hyperintensities , and agenesis of the corpus callosum . all these investigations revealed negative or normal results . 
 on neurological examination , mental retardation was evident in three patients ( patient nos . 4 , 6 and 23 ) . 
 on intellectual quotient test , it was moderate in patient nos . 4 and 6 ( both had developed salt losing crisis during the neonatal period ) and profound in patient no . 23 
 other neurological signs included postural and action tremor in two patients ( patient nos . 4 and 23 ) , 
 1 ) , and static and kinetic cerebellar syndrome in one patient ( patient no . 
 4 ) . at the time of inclusion in the study , mean morning baseline 17-ohp in serum for the whole cohort was 22.51 ng / ml ( range , 1.1 - 77 ) ; mean morning androstenedione levels were 5.25 ng / ml ( range , 1.6 - 24 ) and mean morning plasma acth levels were 118.31 pg / ml ( range , 6.5 - 537 ) . during the treatment period , 
 poor hormonal control , with elevation of 50% or more of 4-androstenedione and 17-ohp serum measurements , was present in 16 patients ( 61.5% ) . 
 these were chronically non - compliant patients , mainly because of ignorance and lack of understanding . the remaining patient ( patient no . 
 eleven patients ( five males and six females , 42.3% ) showed mri abnormalities : one patient was over - treated ( patient no . 
 8) , two patients had good hormonal control ( patient nos . 13 and 15 ) and eight patients were poorly controlled due to non - compliance with their medication . 
 eight patients ( seven with the classic form and one with the nc form ) evidenced white matter hyperintensities on t2-weighted and flair images . 
 these hyperintensities were located in periventricular regions in seven patients [ figure 1a ] and in cerebellar white matter in the remaining patient [ patient no . 
 11 ) with the classical sv form showed moderate right temporal lobe atrophy [ figure 3 ] . indeed 
 21 , hippocampus was globular in shape and its internal structures were not identified [ figure 5 ] . other mri findings included partially empty sella in one patient ( patient no . 
 25 ) and cortico - subcortical atrophy associated with complete agenesis of the corpus callosum in another patient [ patient no . 
 a ) t2-weighted axial mri sequence showing bilateral periventricular white matter hyperintensities and cortico - subcortical atrophy in a 16.5-year - old patient affected by swcah ( patient no . 
 4 ) t1-weighted sagittal mri sequence showing complete agenesis of the corpus callosum in a 16.50 year old patient affected by swcah ( patient no . 
 4 ) axial flair and t2 mri showing bilateral cerebellar white matter hyperintensities in a 21-year - old man affected by sw cah ( patient no . 
 1 ) axial t1-weighted image showing moderate atrophy in the right anterior temporal lobe in a 47.5-year - old man affected by simple virilizingcah ( patient no . 
 11 ) t2-weighted mri , coronal section , showing right hippocampal atrophy in a 36-year - old woman affected by non - classic cah ( patient no . 
 23 ) coronal t2-weighted mri in a 30.5-year - old woman affected by non - classic cah . 
 the right hippocampus is moderately round - shaped and its internal structures are not identified ( patient no . 
 on neurological examination , mental retardation was evident in three patients ( patient nos . 4 , 6 and 23 ) . 
 on intellectual quotient test , it was moderate in patient nos . 4 and 6 ( both had developed salt losing crisis during the neonatal period ) and profound in patient no . 23 
 other neurological signs included postural and action tremor in two patients ( patient nos . 4 and 23 ) , 
 1 ) , and static and kinetic cerebellar syndrome in one patient ( patient no . 
 at the time of inclusion in the study , mean morning baseline 17-ohp in serum for the whole cohort was 22.51 ng / ml ( range , 1.1 - 77 ) ; mean morning androstenedione levels were 5.25 ng / ml ( range , 1.6 - 24 ) and mean morning plasma acth levels were 118.31 pg / ml ( range , 6.5 - 537 ) . during the treatment period , nine patients ( 34.6% ) showed well - controlled cah . 
 poor hormonal control , with elevation of 50% or more of 4-androstenedione and 17-ohp serum measurements , was present in 16 patients ( 61.5% ) . 
 these were chronically non - compliant patients , mainly because of ignorance and lack of understanding . the remaining patient ( patient no . 
 eleven patients ( five males and six females , 42.3% ) showed mri abnormalities : one patient was over - treated ( patient no . 
 . 13 and 15 ) and eight patients were poorly controlled due to non - compliance with their medication . 
 eight patients ( seven with the classic form and one with the nc form ) evidenced white matter hyperintensities on t2-weighted and flair images . 
 these hyperintensities were located in periventricular regions in seven patients [ figure 1a ] and in cerebellar white matter in the remaining patient [ patient no . 
 11 ) with the classical sv form showed moderate right temporal lobe atrophy [ figure 3 ] . indeed 
 21 , hippocampus was globular in shape and its internal structures were not identified [ figure 5 ] . 
 25 ) and cortico - subcortical atrophy associated with complete agenesis of the corpus callosum in another patient [ patient no . 
 a ) t2-weighted axial mri sequence showing bilateral periventricular white matter hyperintensities and cortico - subcortical atrophy in a 16.5-year - old patient affected by swcah ( patient no . 
 4 ) t1-weighted sagittal mri sequence showing complete agenesis of the corpus callosum in a 16.50 year old patient affected by swcah ( patient no . 
 4 ) axial flair and t2 mri showing bilateral cerebellar white matter hyperintensities in a 21-year - old man affected by sw cah ( patient no . 
 1 ) axial t1-weighted image showing moderate atrophy in the right anterior temporal lobe in a 47.5-year - old man affected by simple virilizingcah ( patient no . 
 11 ) t2-weighted mri , coronal section , showing right hippocampal atrophy in a 36-year - old woman affected by non - classic cah ( patient no . 
 23 ) coronal t2-weighted mri in a 30.5-year - old woman affected by non - classic cah . 
 the right hippocampus is moderately round - shaped and its internal structures are not identified ( patient no . 
 our study provides evidence of an increased frequency of white matter abnormalities and temporal lobe structures dysgenesis in cah patients . in the past , only a few studies described brain mri abnormalities in cah.[711 ] table 2 summarizes the major results . 
 overall , these studies documented abnormalities affecting white matter signal , temporal lobe and amygdala structure and function . major previous reports of brain magnetic resonance imaging abnormalities related to congenital adrenal hyperplasia white matter hyperintensities , or leukoencephalopathy , related to cah are an interesting finding in this population . in a previous italian study , 
 disease phenotype does not seem to affect the frequency of such lesions , which may even be present in heterozygous carriers of cyp21 mutations . 
 brain white matter impairment in cah involve typically periventricular regions or , less frequently , cerebellar area as seen in one of our patients . 
 these signal changes are non - specific and without side prevalence,[79 ] but they differ from those due to ischemic causes , as the latter involve namely subcortical and deep structures . 
 white matter hyperintensities in cah are most often subclinical or , less frequently , associated with unremarkable neurological signs . in the present study , 
 the data in the literature concerning the clinical significance of white matter lesions are controversial . 
 it has been suggested that a  threshold  area of white matter lesions must be present before cognitive deficits are installed . 
 the mechanisms that could explain mri lesions in cah remain poorly understood . hormonal imbalance related to a deficiency of cortisol and aldosterone and an overproduction of 17-oh - progesterone and androgen would cause a destabilization of the myelin molecule leading to its degeneration . 
 in addition , exogenous glucocorticoids may alter the process of neuronal maturation and myelination by inhibiting the differentiation of oligodendrocyte precursors . 
 thus both , overdose and discontinuous therapy have been suggested as factors favoring the emergence of brain mri abnormalities . in our patients , 
 chronic hyperandrogenism , a result of non - compliance with treatment , may be the main mechanism involved in the genesis of brain mri abnormalities . 
 aldosterone is provided with a regulating effect on the arterial structure by stimulating fibroblast proliferation and collagen synthesis via direct activation of local receptors . in this 
 regard , aldosterone deficiency could induce changes of cerebral arterial structure , and , as consequence , a subclinical cerebral ischemia . 
 this ischemia could also be indirectly promoted by glucocorticoids that alter glucose and lipid metabolism and reduce platelet aggregability . in our study , 
 similarly , nass , et al . reported some cah patients with white matter lesions , with localizations that were typical of demyelinating diseases ( located in the corpus callosum and cerebellum ) . 
 have described a patient whose condition was diagnosed at birth as the sw subtype of cah in whom relapsing - remitting multiple sclerosis developed . 
 the fact that a susceptibility locus for multiple sclerosis is in the hla region that comprises the cyp21 genes suggests a possible genetic link between multiple sclerosis and cah . 
 the organizational effects refer to permanent changes in brain structure , organization or function in utero or during critical periods of development . 
 the activational effects tend to be transient and refer to the acute effects of circulating steroids during adulthood . 
 two structures are known to have many androgen and glucocorticoid receptors and are particularly susceptible to the effects of excess glucocorticoids : the hippocampus , a central neural substrate of declarative memory , and the amygdala , a key to emotional coding . 
 the hippocampus also plays an important role in the fine - tuning of the hpa axis by participating in its glucocorticoid negative feedback regulation . 
 alterations in amygdala function have been implicated in the pathophysiological characteristics of anxiety and depressive disorders in both adults and children . 
 the effect of hypercortisolism on the amygdala has not been systematically studied ; however , patients with classic cah , who have prenatal glucocorticoid deficiency with possible postnatal iatrogenic glucocorticoid excess , have smaller amygdala volume than healthy age - and sex - matched controls . cah female patients have also enhanced response to negative facial emotions ( anger and fear ) compared with neutral expressions . 
 animal studies and human studies of adult patients with chronic hypercortisolemia have shown that prolonged exposure to glucocorticoid excess could result in extensive hippocampus damage and permanent memory impairment . 
 the mechanisms leading to brain atrophy include impaired neurogenesis , reduction in neurotrophic factors and reduction in cerebral glucose metabolism . 
 interestingly , it has been suggested that at least some of the deleterious effects of prolonged hypercortisolemia on cognitive functioning and hippocampal volume are reversible . 
 our present study showed temporal lobe atrophy in one patient and hippocampal dysgenesis in two patients . 
 this study is the first one demonstrating hippocampal abnormalities on mr imaging in patients with cah , and it showed , also for the first time , brain mri abnormalities in patients with the nc form of cah . studies of cognitive function in cah patients have reported discrepant findings . 
 it was suggested that patients with the most severe form of cah and those who have experienced sw adrenal crises with abnormal electrolytes as neonates are at risk for cognitive impairement . in the present study , 
 the remaining one had the nc form , but also had an uncontrolled congenital hypothyroidism which may also contribute to his altered mental status . in conclusion , 
 brain mri abnormalities are an interesting finding in cah patients and could affect white matter signal and temporal lobe structures 
 . these abnormalities could be the consequence of hormonal imbalance during brain development and corticosteroid treatment . 
 more extensive studies are required to define better the relationships between brain involvement and different cah phenotypes and treatment regimens .","background : congenital adrenal hyperplasia ( cah ) is an inherited recessive disorder of adrenal steroidogenesis . 
 the enzymes most commonly affected are 21-hydroxylase . 
 past reports suggested brain magnetic resonance imaging ( mri ) abnormalities in cah patients , affecting white matter signal , temporal lobe and amygdala structure and function.aims:in the present study , we aimed to investigate the frequency of white matter changes and temporal lobes structures dysgenesis in a population of patients having cah due to 21-hydroxylase deficiency.materials and methods : neurological examination and brain mri were performed in 26 patients.results:neurological examination revealed mental retardation in three patients , tremor in two patients , tendon reflexes asymmetry in one patient , and cerebellar syndrome in one patient . 
 eleven patients ( 42.3% ) showed mri abnormalities : eight of them had white matter hyperintensities , one patient had moderate atrophy in the right temporal , and hippocampal dysgenesis was found in the remaining two patients.conclusions:brain mri abnormalities in cah patients include white matter hyperintensities and temporal lobe structures dysgenesis . 
 the mechanisms involved seem related to hormonal imbalances during brain development and exposure to excess exogenous glucocorticoids . 
 clinical implications of such lesions remain unclear . 
 more extensive studies are required to define better the relationships between brain involvement and different cah phenotypes and treatment regimens .",pubmed
"delivery of the human genome draft sequence by publicly funded   and corporate   projects promises to precipitate significant biomedical advances this century . to rise to this challenge 
 , biologists must become adept at navigating the vast expanses of genomic dna data that may seem , at first glance , to be devoid of features . yet lying beneath this facade of uniformity are rich veins of knowledge awaiting exploitation . surveying and signposting 
 this apparently bland genomic landscape should guide investigators towards experiments that address specific hypotheses about gene function . 
 ? different communities of biologists speak in dialects that are not always mutually comprehensible , particularly with respect to the umbrella term ' function ' . 
 where one investigator might be interested in designing active - site inhibitors using high - resolution protein structural data , another might require information on gene pathways , and another might be focused on relating genotype to phenotype . 
 if the genome is to offer up its secrets to all scientific communities , its surveyors need to adopt universal vocabularies . 
 broadly speaking , there are two common ways of annotating ( assigning names and functions to ) genes . 
 websites such as locuslink , genecards , eugenes   and ensembl   ( see table 1 ) integrate information from diverse sources relevant to individual human genes . 
 thus one may browse sequence information , for example concerning single - nucleotide polymorphisms ( snps ) , alongside both descriptions of molecular and cellular function and information relevant to human disease . 
 such annotation is essential , yet it is currently restricted to the minority of human genes - those that have been characterized experimentally . 
 a key to the databases mentioned in this article the second type of annotation relies not on empirical observations but rather on predicted evolutionary relationships . 
 all genes that are thought to have arisen from a common ancestor are defined as homologs : where additional copies have arisen by gene duplication within a single genome they are defined as paralogs , whereas corresponding genes in different species are orthologs . sometimes homologous gene products have strong sequence similarities , such that an inference of homology is straightforward ; one such example is the drosophila melanogaster gene branchless , which encodes a homolog of human fibroblast growth factor ( fgf ) . on other occasions , 
 protein homologs have subtle or indiscernible sequence similarities that try the patience and expertise of genome wayfarers . 
 for example , human fgf and interleukin-1 have highly similar tertiary structures despite insignificantly similar sequences but , as a result of their similar growth - factor - type functions , they are in fact very likely to be homologs . 
 the importance of annotating the genomic landscape on the basis of homology is that protein homologs invariably have similar tertiary structures and frequently also have similar functions . 
 the surveying and way - marking of each new gene , therefore , need n't always be an arduous process of discovering structure and function from scratch , since clues can be inferred from what has already been experimentally gleaned from its homologs . by applying the concept of homology , the problem of broadly predicting the functions of all genes is brought within the realms of possibility . 
 there is a pitfall to be avoided when annotating genes by homology : homology is defined on the basis of evolution , rather than function . on one hand 
 , homologs may be related only by evolution and not by similarities in molecular mechanism ; relatives of enzymes that now lack catalytic sites are just such examples . on the other hand , examples abound of divergent homologs , or even non - homologs , whose functions overlap . 
 consequently , homology assignment indicates only an approximate direction for future empirical determination of function , perhaps analogous to laying down a compass bearing rather than an exact map reference . 
 the first completely sequenced genomes , such as that of haemophilus influenzae , were annotated following searches of all available sequence databases with each predicted gene product . 
 this approach assumes that matters are straightforward : although homologous genes may not always be similar in function , genes are either homologous or they are not . 
 a major problem in gene annotation arises , however , when one encounters sequence - similar , homologous portions of genes embedded in otherwise sequence - dissimilar , non - homologous contexts . 
 the presence of these domains demonstrates that evolution has constantly fused and divided genes , using a repertoire of pre - existing components ( figure 1 ) . 
 domain architectures of the human signalling proteinsvav , grb2 , nck1 and crk , as predicted using smart . 
 although these proteins share two common domains ( namely src - homology 2 and 3 , or sh2 and sh3 , domains ) they are not all homologous to one another . 
 the complex architectures of these proteinsimply that the co - occurrence of these two domain types has arisen on more than one occasion , and thus that the domain combinations seen in the four proteins are not likely to have arisen from a common ancestor with whom they share a common architecture . 
 domains are homologous portions of sequences that are encoded in different gene contexts and have survived the evolutionary tests of time without fragmentation . in three dimensions , domains are observed to be compact units of structure , often with a hydrophobic interior and a hydrophilic exterior , and they are not divisible into smaller units . 
 consequently , domains represent the finite vocabulary of protein evolution : if domains are words , then multidomain proteins are complete sentences . just as there is a dictionary or lexicon for every language , 
 there is one - or in this case several - for the vocabulary of domains . 
 pfam   is the widest - ranging lexicon of domain families , predicting at least one domain for more than two - thirds of all entries in the swiss - prot   protein database . 
 smart   is a more concise collection , focusing on those domains that are widespread and difficult to detect . 
 prosite   also has a dictionary of domain profiles , as does celera   ( called panther ) and the institute for genomic research , tigr . 
 each of these resources detects domains using numerical representations of multiple sequence alignments , either hidden markov models ( hmms ) or generalized profiles ( gps ) . 
 although the constructions of hmms and gps are very different , formally they are equivalent . 
 homology assignments are guided by comparisons of hmms or gps with protein sequence databases , and by implementation of an upper threshold value for e , the number of unrelated sequences expected purely by chance that are aligned with a particular score , or higher , in the search . 
 this procedure has been shown on many occasions to identify subtle , yet informative , sequence similarities among distant homologs . taken together , pfam , smart   and the other domain resources carry considerable redundancy , although each has its own merits . 
 an additional resource , interpro , has been derived in part to avoid the onerous task of querying each of these domain lexicons separately for each protein sequence of interest . 
 interpro ( release 3.0 ) combines the domain and motif sets of prosite , pfam , prodom , prints   and smart   in a hierarchical manner and is thereby able to provide annotation for 74% of all swiss - prot ( protein database ) and trembl ( translated dna sequence database ) entries . 
 pfam , smart and interpro were recently chosen by the public and private consortia to annotate their human genome draft sequences . to be more precise , 
 annotations were applied to the proteome , the current predicted set of all expressed proteins encoded by the draft . 
 the proportion of the proteome that could be annotated , even minimally , using these resources is low , at approximately 40 - 60% . 
 the resources were used to annotate the proteome according to lists of component domains rather than protein or gene names . 
 thus , protein sequences were identified not , for example , as the product of the lbc oncogene , but rather as containing rhogef ( dbl - homology ) and pleckstrin homology ( ph ) domains ( figure 2 ) . 
 this is protein annotation viewed through the evolutionary lens , rather than faithful extracts from the current body of experimental knowledge . by implication , 
 proteome annotation by domain content implies that a gene product 's function is a synthesis of the generalized functions of its component parts ( see figure 2 ) . 
 although such descriptions of molecular function are approximate and pale in comparison to accounts of experimentally derived characteristics , they provide the best predictions that can be mustered for the uncharacterized majority of human genes . 
 domain architectures of the lbc oncogene product , and the experimentally uncharacterized protein kiaa1415 , as predicted using smart . 
 the predicted domains in kiaa1415 suggest that it facilitates guanine nucleotide exchange on rho - type small gtpases ( it has rho guanine - nucleotide exchange factor , or rhogef , and pleckstrin homology or ph domains ) , it is localized to the plasma membrane ( ph domain ) and it may interact with the carboxyl termini of transmembrane receptors or channels ( it has pdz domains ) . the generalized functions of dep domains are not currently known . 
 it should be noted that prediction of this protein 's domain architecture does not allow prediction of either its cellular function or the phenotypic effect of its deficiency . 
 it is important to emphasize that annotation of individual proteins or complete proteomes using domains is achieved automatically rather than by manual curation . 
 this is relevant , because a newly sequenced genome 's proteome is generally in a high state of flux , with additions and deletions resulting from sequence updates , enhanced understanding of gene structure and identification of previously overlooked genes . 
 the animal genome sequencing projects already underway will proceed in the same manner as the publicly funded human project , through numerous draft stages and on towards completion . providing up - to - date , and necessarily automatic 
 one might suggest that gene annotation by use of domains is a relatively short - term measure that will be made redundant by results from high - throughput studies of non - vertebrate model organisms . 
 it can be argued that detailed predictions of the functions of most human genes can be inferred from studies of their orthologs , as these are the most likely members of a family to have similarities in molecular and cellular roles . 
 in contrast to previous expectations , the human genome draft publications found that the great majority of human genes have no orthologs in each of three important model organisms whose genome sequences are known , namely drosophila ( fruitfly ) , caenorhabditis elegans ( nematode worm ) and saccharomyces cerevisiae ( baker 's yeast ) . 
 thus , the contribution of domain identification to gene - function prediction will remain until such a time as reliable results from high - throughput studies on a more closely related organism , such as the mouse , are available . 
 deconvolution of human proteins into their constituent domains has also played a major role in understanding the evolution of chordates . 
 three significant differences were detected between the repertoires of human domain families and those of the nematode worm , fruitfly , the mustard cress arabidopsis thaliana and yeast . 
 first , only a small proportion ( 7% ) of human domain families are absent from the other proteomes . 
 second , numerous domain families were greatly expanded in terms of the number of members in humans , whereas others were considerably reduced . 
 this finding demonstrated that domain ' invention ' in the chordate lineage has made only a minor contribution to proteome diversity , whereas expansions and contractions of domain families , and domain additions and deletions , have all added greatly to proteome innovation . 
 such studies demonstrate the power of comparative proteomics in relating gene content to the evolution of eukaryotic organisms . 
 but as with the argument that the complexity of organisms is only loosely coupled to total gene number , the question of whether the number of representatives of a domain family in a proteome is directly related to that family 's contribution to cellular and organismal function remains open . 
 it is hoped that future proteome comparisons will progress beyond the simple enumeration of homologous genes and domains towards an understanding of the biology that underlies the variability of domain family sizes . 
 although it provides information about molecular structure , function and evolution , by and large it is unable to predict functional aspects , such as cellular or organismal role , protein - binding partners or post - translational modifications . 
 fortunately , other views that address these aspects have been incorporated with domain predictions into web - based resources such as locuslink , genecards , eugenes   and ensembl . 
 each of these sites represents a confluence of diverse information sources that are mapped to specific regions of genomic sequence . 
 whilst navigating these sites it should be borne in mind that they often fail explicitly to distinguish between annotations that are experimentally derived and those that are predicted by homology - based methods . 
 nevertheless , these sites are a significant boon to biologists since they provide views of genomes from multiple vantage points , from protein tertiary structure through to snps and on to human disease . 
 as viewed now , the human genome appears to be a relatively featureless landscape , punctuated by islands of annotations for well - characterized and biomedically important genes . as biological sciences progress into a more knowledge - rich , as well as data - rich , era , the cartography of this genome will become more complex with numerous different functional characteristics being assigned to a growing fraction of human genes 
 . it will be increasingly important to restrict descriptions of function to a common and broad vocabulary that is compatible with computational approaches . 
 fortunately , a cross - community approach to dealing with this issue is already underway . 
 the gene ontology project ( go )   has created an initial hierarchy of defined terms that encompasses many of the flavours of ' function ' commonly described in biology . 
 go has begun to permeate throughout genomics and it will do so more rapidly as its scope and attention to detail improves . 
 two further domain - centric approaches look set to guide the efficient navigation of genomes : orthology prediction and the partitioning of a domain family into subfamilies with distinct functions . 
 orthology is a valuable concept from which to infer functional information between species , and orthologs from the genomes of 30 bacteria , archaea and the yeast saccharomyces cerevisiae can be predicted directly using the cogs database . 
 pairs of orthologs from animal genomes are also available on the web ( for example from the jackson laboratory ) . 
 no resource is yet available that accurately predict sets of orthologs for several multicellular eukaryotes , however , such as the fruitfly , worm , arabidopsis , mouse and human . 
 a more difficult problem is the partitioning of a homologous domain family into multiple subfamilies representing multiple functions . 
 homologous proteins with divergent sequences frequently have distinct functions   that are characterized by contrasting patterns of conserved amino acids . 
 one productive approach to the analysis and prediction of functional subtypes identified key sites in multiple protein sequence alignments that specify the different functional subtypes . 
 this method has performed well in defining functional subtypes with prediction accuracies of up to 96% . a key element in ensuring 
 a central function will be provided by sophisticated web forums that accumulate and automatically present integrated functional data . 
 the best example so far is the distributed annotation system , or das , which seeks to amalgamate annotations donated by experimentalists worldwide . 
 even these schemes , however , will face a major challenge in integrating functional information from the huge datasets that arise , for example , from microarray   and proteomics   experiments . 
 the human genomic landscape , relatively featureless now , will soon be teeming with evidence , pointers , and clues . 
 ultimately , the success of the genome projects will be measured not in the completion of sequences , but in how access to integrated data opens up new avenues of research and therapy .","the evolutionary history of eukaryotic proteins involves rapid sequence divergence , addition and deletion of domains , and fusion and fission of genes . 
 although the protein repertoires of distantly related species differ greatly , their domain repertoires do not . to account for the great diversity of domain contexts and an unexpected paucity of ortholog conservation 
 , we must categorize the coding regions of completely sequenced genomes into domain families , as well as protein families .",pubmed
"over the last 25 years , heart rate monitors have been widely used in sports and sports science . since the development of handy heart rate monitors ( hrms ) with the size of a watch and appropriate flexible chest belts with electrodes to transmit signals wirelessly to the watch , measurement of heart rate ( hr ) 
 has become an easily accessible and valuable tool for training and coaching purposes and conducting laboratory or field studies . 
 validity and reliability have been shown to be high for these tools compared to ecg measurements for both hr and heart rate variability ( hrv ) [ 16 ] . owed to its widespread use in physical activity not only for individuals but also for team sports , recent technical development focused on real - time monitoring instead of post - exercise evaluation of recorded data . 
 currently , several different systems are available on the market , of which four were tested in the present study : polar team pro , acentas team monitoring system , suunto pro team pack and activio sport system . 
 all systems allow direct and real - time monitoring of several individuals at the same time . 
 data are transmitted wirelessly to a receiver which is connected with a standard laptop with relevant software . however , a direct comparison of these systems and validity of recorded data are missing in literature 
 . therefore the main purpose of this study was to compare all systems in a field test against each other . 
 operating distance and validity against a commercially available ecg were evaluated as well as the installation and use of software and hardware of the above - mentioned systems . 
 a total of 12 active people ( 11 men and 1 woman ; age : 26.4  3.2  yr ; height : 177.9  8.7  cm ; mass : 78  7  kg ) participated in the study . 
 ten men of an amateur soccer team conducted an online field test during a real football match . 
 one male subject participated for validity of hr monitoring systems against ecg whereas the female subject was only involved in operating distance measurement . because of the low case number of the last two tests , data can only be considered as a pilot study for ecg validation and distance measurement . 
 the following four commercially available systems were used for all tests : polar team pro ( polar electro oy , kempele , finland ) , acentas team monitoring system ( acentas gmbh , hrgertshausen , germany ) , suunto pro team pack ( suunto oy , vantaa , finland ) , and activio sport system ( activio ab , stockholm , sweden ) . a quality rating system similar to rating systems used in trade journals 
 which is specialised on the testing of mobile and electronic communication tools . for this purpose , 
 the authors decide to subdivide the rating system into three main categories : measurement , software , and hardware . at this , the core area of the study focused on the adequate online measurement of the heart rate . 
 therefore , the rating system was set in favour of the measurement which was represented by 200 scores of maximal 500 . 
 the latter 300 were equally divided on quality of the hardware and the usability of the software . 
 subcategories were weighted subjectively by the authors in consideration of heart rate monitoring of team sports including the monitoring of 10 or more subjects in parallel . 
 in addition , the following subcategories were rated once by an independent observer who was not familiar with any of the monitor systems to get a first impression : instruction manual , packing , chest belts ( quality ) , and initial software handling . 
 the remaining categories were judged once by an experienced person . with exception of wearing comfort and failing quota in field , 
 all subcategories were judged and quantified by using a ten - stepped visual analogue scale which is known for pain assessment . 
 visual analogue scaling was weighted by multiplying itself with factor f : ( f = maximal score of subcategory/10 ) . 
 parameters leading the judgement of the subcategories are described in tables 2 and 4 . 
 wearing comfort of the chest belts was evaluated via a short anonymous questionnaire which all 12 participants ( see section 2.1 ) had to complete directly after wearing . 
 the questionnaire contained five questions about the handling and individual fitting of the chest belts . 
 the questions one and two are positively formulated whereas questions three to five are negative ( see table 3 ) . 
 all questions had to be answered by an even scaled rating system including six categories . 
 maximum score for the whole questionnaire was 25 ; hence , each question was scored with maximal 5 points . 
 the main field test was measurement of failure ratio during the half time of a real soccer match . 
 all players apart from goal keeper were equipped with chest belts of the relevant system and measured during first half (= 45  min ) of a league soccer game during four home matches in summer 2009 . 
 temperature range was within 9c , and weather conditions were similar ( no precipitation ) for all measurements . 
 the receiver of the relevant system was always positioned three meters in projection of midline of the soccer ground . in order to improve transmission distance , receiver 
 could not be excluded a priori , a mean failure ratio was determined only with transmitters sending valid data . each deviation / loss of one percent of whole duration measurement of 45  min (= 2700  s ) 
 was subtracted with 10 scores , and a maximal score of 100 could be obtained . 
 if , for example , a total loss time of all ten transmitters of a system was 270  s , a total score of 90 would be credited to this system . 
 a failure ratio of 10 percent of whole duration measurement would be credited with a zero score . 
 distance measurement was conducted on a uphill street with a constant slope and in far distance to high - voltage line or radio tower to reduce possible interference . 
 receiver antenna was placed on highest elevation of the hill one meter above ground , and unit was connected to a laptop ( toshiba satellite series ; toshiba inc , ny , usa ) . 
 the participants wearing the chest belts were equipped with a gps device ( nokia n79 , nokia group , espoo , finland ) to measure distance . by moving backwards 
 additionally , the participants were instructed to move arms to keep hr variable with time . 
 attempts were only started when both valid signals of gps and hr were available and stopped when hr signal was lost . 
 tests were conducted twice , and mean value of both attempts was calculated and taken as maximal range . 
 maximal range distance was credited with 70 scores whereas the system with the absolute maximal covered range was taken as reference system and credited maximum score . 
 percentage deviation of distance of other systems was analogously deducted as percentage of scores . if , for example , reference system had 100  m range , it was credited 70 scores . if a second system had only 50  m range , it would be credited 50% of maximum score , hence 35 . comparison against ecg 
 was conducted in a laboratory by using ecg tool vicardio ecp-6 standard ( energy - lab technologies , hamburg , germany ) . 
 a maximum score of 30 could be obtained at this test . a deviation of 1 beat per minute ( bpm ) 
 was deducted with two scores . only complete accordance of both values was credited with maximum score . 
 a plus ( + ) is a positive feature ; a minus (  ) is a negative one . 
 a plus / minus (  ) indicates aspects which are both positive and negative . in context of the supplied hardware acentas and polar lead the field of monitor systems , both reaching 100 and 106 scores in sum for the hardware . 
 the leading position of polar depends on the storage capacity of the chest belt increasing data safety but also decreasing wearing performance which is indicated by the chest - belt questionnaire presented in table 3 . in case of wearing comfort , it has to be noted that both chest belts of activio and acentas were supplied by the identical manufacture . 
 both polar and acentas delivered high - quality receivers . results of software evaluation are summarized in table 4 . 
 again , similar to hardware evaluation , a plus ( + ) is a positive feature , and a minus (  ) is a negative one . if all systems were similar or identical , there is no separate classification . 
 a plus / minus (  ) indicates aspects which are both positive and negative . here , activio offered the harmonious concept which led to the highest ranking . 
 the installation procedure was easiest with the acentas software , but acentas did not focus on extensive statistical analysis . in this case , 
 polar as well as activia provide a more developed software tool . as data of ecg comparison 
 were only obtained with a single subject , they have to be regarded with caution . 
 suunto 's deviation was 80 to 85  bpm , activio 's deviation was 81 to 85  bpm , and polar 's deviation was 82 to 85  bpm , respectively . 
 same caution has to be taken as only one subject 's data were included in analysis . 
 nevertheless , acentas gave reference with 349  m , followed by activio with 141  m and suunto with 107.5  m. it has to be noted that activio 's system was supposed to reach 300  m. last but not least , polar reached 102  m which corresponded to the manufacturer information for this system . 
 nevertheless , all other systems had a wider range of data acquisition , and hence score distribution was credited as follows : acentas 70 , activio 28 , suunto 21 , and polar 20 . 
 regarding failure quota , average downtime was 148  s ( 0,6% ) for acentas , 286  s ( 1,0% ) for polar , and 552  s ( 2,0% ) for activio . 
 single belts were recognised from time to time , but no consistent data acquisition could be accomplished . 
 similarly , five out of ten polar chest belts had signal loss and could not be reactivated whereas activio managed 10 out of 10 signals , and for the acentas system 9 out of 10 were used for data evaluation as one person was injured during match play . 
 the purpose of the present study was to compare four commercially available online heart rate monitoring systems and especially their use in everyday situation in team sports . 
 the system of polar could score especially with a clear design , useable features of the software , and a convincing product quality . 
 only polar uses wlan for data transmission and also adds a mobile terminal in pocket format which can easily be used outdoor and with bad weather conditions . 
 but the high energy consumption of wlan transmission would terminate the battery capacities and additionally the duration of measurement . 
 the system of activio has a functional and useful software and the instruction manual was very detailed and easily comprehensible . 
 acentas could score with a huge operating distance , a valid heart rate signal compared with ecg , a low failure quota , and an easy handling . 
 in contrast , the system of suunto had a total breakdown during competition measurements and could not be reactivated . 
 prospectively , it would be a great challenge to realise online monitoring for swimmers , possibly leading to new opportunities for the training regime . 
 in this context the system of polar and suunto had the advantage that data are also recorded on the chest belt memory during water immersion and could be evaluated after exercise . 
 meanwhile , some of the tested heart rate monitoring systems are updated and upgraded with new functions , respectively . 
 for example , acentas included an offline memory in the chest belts and added a wrist monitor for direct heart rate analysis from up to twenty athletes by the coach . in summary , 
 acentas system reached the best mark of all systems , but every system has its advantages and disadvantages depending on the using purpose , location , and weather . so this present comparative study can not recommend a single system but rather shows strength and weakness of all systems and additionally can be used for further system improvements . 
 the investigators were responsible for the study design , data collection , data analysis , data interpretation , and submission of the paper for publication , independently of all funding sources . 
 the authors declare that they have no financial conflict of interests to disclose with any of the companies named in this paper .","recent technical development focused on real - time heart rate monitoring instead of postexercise evaluation of recorded data . 
 there are several systems on the market that allow direct and real - time monitoring of several individuals at the same time . 
 the present study compared the systems of polar , acentas , activio , and suunto in a field test with twelve subjects regarding failure quota , operating distance , and ecg validity . moreover , 
 the installation and use of software and hardware were evaluated with a quality rating system . 
 chest belts were evaluated with a questionnaire , too . 
 overall the system of acentas reached the best mark of all systems , but detailed results showed that every system has its advantages and disadvantages depending on using purpose , location , and weather . 
 so this evaluation can not recommend a single system but rather shows strength and weakness of all systems and additionally can be used for further system improvements .",pubmed
"in the 1960s , mcculloch and till proposed three functional characteristics that came to form the definition of stem cells . 
 their findings supported the hypothesis that a specific population of cells was responsible for propagation and sustained growth in a hierarchical manner , a theory first proposed in the middle of the 19 century by virchow . 
 first , stem cells have the ability to make identical copies of themselves ( self - renewal ) . 
 assessment of a cell 's ability to proliferate extensively and self - renew requires the induction of these behaviors . 
 thus , the cell must be isolated and the behaviors induced in vitro for the cell to be classified as a stem cell . to more efficiently isolate candidate stem cells , much work has turned to the use of cell surface proteins unique to stem cells , which permit isolation by flow cytometry , magnetic bead isolation , fluorescent protein tagging , and immunostaining . 
 these markers also enable stem cells to be tracked and their functions to be characterized . 
 advances in stem cell biology have provided insight into the role of stem cells in the lung . due to the relative quiescence of the lung epithelia , with a proliferative fraction of just 0.06% to 1.3% in the bronchiolar and tracheal epithelia , identification of putative stem cells 
 has been more difficult than in tissues with higher rates of turnover , such as the epithelia of the gut or skin . despite these challenges , populations of stem cells 
 have been identified throughout the lung by way of their role in the response to injury of the airway . in each case , the functional tenants of stem cell behavior  namely , self - renewal , proliferation , and multipotency  have been observed . 
 a growing body of evidence now indicates that the earliest stages of lung malignancies may be related to a deregulation of this stem cell - based repair mechanism . 
 the epithelial lining of the adult airway progresses from proximal to distal in a stereotyped fashion . 
 proximal airways consist of pseudostratified epithelium , middle airways consist of a simple columnar epithelium , and the most distal airways consist of a cuboidal epithelium . 
 multiple studies indicate that cells capable of stem - like behavior in response to airway injury are present throughout the airway . in proximal airways , studies of airway repair in the trachea 
 identified a population of tracheal basal cells that are pluripotent , retain bromodeoxyuridine , and express keratin-5 ( k5 ) . 
 a second study using a naphthalene inhalation injury model identified an additional population of tracheal basal cells , this time expressing the keratin-14 ( k14 ) marker . 
 these k14 cells were able to proliferate to give rise to cells of multiple phenotypes in response to airway injury . 
 it appears that in the trachea , basal cell populations expressing either k5 or k14 are capable of the functional aspects of stem cell niches . 
 squamous cell carcinoma frequently arises from the more proximal tracheobronchial airways , yielding centrally located tumors that often exhibit elevated levels of k5 basal cells . as such 
 , it appears that cancers with histology consistent with squamous cell carcinoma are more likely to arise from k5 stem cells . 
 although there is no targeted therapy available against k5 cells to date , this stem cell niche of squamous cell carcinoma may provide a worthwhile direction for targeted research , providing a unique biomarker that can be used to target the cell of origin in these tumors . in the middle airways , 
 a rat model of nitrogen dioxide ( no2)/ozone ( o3 ) inhalation injury revealed that mature rat clara cells could proliferate into phenotypically diverse progeny . this ability to move from quiescent and fully differentiated epithelial cells to actively proliferating multipotent cells led evans et al . to deem these clara cells 
 facultative progenitor cells ,  as they are capable of but not limited to behavior indicative of a stem cell . in the setting of injury to a clara cell - depleted airway , as is the case with the naphthalene model of injury , clara - cell secretory protein ( ccsp)-positive cells located within neuroepithelial bodies ( neb ) have been shown to proliferate to repair the airway . 
 these neb ccsp cells are able to independently repopulate the middle airways with phenotypically diverse progeny after naphthalene injury , characteristic of true bronchial airway stem cells . 
 small cell lung carcinomas , frequently arising in the mid - level bronchioles , have been shown to contain fractions of cells expressing cell surface markers associated with mid - airway stem cell subpopulations . 
 in particular , the cell surface markers cd44 and multidrug resistance 1 ( mdr1 ) have previously been identified in a series of small cell lung carcinoma cell lines . 
 cells expressing cd44 have also very recently been implicated in lung cancer progression and metastasis by the epithelial - to - mesenchymal transition ( emt ) . in their 2011 study , 
 tellez et al . found that bronchial epithelial cells exposed to carcinogens gained stem - like properties and that emt led to a progressively motile and invasive phenotype . 
 again , while no targeted therapies are presently available to target cells expressing cd44 and mdr1 , the potential exists for improved delivery by using agents directed at this cell or origin in conjunction with the current standard of care . for the most distal airway 
 , there is no consensus on the cell surface marker phenotype of representative lung stem cells . 
 first identified pluripotent , neb - independent , ccsp cells in the bronchoalveolar duct junction ( badj ) that could , in response to naphthalene injury , yield diverse populations of cells 
 . cells expressing surface markers surfactant protein c ( sp - c ) , stem cell antigen-1 ( sca-1 ) , and cd34but not expressing cd45 or pecam  were shown to act as pluripotent and to have the ability to proliferate and self - renew in culture . 
 however , others have argued that these ccsp / sp - c cells are insufficient as the distal lung stem cell population because they are unable to differentiate into type ii alveolar cells . 
 instead promoted id2 as a more suitable cell surface marker for distal lung epithelial stem cells . 
 thus , while there is consensus that stem cells form a niche population in the badj , the cell marker phenotype remains under debate . 
 carcinomas of the distal airway  most frequently lung adenocarcinoma and bronchioloalveolar carcinoma  have been associated with stem cells from the badj region . 
 the hierarchical or cancer stem cell hypothesis has proven to be an important theory with which to understand lung tumorigenesis , and may have important implications for future management of lung tumors . in this model , 
 relatively rare , multipotent cells gain unlimited proliferative potential that may then drive tumor growth . 
 the multipotent nature of these cells leads to heterogeneity within the tumor by aberrant differentiation and epigenetic changes in the progeny . 
 it follows , then , that a stem - like cell , with the ability to rapidly proliferate into phenotypically diverse progeny , could act as this cancer stem cell . 
 stem cells are also known to express high levels of anti - apoptotic and drug - resistant proteins , which may explain why chemotherapy is extremely effective in shrinking the size of primary tumors but frequently ineffective against localized recurrence and metastatic disease . 
 proponents of the theory of lung cancer stem cells propose that although chemotherapy is able to effectively kill non - stem cell cancer cell types in these settings , chemoresistant lung cancer stem cells to continue to proliferate unchecked . 
 this understanding has sparked new energy in the development of targeted therapies against specific cell types within the heterogeneous tumor . as introduced above , common tumors from each representative airway zone 
 have been associated with stem cell niches based on shared cellular expression of surface markers . 
 in addition , recent studies have successfully identified cells within lung cancer specimens that exhibit stem - like properties . 
 identified tumor cell subpopulations that exhibited several key functional properties  multidrug resistance , increased telomerase expression , and self - renewal  that are characteristic of stem cells . 
 in addition , cd133 cells isolated from freshly resected human lung cancer specimens can grow indefinitely in culture , a characteristic usually only seen in transformed cell lines . 
 cd133 cells are also able to proliferate into phenotypically diverse tumors , both in culture and when injected into immunodeficient mice . of high clinical interest 
 are recent observations that patients with lung tumors enriched with these cd133 lung cancer stem cells tend to have worse prognosis than those with fewer cd133 lung cancer stem cells . 
 in addition , these cd133 subpopulations exhibit increased resistance to cisplatin - based therapies , suggesting that the presence of cd133 cell populations may warrant changes to chemotherapeutic regimens in these patients . that lung cancer stem cells impact prognosis raises the possibility , then , that major therapeutic advances in lung cancer treatment , through the specific targeting of lung cancer stem cells or the pathways that confer stem cell - like properties to lung cancer cells , are possible in the near future . 
 in addition , measurement of the lung cancer stem cell fraction in a tumor could prove a useful modality for better differentiating between transient tumor debulking effects of chemotherapy and longer lasting therapies . 
 the study of the lung epithelial response to airway injury has shown compelling evidence for the role of stem cells as a component of airway repair . 
 while there is still controversy in the field , there is also a growing body of evidence to suggest that these same lung stem cells may play a key role in the initiation and proliferation of human lung cancers . 
 two primary pieces of evidence reviewed here support the hypothesis that lung stem cells may play a role in lung tumorigenesis : 1 ) cells sharing the functional properties of stem cells are present in many tumors , and 2 ) many tumor sites of origin match known stem cell niches . because characterization of putative stem cells requires manipulation of the cells themselves to induce a specific phenotype , it remains exceptionally difficult to discriminate between potential or facultative and true stem cells . on account of the inducible abilities that form the critical features of stem cells , it remains possible that these cancer stem cells may exist on a continuum of inducible features 
 that is , if genetic and epigenetic changes can confer stem cell characteristics upon a given tumor cell , then any tumor cell may in fact have the potential to drive tumor growth and resistance 
 . this spectrum of qualities may yield a mixed model , as proposed by rahman et al . 
 , in which stem - like cells may have variable potential to drive tumor growth , depending on the conditions presented to the cell . 
 several key questions remain unanswered with respect to the role of stem cells in lung tumorigenesis . 
 it remains to be conclusively determined whether healthy adult stem cells undergo oncogenic transformation leading to the initiation of tumorigenesis , or whether healthy airway cells undergo oncogenic transformation leading to their de - differentiation and subsequent adoption of stem cell - like properties . 
 in addition , the relationship between the tumor microenvironment and the lung cancer stem cell remains poorly characterized . 
 however , the sum of the available evidence to date , in conjunction with ever - improving modalities for the identification and isolation of putative stem cell populations , should provide a wealth of future directions that hold great promise in improving the management of patients with lung tumors .","lung cancer is the leading cause of cancer - related deaths worldwide . 
 recently , advancements in our ability to identify and study stem cell populations in the lung have helped researchers to elucidate the central role that cells with stem cell - like properties may have in lung tumorigenesis . much of this research has focused on the use of the airway repair model to study response to injury . in this review , 
 we discuss the primary evidence of the role that cancer stem cells play in lung cancer development . 
 the implications of a stem cell origin of lung cancer are reviewed , and the importance of ongoing research to identify novel therapeutic and prognostic targets is reiterated .",pubmed
"tubularized incised plate ( tip ) repair is recommended as the ideal repair for distal penile hypospadias ; it has shown excellent results in the management of primary , redo , distal , and proximal hypospadias . 
 a number of groups have recently been engaged in studying the plate characters and its impact on the success of tip . 
 while some authors have shown the importance of the plate characters in the success of tip repair , others have shown that the technique could be used for all cases of hypospadias without concern for the plate configurations . 
 these previous studies have inspired us to undertake the current study to examine the role of the plate character in the success of tip repair among the study population in yemen . 
 here , we present a study evaluating the urethral plate characters , such as shape and the width in the success of tip urethroplasty for the management of primary anterior penile hypospadias in 100 children . 
 between january 2010 and december 2013 , a total of 100 children with primary anterior penile hypospadias were undergone tip repair . 
 all cases were undergone in mayo hospital , hodiedah , yemen . we used snodgrass technique with modifications for our current study . 
 all children were examined under general anesthesia ; the type of the plate is defined and reported by a single surgeon . 
 the plates were measured at the widest area ( in mm ) at the time of surgery . 
 we divided the study population according to the width of urethral plate > 8 mm ( group - a ) , while patients with urethral plate < 8 mm ( group - b ) . following the classification made by nguyen et al . 
 , the plates were categorized as flat , cleft , and deeply grooved [ figures 13 ] . 
 in addition , examination of the penis was also carried out to evaluate the presence or absence of chordee . 
 first a u - shaped incision was made around the plate associated with circumferential incision for complete degloving of the penis . 
 an artificial erection was achieved by saline injection , and the intrinsic ventral curvature was corrected with a dorsal plication if needed . 
 one deep midline incision in the urethral plate was made from the tip of the penis to the urethral meatus as described by snodgrass . 
 the plate was tubularized around 6 or 8 french ( fr ) nelaton catheter using polyglycolic acid 6/0 subcuticular running suture . 
 glanuloplasty was performed in a standard manner with interrupted 6 - 0 monofilament polyglyconate synthetic absorbable sutures . 
 postoperative urethral feeding tube or silicone foley catheter 8 fr was used for urinary drainage for average 1 week . 
 intraoperative single dose antibiotic was used ( ampicillin / gentamycin weight - adjusted dose in all cases ) . 
 postoperative management included ampicillin / gentamycin weight - adjusted dose in all cases and oxybutynin for relieving bladder spasms . 
 nonsteroidal antiinflammatory drugs , oral or suppository , were used in weight - adjusted doses . 
 success of surgery and postoperative complications were assessed in patients categorized on the basis of the shape of the plate or its width . 
 postoperatively , patients were followed - up for evaluating meatal stenosis , fistula formation , and partial or complete glandular dehiscence at the 1 , 3 and 6 months . 
 patients were followed - up for urethral calibration by urethral sound 8 fr at the third and 6 months of follow - up . 
 the flow rate was selectively indicated for patients with voiding difficulty and/or a narrow meatal orifice or weak stream . 
 a meatal stenosis was diagnosed when the flow rate was < 5 ml / s . 
 success was defined as the presence of an anatomically positioned vertical slit - like meatus , a normal urinary stream , and a satisfactory cosmetic appearance resembling a circumcised penis . 
 data were statistically analysed using epi info program ( centers for disease control and prevention , atlanta , georgia , usa ) . 
 fischer exact test for 2  2 tables was performed when expected cell count in > 25% of cases was < 5 . 
 first a u - shaped incision was made around the plate associated with circumferential incision for complete degloving of the penis . 
 an artificial erection was achieved by saline injection , and the intrinsic ventral curvature was corrected with a dorsal plication if needed . 
 one deep midline incision in the urethral plate was made from the tip of the penis to the urethral meatus as described by snodgrass . 
 the plate was tubularized around 6 or 8 french ( fr ) nelaton catheter using polyglycolic acid 6/0 subcuticular running suture . 
 glanuloplasty was performed in a standard manner with interrupted 6 - 0 monofilament polyglyconate synthetic absorbable sutures . 
 postoperative urethral feeding tube or silicone foley catheter 8 fr was used for urinary drainage for average 1 week . 
 intraoperative single dose antibiotic was used ( ampicillin / gentamycin weight - adjusted dose in all cases ) . 
 postoperative management included ampicillin / gentamycin weight - adjusted dose in all cases and oxybutynin for relieving bladder spasms . 
 nonsteroidal antiinflammatory drugs , oral or suppository , were used in weight - adjusted doses . 
 success of surgery and postoperative complications were assessed in patients categorized on the basis of the shape of the plate or its width . 
 postoperatively , patients were followed - up for evaluating meatal stenosis , fistula formation , and partial or complete glandular dehiscence at the 1 , 3 and 6 months . 
 patients were followed - up for urethral calibration by urethral sound 8 fr at the third and 6 months of follow - up . 
 the flow rate was selectively indicated for patients with voiding difficulty and/or a narrow meatal orifice or weak stream . 
 a meatal stenosis was diagnosed when the flow rate was < 5 ml / s . 
 success was defined as the presence of an anatomically positioned vertical slit - like meatus , a normal urinary stream , and a satisfactory cosmetic appearance resembling a circumcised penis . 
 data were statistically analysed using epi info program ( centers for disease control and prevention , atlanta , georgia , usa ) . 
 fischer exact test for 2  2 tables was performed when expected cell count in > 25% of cases was < 5 . 
 mean age of the patients underwent surgery was 4.3 years , ranging between 9 months and 12 years . 
 mean operative time was 115 min ( ranging 70 - 140 min ) and the average period of hospital stay was 4.6 days ( ranging 1 - 15 days ) . 
 urethral stenting for urine diversion was used for an average 7.7 days ( ranging 3 - 14 days ) , and the patients were followed - up for an average period of 6.4 months ( ranging 2 - 24 months ) . 
 we found mild insignificant chordee in 15 cases , improved after complete degloving of the penile skin without any additional procedures . 
 five infants had subcoronal hypospadias along with unilateral undescended testes ; these infants underwent orchiopexy at the same time along with hypospadias surgery . 
 location of the meatus was reported as coronal in 46 , subcoronal in 50 and anterior penile in 4 patients . 
 we divided the study population according to the width of urethral plate as 74 cases ( 74% ) with urethral plate > 8 mm ( group - a ) , whereas 26 cases ( 26% ) with urethral plate < 8 mm ( group - b ) . 
 table 1 shows that group - b patients with urethral plate < 8 mm had a statistically significant higher fistula rate compared with the group - a patients with urethral plate > 8 mm ( p = 0.004 ) . 
 similarly , group - b patients also had significantly higher incidences of failed 8 fr calibration compared with group - a ( p = 0.01 ) [ table 1 ] . 
 complications correlated to urethral plate width we also divided our study population ( 100 patients ) according to the character of urethral plate as flat ( 26 patients ) , cleft ( 52 patients ) , and deeply grooved ( 22 patients ) and monitored for postoperative complications . 
 table 2 shows that higher fistula rate as well as failed 8 fr calibrations is associated with patients having flat urethral plate compared with the patients with either cleft or deeply grooved urethral plates , although not statistically significant . 
 irrespective of urethral plate character , overall 8% patients developed fistula , 12% patients had failed 8 fr calibration , while partial dehiscence occurred in 1 case ( 1% ) . in addition , redo surgery was also indicated in 1 patient and fistulectomy in 8 patients . 
 the aim of hypospadias surgery is the creation of a straight penis with slit - like meatus at the tip of a glans with enough skin coverage . 
 tip urethroplasty is the most common procedure used for hypospadias repair since its introduction in 1994 by snodgrass . 
 tip repair is versatile and can be used for creation for neourethra with satisfactory slit - like meatus . the urethral plate shape and width 
 are generally evaluated when the tip repair is opted for the management of hypospadias . in this study 
 , we also categorised our study population based on both urethral plate width and shape . 
 based on the plate width , patients were divided into group - a with width > 8 mm and group - b < 8 mm . 
 we found significantly higher incidences of fistula in group - b patients when compared to the group - a patients ( p = 0.004 ) . 
 ideal urethral plate allows the creation of the neourethra > 10 fr in children after a midline incision to widen the urethral plate . 
 however , a narrow plate < 8 mm is inadequate for the creation of neourethra , which might be the cause of the statistically significant higher fistula rate in group - b patients with urethral plate width < 8 mm . 
 our findings are in accordance with the earlier reports by both holland and smith and nguyen et al . where statistically significant higher fistula was found in patients with urethral plate < 8 mm width 
 however , nguyen et al . reported 1 case ( 3.3% ) over 30 cases with narrow urethral plate versus one case ( 0.7% ) over 129 cases with urethral plate > 8 mm ; therefore , the result was statistically insignificant 
 . the higher fistula rate with narrow urethral plate occurs because of the fact that midline incision of the urethral plate is not enough to create a neourethra with too much width around 8 fr catheter ; as a consequence , the urethral plate makes the anastomoses under tension . 
 therefore , tubularisation with tension on suture line increases the incidence of fistula formation in cases with narrow urethral plate . 
 failure in 8 fr calibration in association with fistula formation could be resolved with urethral dilation . in our study , two of the children who had fistula resolved with urethral dilation . 
 modification to midline incision of the plate by extending it beyond to the tip of glans prevents meatal stenosis as described by khairallah and bader - eldin ; however , it only decreases fistula rate and meatal stenosis to < 1% . 
 we also divided our study population based on the urethral plate character as flat , cleft , and deeply grooved . 
 a higher fistula rate was noted in patients with flat urethral plate compared to the patients with cleft or deeply grooved ; however , the result is not statistically significant ( p = 0.4 ) . 
 this is also in agreement with the previous reports , where no significant relation was found between urethral plate depth and incidence of fistula . 
 although higher failed 8 fr calibration rate was observed in patients with flat urethral plate compared with the other two groups cleft and deeply grooved , the result is statistically insignificant ( p = 0.09 ) [ table 2 ] . 
 similar to earlier reports , our study also provide evidences of excellent outcome in hypospadias management through tip repair with overall 8% patients developing fistula , 12% patients with failed 8 fr calibration , while partial dehiscence occurred in 1 case ( 1% ) ; redo surgery was indicated in only 1 patient . in the current study , we found patients with flat and narrow urethral plate developed more complications . in this 
 regard , tavakkoli tabassi and mohammadi rana advocated the use of buccal mucosal graft ( bmg ) with tip repair to augment the narrow urethral plate ( 6 - 8 mm ) and they concluded that the use of mucosal graft decreased fistula rate formation and meatal stenosis . 
 ye et al . also combined tip with bmg in 53 patients and found better outcome . 
 therefore , urethral plate augmentation is recommended when the urethral plate is narrow and inelastic requiring augmentation with free graft . 
 based on our findings , we conclude that the tip urethroplasty has good cosmetic and functional results and offer a modality for hypospadias repair that has the following advantages : preservation and utilisation of urethral plate without the need for additional flaps , single suture line of the neourethra , creation of glandular and penile urethra , short operative time and low complication rate . 
 this technique can be used for primary anterior penile hypospadias with satisfactory results . using this technique with plates 
 flat plates may need augmentation by bmg as advised by some authors to improve the results in this group of patients .","background : today , tubularized incised plate ( tip ) urethroplasty is the most commonly performed operation for distal and mid - penile hypospadias . 
 reports from different centers worldwide confirm its nearly universal applicability and low complications rate.aim:evaluation of the urethral plate characters and its effect on the outcome of tip urethroplasty.materials and methods : between 2010 and 2013 , 100 children with primary distal penile hypospadias underwent tip urethroplasty . 
 urethral plates were categorized as flat , cleft , and deeply grooved . 
 postoperatively , patients were followed - up for evaluation of meatal stenosis , fistula formation , and glandular dehiscence at 1st , 3rd and 6th months . 
 patients were followed - up for urethral calibration by urethral sound 8 fr at 3rd and 6 months follow - up . 
 data were statistically analysed using epi info program to correlate between the width , plate shape , and complications.results:mean age at surgery was 4.3 years . 
 patients were followed - up for an average period of 6.4 months . 
 pre - operative location of the meatus was reported as coronal in 46 , subcoronal in 50 and anterior penile in 4 cases . 
 urethral plate characters were flat in 26 cases , cleft in 52 , and deeply grooved in 22 . 
 urethral plate width was > 8 mm in 74 cases and < 8 mm in 26 . 
 patients with urethral plate < 8 mm had a statistically significant higher fistula rate ( p = 0.004 ) and failed 8 fr calibrations in 26.9% ( p = 0.01 ) compared with the patients with urethral plate > 8 mm . in addition , we also founds higher fistula rate and failed 8 fr calibrations in flat urethral plate.conclusions:an adequate urethral plate width ( > 8 mm ) is essential for successful tip repair . 
 lower success rates with flat plates may need buccal mucosal augmentation to improve the results .",pubmed
"a biodegradao de materiais que ocorre na cavidade bucal pode interferir na resistncia de unio entre o braquete e o esmalte , causando baixos valores de resistncia de unio in vivo , em comparao aos valores in vitro . 
 o objetivo do presente estudo foi desenvolver um prottipo para mensurar , in vivo , a fora de descolagem de braquetes e avaliar , in vitro , a resistncia de unio obtida com o uso do prottipo . 
 um alicate ( 3 m unitek ) foi modificado por meio da adio de um extensmetro diretamente conectado  garra do alicate . um circuito eletrnico realizou a leitura do extensmetro , e um software instalado em um computador registrou os valores de fora de descolagem do braquete em kgf . 
 os braquetes ortodnticos foram colados com material adesivo na face vestibular de 30 dentes incisivos bovinos . no grupo 1 ( n = 15 ) , a descolagem foi realizada com o prottipo , enquanto no grupo 2 ( n = 15 ) foi realizado teste de resistncia de unio  trao , utilizando - se uma mquina universal de ensaios . 
 de acordo com o teste de t de student (  = 0,05 ) , os grupos 1 ( 2,96 mpa ) e 2 ( 3,08 mpa ) no foram estatisticamente diferentes 
 o prottipo mostrou - se confivel para se obter , in vivo , os valores de resistncia de unio dos braquetes ortodnticos . 
 a stable interface between the bonding material and the bracket , as well as between the bonding material and the enamel , is important to withstand the loads generated by an activated arch 
 . 

  using universal testing machines , direct bracket bonding to dental enamel and the forces required to debond the brackets have been studied , in vitro , by means of shear or tensile strength . 
 regardless of the precision obtained in the measurement of the debonding forces , the universal testing machines are expensive , large and complex to handle . 
 due to the proportions , it may be impossible to use it for in vivo studies . 
 furthermore , the complex interaction processes that exist in the oral cavity can not be completely reproduced by in vitro studies . 
 the oral cavity is a challenging environment for dental materials , especially regarding the adhesion process . 
 the contact of adhesive systems and composite resins with the oral medium is associated with an ageing pattern , which may alter the composition and the mechanical properties of the polymers . 
 material biodegradation may interfere in the bonding between the bracket and the enamel , 
  causing lower bond strength values in vivo , in comparison with in vitro values . 
 because in vitro bond strength can not be correlated with clinical failure indexes , 
  and facing the necessity of creating a method to test the effects of the oral medium on the composite resins used in orthodontics , different devices have been developed to measure the bracket debonding force in vivo . 
 usually , strain gauges are bonded to the handle 
  or to the arm of the pliers . 
 the force obtained is a measure of the deformation that occurs in the handle or arm of the pliers during the bracket debonding procedure , and the results can be influenced by the way pliers are holded . 
  no study has developed a device using strain gauges connected directly to the part of the pliers where the bracket is attached for the debonding procedure .   thus , the aim of the present study was to develop a prototype to measure the bracket debonding force in vivo , using a strain gauge connected directly to the extremity of the pliers where the bracket debonding occurs . 
 the bond strength obtained with the prototype was also compared in vitro by using the tensile bond strength test in a universal testing machine . 
 a polypropylene plier ( debracketing instrument - 444 - 761 , instrument kit - 3m / unitek , monrovia , ca , usa ) was modified to develop the prototype ( fig 1a ) , which consists of three basic parts :  
 figure 1a ) original plier . 
 b ) exploded image of the intermediate part of the prototype ( the arrow indicates the localization of the strain gauges ) . 
 1 ) modified plier : the pliers have undergone changes from its original design . to install the strain gauge , 
 the original pliers were cut to receive an intermediate part positioned between the active arm of the pliers and the claw ( fig 1b ) . 
 one strain gauge ( 45% ni ; 55% cu ) with 10 mm of a sheet type grid and with temperature compensation for steel ( kyowa , chofu , tokyo , japan ) was directly connected to the claw . 
 figure 1c shows a photograph of the prototype.2 ) measurement and control circuit : corresponds to the electronic circuit that performs the reading of the strain gauge mounted on the intermediate part of the prototype and changes the obtained value into an 8-bit binary number . 
 the value 0 ( zero ) represents a strength of 0 kgf , and the value 255 ( 8 bits ) is the maximum measured force of 6.8 kgf . 
 prior adjustments are necessary for the required performance of the circuit ( wheatstone bridge , amplifier gain and the ad converter range ) . 
 3 ) pc : a specific software was developed to record the values ( in kgf ) of the bracket debonding force . 
 the software was programmed in c ( programming language ) and was designed to enable appropriate use in a research environment . 
 the software has a calibration system , and calibration was performed with weights ranging from 0.5 kg to 6 kg . a known load ( kg ) 
 a simple linear regression analysis was applied to adjust the data ( r = 0.99 ) . 
  ) 
 modified plier : the pliers have undergone changes from its original design . to install the strain gauge , 
 the original pliers were cut to receive an intermediate part positioned between the active arm of the pliers and the claw ( fig 1b ) . 
 one strain gauge ( 45% ni ; 55% cu ) with 10 mm of a sheet type grid and with temperature compensation for steel ( kyowa , chofu , tokyo , japan ) was directly connected to the claw . 
 measurement and control circuit : corresponds to the electronic circuit that performs the reading of the strain gauge mounted on the intermediate part of the prototype and changes the obtained value into an 8-bit binary number . 
 the value 0 ( zero ) represents a strength of 0 kgf , and the value 255 ( 8 bits ) is the maximum measured force of 6.8 kgf . 
 prior adjustments are necessary for the required performance of the circuit ( wheatstone bridge , amplifier gain and the ad converter range ) .   ) 
 pc : a specific software was developed to record the values ( in kgf ) of the bracket debonding force . the software was programmed in c ( programming language ) and was designed to enable appropriate use in a research environment . 
 the software has a calibration system , and calibration was performed with weights ranging from 0.5 kg to 6 kg . 
 a known load ( kg ) was applied to the claw , and the measurements were recorded in kgf . a simple linear regression analysis was applied to adjust the data ( r = 0.99 ) . 
 figure 2a ) prototype after electronic instrumentation ; b ) measurement and control circuit ; c ) usb communication cable . 
 figure 3representative diagram of the parts of the prototype . 
  to measure the accuracy and precision of the prototype , 
 the following weights were used : 1 , 2 , 3 , 4 , 5 , and 6 kg . 
 each weight was connected to the claw of the prototype by a stainless steel wire , and the measurement was done by means of displacing the active arm and lifting the weight . 
 the measurements were undertaken by two examiners , who did not standardize the position of the hand on the pliers . 
 twenty repetitions were performed for each weight . the numerical value recorded by the software installed on the computer was noted , and the arithmetic mean was calculated . 
 the mean temperature in the laboratory during the experiments was 23  2c and the humidity was 70% . 
 the exposed enamel surface was lightly abraded with # 600 grain silicon carbide abrasive paper in a polisher to obtain a flat enamel surface . 
 the maxillary central incisor brackets ( eurodonto , curitiba , pr , brazil ) were bonded to the enamel using the following procedure : a ) prophylaxis with pumice stone and water ; b ) etching with phosphoric acid at 37% for 15 s , followed by washing and drying ; c ) application of scotchbond multi - purpose adhesive ( bond ) ( 3 m , st . 
 paul , mn , usa ) on the enamel ; d ) application of z100 composite resin ( 3 m , st . 
 paul , mn , usa ) at the base of the bracket and positioning on the tooth with light manual pressure , followed by removal of the excess with a periodontal scaler ; e ) light curing for 40 s ( 10 seconds on each side ) with the optilight plus appliance ( gnatus , ribeiro preto , brazil ) . 
 the specimens were stored in 100% relative humidity at 37c for 24 hours . after storage 
 , specimens were divided into two equal groups : group 1 - bracket debonding with the prototype : each specimen was placed on a bench lathe . 
 a single operator held the prototype , which was placed on the bracket with the supports aligned in an occlusal - gingival direction in contact with the enamel . 
 with the claw hitched onto one of the bracket wings , the prototype was activated by moving the active arm until bracket debonding occurred . 
 the value of the force necessary to remove the bracket was recorded by the software in kgf , transformed into newtons and divided by the bracket area ( 14.12 mm ) , to obtain resistance values in mpa . 
 group 2 - bracket debonding by tensile testing in a universal testing machine : the specimen was fixed in a metal sleeve on the bottom part of the emic - dl 2000 universal testing machine ( so jos dos pinhais , paran , brazil ) . for tensile testing 
 , a claw made of stainless steel wire , 0.40 mm in diameter and 5 cm in length , was connected to one of the bracket wings . at the top part of the claw , a 2 mm thick and 20 mm long wire segment 
 was welded , which was hitched onto the top part of the universal testing machine . 
 , tokyo , japan ) at 10x magnification to assess the adhesive remnant index ( ari ) : score 0 = no composite resin left on the tooth ; score 1 = less than half of the composite resin left on the tooth ; score 2 = more than half of the composite resin left on the tooth ; score 3 = all composite resin left on the tooth , with a distinct impression of the bracket mesh . 
 student s t test (  = 0.05 ) for paired samples was used for comparison between the examiners . 
 student s t test (  = 0.05 ) was performed to compare the groups . 
 a polypropylene plier ( debracketing instrument - 444 - 761 , instrument kit - 3m / unitek , monrovia , ca , usa ) was modified to develop the prototype ( fig 1a ) , which consists of three basic parts :  
 figure 1a ) original plier . 
 b ) exploded image of the intermediate part of the prototype ( the arrow indicates the localization of the strain gauges ) . 
 1 ) modified plier : the pliers have undergone changes from its original design . to install the strain gauge , 
 the original pliers were cut to receive an intermediate part positioned between the active arm of the pliers and the claw ( fig 1b ) . 
 one strain gauge ( 45% ni ; 55% cu ) with 10 mm of a sheet type grid and with temperature compensation for steel ( kyowa , chofu , tokyo , japan ) was directly connected to the claw . 
 figure 1c shows a photograph of the prototype.2 ) measurement and control circuit : corresponds to the electronic circuit that performs the reading of the strain gauge mounted on the intermediate part of the prototype and changes the obtained value into an 8-bit binary number . 
 the value 0 ( zero ) represents a strength of 0 kgf , and the value 255 ( 8 bits ) is the maximum measured force of 6.8 kgf . 
 prior adjustments are necessary for the required performance of the circuit ( wheatstone bridge , amplifier gain and the ad converter range ) . 
 3 ) pc : a specific software was developed to record the values ( in kgf ) of the bracket debonding force . 
 the software was programmed in c ( programming language ) and was designed to enable appropriate use in a research environment . 
 the software has a calibration system , and calibration was performed with weights ranging from 0.5 kg to 6 kg . a known load ( kg ) 
 a simple linear regression analysis was applied to adjust the data ( r = 0.99 ) . 
  ) 
 modified plier : the pliers have undergone changes from its original design . to install the strain gauge , 
 the original pliers were cut to receive an intermediate part positioned between the active arm of the pliers and the claw ( fig 1b ) . 
 one strain gauge ( 45% ni ; 55% cu ) with 10 mm of a sheet type grid and with temperature compensation for steel ( kyowa , chofu , tokyo , japan ) was directly connected to the claw . 
 measurement and control circuit : corresponds to the electronic circuit that performs the reading of the strain gauge mounted on the intermediate part of the prototype and changes the obtained value into an 8-bit binary number . 
 the value 0 ( zero ) represents a strength of 0 kgf , and the value 255 ( 8 bits ) is the maximum measured force of 6.8 kgf . 
 prior adjustments are necessary for the required performance of the circuit ( wheatstone bridge , amplifier gain and the ad converter range ) .   ) 
 pc : a specific software was developed to record the values ( in kgf ) of the bracket debonding force . the software was programmed in c ( programming language ) and was designed to enable appropriate use in a research environment . 
 the software has a calibration system , and calibration was performed with weights ranging from 0.5 kg to 6 kg . 
 a known load ( kg ) was applied to the claw , and the measurements were recorded in kgf . a simple linear regression analysis was applied to adjust the data ( r = 0.99 ) . 
 figure 2a ) prototype after electronic instrumentation ; b ) measurement and control circuit ; c ) usb communication cable . 
 to measure the accuracy and precision of the prototype , the following weights were used : 1 , 2 , 3 , 4 , 5 , and 6 kg . 
 each weight was connected to the claw of the prototype by a stainless steel wire , and the measurement was done by means of displacing the active arm and lifting the weight . 
 the measurements were undertaken by two examiners , who did not standardize the position of the hand on the pliers . 
 twenty repetitions were performed for each weight . the numerical value recorded by the software installed on the computer was noted , and the arithmetic mean was calculated . 
 the mean temperature in the laboratory during the experiments was 23  2c and the humidity was 70% . 
 the exposed enamel surface was lightly abraded with # 600 grain silicon carbide abrasive paper in a polisher to obtain a flat enamel surface . 
 the maxillary central incisor brackets ( eurodonto , curitiba , pr , brazil ) were bonded to the enamel using the following procedure : a ) prophylaxis with pumice stone and water ; b ) etching with phosphoric acid at 37% for 15 s , followed by washing and drying ; c ) application of scotchbond multi - purpose adhesive ( bond ) ( 3 m , st . 
 paul , mn , usa ) on the enamel ; d ) application of z100 composite resin ( 3 m , st . 
 paul , mn , usa ) at the base of the bracket and positioning on the tooth with light manual pressure , followed by removal of the excess with a periodontal scaler ; e ) light curing for 40 s ( 10 seconds on each side ) with the optilight plus appliance ( gnatus , ribeiro preto , brazil ) . 
 after storage , specimens were divided into two equal groups : group 1 - bracket debonding with the prototype : each specimen was placed on a bench lathe . 
 a single operator held the prototype , which was placed on the bracket with the supports aligned in an occlusal - gingival direction in contact with the enamel . 
 with the claw hitched onto one of the bracket wings , the prototype was activated by moving the active arm until bracket debonding occurred . 
 the value of the force necessary to remove the bracket was recorded by the software in kgf , transformed into newtons and divided by the bracket area ( 14.12 mm ) , to obtain resistance values in mpa . 
 group 2 - bracket debonding by tensile testing in a universal testing machine : the specimen was fixed in a metal sleeve on the bottom part of the emic - dl 2000 universal testing machine ( so jos dos pinhais , paran , brazil ) . for tensile testing , a claw made of stainless steel wire , 0.40 mm in diameter and 5 cm in length , was connected to one of the bracket wings . at the top part of the claw , a 2 mm thick and 20 mm long wire segment was welded , which was hitched onto the top part of the universal testing machine . 
 tokyo , japan ) at 10x magnification to assess the adhesive remnant index ( ari ) : score 0 = no composite resin left on the tooth ; score 1 = less than half of the composite resin left on the tooth ; score 2 = more than half of the composite resin left on the tooth ; score 3 = all composite resin left on the tooth , with a distinct impression of the bracket mesh . 
 student s t test (  = 0.05 ) for paired samples was used for comparison between the examiners . student s t test (  = 0.05 ) was performed to compare the groups . 
 table 1 compares the mean values obtained by the two examiners , with each weight , during the gauging procedure of the prototype . according to student s t test for paired samples 
 , there were no significant differences ( p < 0.05 ) between the examiners at any weight . 
 table 1comparison between the examiners in each weight.comparisonnmean ( kgf)standard deviationp valuea - 1 kg 201.040.080.30b - 1 kg 201.070.08a - 2 kg 202.030.070.20b - 2 kg 
 202.060.09a - 3 kg 202.950.060.08b - 3 kg203.010.09a - 4 kg 204.080.080.44b - 4 kg 204.040.09a - 5 kg205.010.090.16b - 5 kg 205.050.07a - 6 kg 206.080.080.32b - 6 kg 206.050.09*p > 0.05 indicates no significant difference between the examiners a - examiner a.b - examiner b. 
  * p > 0.05 indicates no significant difference between the examiners   according to student s t test , the mean bond strength obtained in group 1 ( 2.96 mpa ) and in group 2 ( 3.08 mpa ) were statistically similar ( table 2 ) . 
 table 2comparison of mean bond strength ( mpa ) obtained in each group.groupnmeansdpgroup 1152.960.32 > 0.05group 2153.080.40group 1 : prototype.group 2 : tensile test in a universal testing machine . 
 adhesive remnant index score 3 was predominant in the two groups ( table 3 ) . 
 score 0score 1score 2score 3group 1 - 1 ( 7%)1 ( 7%)13 ( 86%)group 2- -2 ( 13%)13 ( 86%)group 1 : prototype.group 2 : tensile test in a universal testing machine . 
 the prototype of the present study was an evolution of a device which had two strain gauges bonded to the anterior and posterior 2/3 surfaces of the active arm of the pliers . 
 because the strain gauges were placed in the active arm of the pliers , they suffered from the influence of the operator s hand . 
 the present prototype has been developed in order to control this problem by placing the strain gauge near the claw , and not in the active arm of the pliers . 
 strain gauges are defined as localized mechanical deformation sensors , since all and any phenomenon responsible for mechanical deformation can be analyzed . 
 therefore , strain gauges are transducers that convert mechanical displacement into a range of electrical resistance . 
 strain gauges are very sensitive and temperature alterations are able to influence the measurements . 
  to compensate for any possible temperature alterations , 
 the following procedures were adopted : a ) use of an electrical resistance strain gauge with temperature compensation for steel that self - compensates eventual temperature variations , and ; b ) control of the laboratory mean temperature during the experiments .   in the present study , 
 the strain gauges were bonded in the intermediate part positioned between the active arm of the plier and the claw . 
 one strain gauge was directly connected to the claw . when the plier was used to debond the brackets , the strain gauge metal grid resistance generated a tension ( mv ) . 
 the small amount of tension generated was multiplied by an amplifier circuit in the electronic circuit , which performed the reading of the strain gauge , into binary numbers that were converted into kgf . 
 the mode of holding the prototype was not standardized between the two examiners , and there was no statistically significant difference in the values obtained between the examiners at any weight . 
 this means that the prototype was not influenced by the way that it was held - as in another study 
 - , confirming that the new prototype is superior to the previous one .   after gauging the prototype , in vitro orthodontic bracket debonding 
 the bracket debonding performed with a universal testing machine is not dependent on operator variation , the values obtained with this method were compared to the values obtained with the prototype to evaluate its reliability . 
 the tensile test on the universal testing machine ( group 2 ) tried to reproduce the same conditions as debonding with the prototype . for this purpose 
 , a 0.40 mm in diameter stainless steel wire claw was made reproducing the same thickness as the prototype claw ( group 1 ) . 
 along with that , the claw made for the test machine was also connected on a single wing of the bracket at the testing time , reproducing the same position used by the prototype claw at the debonding moment . 
 although it is named tensile test , it is known that not only tensile forces occur at the debonding moment of the bracket , but that there is also an interaction of tensile , shear and compressive forces . 
 debonding of the bracket was performed under a constant crosshead speed of 0.5 mm / min in the universal testing machine . 
 this load speed is generally used in spite of not corresponding to clinical conditions due to the fact that in vivo debonding occurs at a higher speed . 
 therefore , when the prototype was applied , a speed higher than 0.5 mm / min was probably used for bracket removal . despite the fact that differences existed between the methodologies used for debonding 
 , the bond strength did not differ statistically between the prototype ( 2.96 mpa ) and the tensile test on the universal testing machine ( 3.08 mpa ) . 
 this suggests that the prototype might be a useful tool for measuring bond strength in vivo . 
 orthodontic bracket bonding to teeth requires that the bond system applied must provide sufficient bond strength to withstand the forces that occur during the orthodontic mechanics and chewing . for clinical situations 
 bond strength values above 10 mpa are potentially dangerous since they may cause enamel fractures during bracket debonding . 
 however , it is difficult to establish a numerical value because these values depend on several factors , such as the condition of the substrate , 
  the material involved , 
  the devices chosen for the test , 
  cure time , 
  and the location where force is applied to the bracket . 

  due to the difficulty of comparing and extrapolating the results obtained from in vitro studies to the clinical situation , it remains unknown what minimum bond strength values are required for safe clinical performance of orthodontic bonding procedures . 
 thus , it would be useful to determine the bracket bond strength values in vivo , by using a device to measure the bracket debonding force directly in the oral cavity . 
 the ari scores showed that the fracture pattern was predominantly score 3 in both groups . 
 the composite resin remained completely bonded to the enamel after bracket debonding , which is advantageous since score 3 has the least probability of harming the enamel . 
 therefore , debonding with the prototype was shown to be a safe method for preserving the enamel surface . 
 ","abstractintroduction : material biodegradation that occurs in the mouth may interfere in the bonding strength between the bracket and the enamel , causing lower bond strength values in vivo , in comparison with in vitro studies . objective : to develop a prototype to measure bracket debonding force in vivo and to evaluate , in vitro , the bond strength obtained with the prototype.methods : a original plier ( 3 m unitek ) was modified by adding one strain gauge directly connected to its claw . 
 an electronic circuit performed the reading of the strain gauge , and the software installed in a computer recorded the values of the bracket debonding force , in kgf . 
 orthodontic brackets were bonded to the facial surface of 30 bovine incisors with adhesive materials . in group 1 
 ( n = 15 ) , debonding was carried out with the prototype , while tensile bond strength testing was performed in group 2 ( n = 15 ) . 
 a universal testing machine was used for the second group . the adhesive remnant index ( ari ) 
 was recorded.results : according to student s t test (  = 0.05 ) , group 1 ( 2.96 mpa ) and group 2 ( 3.08 mpa ) were not significantly different . 
 ari score of 3 was predominant in the two groups.conclusion : the prototype proved to be reliable for obtaining in vivo bond strength values for orthodontic brackets .",pubmed
"trichomonas vaginalis   infection is one of the most common curable sexually transmitted infections . who estimates that 173 million of new cases occur annually . 
 trichomonal infection has been associated with adverse reproductive health outcomes including pelvic inflammatory disease , low infant birth weight and premature delivery [ 36 ] . 
 furthermore , trichomoniasis has been shown to increase transmission and acquisition of other sexually transmitted diseases , including hiv infection [ 1 , 8 , 9 ] . 
 the frequency of trichomonal infection is not monitored in most countries nor do control programs exist . 
 peruvian sti guidelines do not include trichomonas diagnostic testing ; the syndromic management of suspected vaginal trichomoniasis is the current standard for clinical care . 
 trichomonal infection is most likely more common among women and men than is generally understood , and long standing , asymptomatic infections may lead to continued community level transmission . as trichomonas prevalence depends on many factors such as age , sexual activity , and 
 the number of sex partners , a better understanding of the epidemiology of this infection in women is needed to foster disease control programs in populations at risk for reproductive health complications . 
 this study was a cross - sectional analysis of the behavioral and biological data from the baseline assessment of a cohort subsequently enrolled in the nimh collaborative hiv / std prevention trial collected from 2003 to 2005 in peru . 
 we enrolled participants from low - income neighborhoods in three coastal peruvian cities : lima , trujillo , and chiclayo . 
 we chose neighborhoods based on data from the peruvian national institute of statistics and informatics . 
 socially marginalized women were characterized through informal interviews and participant observation , and based on ethnographic information collected prior to the study baseliness were shown to have high levels of sexual risk behaviors . we defined socially marginalized women   as those women living in low - income neighborhoods  who contradicted peruvian social norms by spending time with and having sex with poor unemployed men . 
 most of these women were also unemployed , and although the majority reported a stable sex partner , most of them also reported having multiple sex partners , further defying the social norms of peru . 
 eligible women were recruited by outreach workers from venues such as volleyball fields , hair salons , parks , bars , and street corners . 
 the number of venues selected per city was approximately proportional to the city 's overall population . 
 eligible participants were restricted to women between 18 and 40 years of age who reported having had sex in the past six months , intended to stay in the neighborhood for at least two years , and who frequented the target venue at least twice a week . 
 participants were invited to a temporary project office in their neighborhood , where we privately administered a 30-minute structured survey in spanish , wherein trained interviewers read questions to participants and entered their answers into a portable computer . 
 the survey instrument collected sociodemographic information as well as detailed sexual risk behavior with the last five sex partners , number of sex partners , sti - related symptoms , alcohol and drug , use and hiv status . upon completing the survey , participants underwent pretest counseling for stis including hiv infection , conducted by a trained counselor . trained laboratory technicians then explained to each participant how to self - collect a vaginal specimen using a dacron swab . 
 a second dacron swab was stored for chlamydia trachomatis   and neisseria gonorrhoeae pcr testing ( ct / ng amplicor pcr , roche diagnostics , nj , usa ) , and a blood sample was also collected for hiv-1 ( genetic systems bio - rad laboratories , hercules , calif , usa ) , herpes virus simplex type 2 antibodies ( herpeselect ; focus technologies , cypress , calif , usa ) , and syphilis testing ( rprnosticon , shield diagnostics , dundee , uk and serodia - tppa , fujirebio diagnostics inc , tokyo , japan ) . participants returning for results underwent posttest counseling to ensure their understanding of the meaning of both positive and negative results and for treatment in case of a positive result . 
 treatment for trichomoniasis consisted of 2 g , single dose metronidazole by mouth administered by trained health personnel . 
 all participants with positive results were encouraged to share test results with recent sex partners . 
 reimbursement for travel costs and time spent during the assessment was given to each participant at the rate of 15 peruvian soles ( approximately $ 5 ) at their first visit and an additional 10 peruvian soles ( approximately $ 3 ) at their posttest visit . 
 all vaginal swab specimens were immediately inoculated and transported at ambient temperature using the in pouch tv culture media ( biomed diagnostics , white city , ore , usa ) to the cayetano heredia university  sexual health laboratory in lima , chiclayo , or trujillo . 
 cultures were transported to the laboratory within 68 hours after sample collection and incubated at 37c once they arrived at the laboratory . to monitor the performance of test and as part of the quality control of the study , 
 known positive samples were inoculated in the field , transported , and cultured under the same conditions . 
 all samples , including the controls , were observed microscopically at 400x power for the presence of motile , oval flagellated protozoan daily for 5 days . 
 the observation of motile trichomonads was reported as positive , and specimens showing no organisms after 5 days of incubation were considered negative for t. vaginalis . 
 laboratory work was performed in accordance with the college of american pathology quality assurance and quality control program . 
 as odds ratios overestimate the estimated risk when the prevalence is high , we calculated prevalence ratios to avoid that bias . to model the prevalence ratios we used poisson family generalized linear models and adjusted the standard errors to account for the clustered nature of sample . 
 multivariate models were created taking into account all variables that were significant at p - value < .1 in bivariate analysis . 
 the variables added to the multivariate model were selected based on likelihood ratio tests comparing nested to saturated models and included only if the likelihood ratio test had a p - value   < .05 . 
 the study was approved by the committee on human research of the university of california , san francisco ; university of california , los angeles ; naval medical research center detachment and cayetano heredia university of peru . 
 we collected data only from participants that gave written informed consent to participate in the study . 
 between 2003 and 2005 ,   we recruited and assessed 308 women from 20 low - income communities in coastal peru . the overall prevalence of t . 
 women with t. vaginalis   infection were more likely to be older ; the highest prevalence was found in the oldest age group , 3640 years ( figure 1 ) . those with less education were 3 times more likely to have trichomonal infection compared to those who completed high school , and being single 
 the prevalence of trichomonal infection among women with vaginal discharge and painful urination in the prior six months was lower than the prevalence among those who did not report vaginal discharge during the same period , although the difference was not statistically significant . 
 women with hsv-2 infection and syphilis infection were twice as likely to have trichomonal infection . in the multivariate model , because only the number of partners in the past 6 months significantly improved the model , no other variables were entered into the model . 
 vaginalis   infection among socially - marginalized young women from low - income communities in coastal peru . 
 t. vaginalis   infection occurred more often among older women ( see figure 1 ) , women who had not completed high school , single women , women who reported unprotected sex with a nonsteady partner , and women who reported multiple sex partners . 
 no association was found between any vaginal symptoms and t. vaginalis . while most epidemiological studies on trichomonal infection have been focused on pregnant women , adolescents , and female sex workers accessing care , there are few studies at the population - level and in particular , socially - marginalized women [ 5 , 1519 ] . 
 our findings are important because such hard to reach populations are often under - represented because they are not seeking health care and represent a high - risk target population and potential core population that maintain endemic trichomonal infections at the population level . in latin america trichomonal prevalence 
 has varied from 2.9% to 16.5% , where the highest prevalence was found in population samples in the jungle and the highlands of per , and the lower prevalences were found in clinical settings such as family planning clinics , pharmacies , and obstetrics services . in general , tested populations in those studies were most often symptomatic women [ 15 , 20 , 21 ] . 
 the association between higher prevalence of trichomonal infection and older age is unusual for nonviral sexually transmitted infections . 
 the increased prevalence of trichomonal infection in older women suggests long - term prevalent infection that does not spontaneously resolve and would be missed by screening programs focused on younger age women . 
 the prevalence of trichomonal infection was not different between women with or without presence of recent painful urination or vaginal discharge . 
 that finding suggests that syndromic management of t. vaginalis   may result in both over and under treatment . 
 the asymptomatic nature of trichomonal infection also could contribute to a long duration of infection and continued spread of infection in the population [ 1 , 7 , 23 ] . 
 our data , and others [ 4 , 21 , 24 ] , attest to the asymptomatic nature of t. vaginalis   and the need for screening and treatment programs to control the spread of trichomonal infection . 
 results from other peruvian studies as well as the results in the present study indicate that there is no direct relationship between t. vaginalis   infection and symptoms making syndromic treatment unlikely to impact the population burden of infection [ 20 , 21 ] . 
 first , the sample size was small resulting in limited power to assess multivariate models between demographic and behavioral characteristics and t. vaginalis . 
 furthermore , the results presented here may not be generalizable to other female populations . while response bias is always a concern , we believe that the use of standardized interviews by trained staff helped to minimize social desirability and other information biases . 
 importantly as all epidemiologic information was collected prior to testing , there was no reason to suspect differential bias associated by trichomonal infection . 
 selection bias was also a concern , but because most women were asymptomatic we do not believe that those with potential trichomonal infection were more likely to participate compared with those without infection . since t. vaginalis   is a common sexually transmitted pathogen , the screening and treatment of sex partners of infected women must be also prioritized as a public health measure to prevent reinfection and reduce infection prevalence and complications in untreated males [ 23 , 25 ] . as the majority of the assessed population in this study was in a fertile age range , 
 our findings support the need for improved disease control activities to reduce adverse trichomoniasis - associated reproductive health outcomes such as ectopic pregnancy , low infant birth weight , preterm labor , and non - hpv associated cervical neoplasia [ 14 , 26 , 27 ] . 
 as t. vaginalis is widely prevalent and one of the easiest infections to treat with an inexpensive antimicrobial , further studies are needed to determine if screening and treatment of trichomoniasis will improve the reproductive health and birth outcomes of women .","objective . the epidemiology of trichomonas vaginalis infection among sexually active socially - marginalized women in three urban , 
 coastal peruvian cities was examined in order to quantify the prevalence of trichomonas infection and identify associated risk factors . 
 methods . 
 we conducted a cross - sectional , venue - based study of women from socially - marginalized populations in three coastal peruvian cities . results . among the 319 women enrolled , the overall prevalence of trichomonal infection was 9.1% ( 95% ci , 5.9%12.3% ) . 
 the mean age was 26.3 years , and 35.5% reported having had unprotected intercourse with nonprimary partners and 19.8% reported two or more sex partners in the last three months . 
 trichomonal infection was associated with increased number of sex partners ( pr 2.5 , 95% ci 1.44.6 ) and unprotected sex with nonprimary partner in the last three months ( pr 2.3 , 95% ci 1.14.9 ) . 
 conclusions . a moderately high prevalence of trichomonal infection was found among women in our study . 
 trichomonal infection was associated with unprotected sex and multiple sex partners . 
 efforts to control the continued spread of trichomonal infection are warranted .",pubmed
"lennox - gastaut syndrome ( lgs ) , one of the catastrophic epilepsies of childhood , is classified by the international league against epilepsy as a symptomatic generalized epilepsy syndrome . originally described in 1966 , this 
 tonic seizures ( especially during sleep ) , atonic ( astatic or drop attacks ) seizures , and atypical absence seizures are most commonly observed ; some patients also develop myoclonic , generalized tonic - clonic , or partial seizures . 
 the second feature required by the definition is an interictal awake electroencephalogram ( eeg ) pattern consisting of slow spike wave discharges ( less than 3 hz ) , usually with a generalized distribution . 
 another characteristic eeg feature is paroxysms of low voltage fast activity at about 10 hz during sleep . 
 the third component of the definition of lgs is cognitive impairment involving moderate to severe mental retardation and behavioral disorders including aggression and autistic features . over 75% of children with lgs 
 these include numerous congenital or acquired etiologies , such as cortical maldevelopment , perinatal hypoxia - ischemia , cns infection , or neurometabolic disorders . 
 about 20% of children with lgs have prior infantile spasms ( west syndrome ) and evolve into lgs with age . 
 the typical age of onset of lgs is between 2 and 5 years ; boys are affected about 5 times more often than girls.5 the prognosis of lgs is poor , with regard to both seizures and cognitive outcome . 
 risk factors for a poor cognitive prognosis include symptomatic etiology , history of nonconvulsive status epilepticus , prior infantile spasms , and early age of seizure onset.6 due to the encephalopathic nature and multiple seizure types , lgs is notoriously difficult to treat.7 many drugs reduce seizures initially , only to lose effectiveness over time . 
 children often end up on polypharmacy with numerous anticonvulsants , which adds to the cumulative side effects and drug 
 drug interactions.3 furthermore , the seizures themselves are thought to contribute to the cognitive impairment and behavioral comorbidities . 
 difficulties in diagnosing lgs are discussed in detail in a recent review.4 sometimes the classic clinical and eeg features are not present at the onset of the syndrome . due to the heterogeneous causes , 
 for example , it can be difficult to differentiate between spasms and tonic seizures and to identify and quantify atypical absence seizures accurately . 
 systematic difficulties complicate performance of drug trials in lgs , including the very frequent occurrence ( often nearly uncountable ) of atypical absence seizures , the inaccuracy of parental reports of seizure semiology and frequency , the wide range of etiologies , and the evolution of seizure types over time . 
 a few randomized , double blind , placebo - controlled trials of single agents have been performed in lgs.5,7 felbamate,8 lamotrigine,9 and topiramate10 reduced the occurrence of atonic and tonic - clonic seizures in children with lgs . 
 all of these studies entailed addition of the study drug to other medications , and the studies varied considerably in their experimental design and patient selection criteria . 
 no head - to - head trial comparing more than one drug has been published . 
 even with the new generation of anticonvulsants , valproic acid is considered the most useful initial medication of choice for drop attacks , atypical absences and myoclonic seizures in lgs.5,11,12 although there are no controlled studies , valproic acid is reportedly effective against multiple lgs seizure types including atypical absence , myoclonic , and other situations in which slow spike - wave discharges are found on eeg . 
 caution must be exercised in using valproic acid in children under the age of 2 years , especially if they are receiving several other anticonvulsants , because of the risk of hepatotoxicity . in that age group , it has been recommended to try topiramate or lamotrigine first.11 felbamate could also be considered as an alternative to valproic acid , because felbamate lacks the sedative side effect seen with other anticonvulsants ( eg , topiramate , benzodiazepines ) , which exacerbates seizure occurrence . 
 owing to the risk of aplastic anemia and hepatoxicity with felbamate use , this medication must be used with caution and appropriate patient monitoring of blood levels , liver function , and hematologic indices . 
 a recent study showed that clobazam significantly reduced both drop and non - drop seizures in a dose - dependent manner in patients with lgs.13 clobazam reportedly has less sedative effects than other benzodiazepines , making it an attractive potential adjunctive treatment for lgs . 
 an animal model of lgs does not exist , hampering progress in design of therapeutics . 
 it is not surprising that there is no experimental model , since lgs comprises so many distinct seizure types and lacks a consistent underlying etiology . 
 treatment of rats with a cholesterol synthesis blocker produces atypical absence seizures with slow spike waves , providing an opportunity to study the mechanism of this specific seizure type , which appears to involve gabab receptors.14 these observations have not yet been exploited therapeutically . 
 some general treatment considerations include recommendations to use as few anticonvulsants concurrently as possible to avoid side effects from polytherapy , avoid excessive drowsiness which exacerbates several of the seizure types in this syndrome , and consider the cognitive and psychological comorbidities which result from both lgs and its treatment . 
 clearly , a multidisciplinary approach is required to address the medical and psychosocial aspects of lgs . at present , authorities recommend valproic acid as the first line medication , followed by one or two of the second - line agents ( lamotrigine , rufinamide , topiramate , clobazam , felbamate , 
 levetiracetam).5,12 if those therapies fail to achieve treatment goals , zonisamide , the ketogenic diet , vagus nerve stimulation , or corpus callosotomy can be tried ( the latter targeting drop attacks ) . of note 
 , carbamazepine and gabapentin can exacerbate some of the seizure types in lgs.11,15 the role of newer anticonvulsants such as vigabatrin and zonisamide remain undetermined in lgs . 
 rufinamide [ 1-(2,6-difluoro - phenyl)methyl-1h-1,2,3-triazole-4-carboxamide ] is a triazole derivative granted orphan drug status for the adjunctive treatment of lgs in the united states in 2004 . 
 , rufinamide was approved by the united states food and drug administration for treatment of lgs in children 4 years of age and older . 
 it is also approved for adjunctive treatment of partial seizures in adults and adolescents.16 rufinamide is structurally unrelated to other anticonvulsants . 
 its mechanism of action reportedly involves decreased firing of high frequency sodium - dependent action potentials and prolongation of sodium channel inactivation.17 enhancing sodium channel inactivation would prevent a neuron from generating subsequent bursts of high frequency action potentials . 
 however , data implicating an effect of rufinamide on sodium channels is presently published only in abstract form and , given the preclinical profile , other mechanisms of action are likely to operate.18 rufinamide has a wide spectrum of anticonvulsant effectiveness in animals including the maximal electroshock model ( for generalized tonic - clonic and partial seizures ) and the subcutaneous pentylenetetrazol model ( for clonic seizures).19 in support of the hypothesized action of rufinamide on sodium channels , seizure protection in those in vivo models is also afforded by other anticonvulsants that block sodium channel function . 
 rufinamide also blocks seizures induced by subcutaneous strychnine , bicuculline , and picrotoxin.20 rufinamide has an excellent safety profile in animals and lacks obvious cognitive side effects and behavioral toxicity at clinically relevant doses . on the rotorod test of motor coordination 
 , rufinamide had a higher safety index than phenytoin , phenobarbital , and ethosuximide.19 the drug also appears to be relatively devoid of cognitive side effects in humans . in a multicenter study of 189 adolescent and adult patients with partial seizures , 12 weeks of treatment with 
 rufinamide failed to cause deficits ( compared to placebo ) in several measures of cognitive function , including psychomotor speed and alertness , processing speed , and working memory.21 taken orally , rufinamide is well absorbed ( 85% after an oral dose).22 the absorption rate is slow and the extent of absorption decreases as the dose is increased . 
 after a single 400 mg oral dose in healthy adults , the time to maximum plasma concentration ranges from 1.5 to 10 hours with an average of about 6 hours , with a mean maximum plasma concentration ( cmax ) of 3.03 g / ml.23 rufinamide has low protein binding ( 34% ) and food does not affect the time to maximum plasma concentration or peak plasma concentration.22 the plasma half life is 6 to 10 hours and is unaffected by renal disease . 
 rufinamide is eliminated primarily via metabolism , the principal metabolite being a carboxylic acid derivative . 
 this metabolite primarily appears in the urine , and only about 2% of rufinamide occurs in the urine unchanged . 
 the low plasma binding rate of rufinamide suggests that drug  drug interactions are likely to be minimal . 
 however , in conditions like lgs , in which polypharmacy is usual , careful elucidation of drug  drug interactions is necessary . in all of the clinical trials with rufinamide , 
 , there were no significant effects of rufinamide on the plasma concentrations of valproic acid , lamotrigine , or topiramate.24 there is no study of the effects of rufinamide on felbamate concentration . at average steady state concentrations of rufinamide , 
 this comparison includes population pharmacokinetic analyses of carbamazepine , lamotrigine , phenobarbital , phenytoin , and valproic acid . 
 however , phenytoin clearance was decreased at average steady state levels of rufinamide up to 21% , suggesting that phenytoin levels should be closely monitored in patients on concurrent rufinamide . 
 the clearance of rufinamide is not significantly affected by several other anticonvulsants including carbamazepine , phenytoin , primidone , and phenobarbital . 
 however , rufinamide clearance was decreased by valproic acid with elevated rufinamide levels of up to 70%.20 therefore , valproic acid therapy should be introduced cautiously and at relatively low doses in children already on rufinamide . 
 early sponsored studies , including open label extensions , provided data that rufinamide had a beneficial effect as add - on therapy for partial seizures in adolescents and adults . for lgs , a single double - blind randomized placebo controlled trial has been published.24 this study involved 138 patients between the ages of 4 and 30 years who had lgs diagnoses for a median of 7.5 years . 
 study subjects were randomized to either oral rufinamide ( titrated up to 45 mg / kg / day over 14 days ) ( n = 74 ) or placebo ( n = 64 ) in addition to their other antiepileptic drugs . 
 this study involved a 28-day baseline followed by an 84-day parallel group treatment ( 14 days titration , 70 days maintenance ) . 
 primary end points were the percent change in seizure frequency and the parent / guardian ratings of seizure severity . 
 the investigators found a significant decrease in total seizures compared with placebo ( 32.7% vs 11.7% : p = 0.0015 ) , and tonic - atonic seizures compared with placebo ( 42.5% vs + 1.4% : p < 0.0001 ) . 
 the patients on rufinamide also had a greater improvement in seizure severity and a higher 50% responder rate compared with placebo for both total seizures and tonic - atonic seizures . 
 adverse effects in this study were modest , with 24% of treated patients experiencing sedation and 21% experiencing vomiting . eight percent of the rufinamide group withdrew because of adverse side effects . 
 cognitive or psychiatric adverse events were less common among rufinamide - treated patients than in the placebo group . 
 it was concluded that rufinamide is well tolerated and efficacious for seizures in lgs . a 3-year open - label followup study , published in abstract form only 
 , reports continued rufinamide effectiveness.25 that study included 124 patients treated for a median of 432 days at a dose of 10 to 45 mg / kg / day . 
 compared to the placebo group , there was a decrease in total seizures at all time points assessed up to 3 years , ranging from 42.6% to 79.3% . 
 furthermore , rufinamide was well tolerated over the long term , with only 12 patients discontinuing the drug because of adverse side effects ( most commonly vomiting , pyrexia , and somnolence ) . a recently published study from europe , using observational retrospective data from multiple centers , examined the effectiveness of rufinamide in children and adults with refractory epilepsy , including lgs.26 in the subgroup with lgs , 17 of 31 patients ( 55% ) had a response rate with greater than 50% reduction in countable seizures . 
 investigators found fatigue , vomiting , anorexia in 10% to 20% of patients but no serious adverse effects . 
 again , this study concludes that rufinamide is effective and well tolerated in patients with refractory epilepsy , including lgs . 
 although promising as a novel anticonvulsant in lgs , several caveats arise regarding rufinamide . in part , issues relate to the study populations . 
 as discussed above , patients with lgs are notoriously difficult to treat , are often treated with multiple anticonvulsant medications , and seizures are of several different types and difficult to monitor and count . therefore , the question arises as to what place rufinamide will hold in overall treatment algorithm for lgs . 
 however , a larger number of patients must be treated with this medication to establish its role in the treatment armamentarium . 
 the role of this new medication in relationship to prior drugs with some reported effectiveness in lgs needs to be further defined . 
 taken orally , rufinamide is well absorbed ( 85% after an oral dose).22 the absorption rate is slow and the extent of absorption decreases as the dose is increased . 
 after a single 400 mg oral dose in healthy adults , the time to maximum plasma concentration ranges from 1.5 to 10 hours with an average of about 6 hours , with a mean maximum plasma concentration ( cmax ) of 3.03 g / ml.23 rufinamide has low protein binding ( 34% ) and food does not affect the time to maximum plasma concentration or peak plasma concentration.22 the plasma half life is 6 to 10 hours and is unaffected by renal disease . 
 rufinamide is eliminated primarily via metabolism , the principal metabolite being a carboxylic acid derivative . 
 this metabolite primarily appears in the urine , and only about 2% of rufinamide occurs in the urine unchanged . 
 the low plasma binding rate of rufinamide suggests that drug  drug interactions are likely to be minimal . 
 however , in conditions like lgs , in which polypharmacy is usual , careful elucidation of drug  drug interactions is necessary . in all of the clinical trials with rufinamide , 
 , there were no significant effects of rufinamide on the plasma concentrations of valproic acid , lamotrigine , or topiramate.24 there is no study of the effects of rufinamide on felbamate concentration . at average steady state concentrations of rufinamide , 
 this comparison includes population pharmacokinetic analyses of carbamazepine , lamotrigine , phenobarbital , phenytoin , and valproic acid . 
 however , phenytoin clearance was decreased at average steady state levels of rufinamide up to 21% , suggesting that phenytoin levels should be closely monitored in patients on concurrent rufinamide . 
 the clearance of rufinamide is not significantly affected by several other anticonvulsants including carbamazepine , phenytoin , primidone , and phenobarbital . 
 however , rufinamide clearance was decreased by valproic acid with elevated rufinamide levels of up to 70%.20 therefore , valproic acid therapy should be introduced cautiously and at relatively low doses in children already on rufinamide . 
 results from randomized controlled clinical trials of rufinamide are just appearing . early sponsored studies , including open label extensions , provided data that rufinamide had a beneficial effect as add - on therapy for partial seizures in adolescents and adults . for lgs , a single double - blind randomized placebo controlled trial has been published.24 this study involved 138 patients between the ages of 4 and 30 years who had lgs diagnoses for a median of 7.5 years . 
 study subjects were randomized to either oral rufinamide ( titrated up to 45 mg / kg / day over 14 days ) ( n = 74 ) or placebo ( n = 64 ) in addition to their other antiepileptic drugs . 
 this study involved a 28-day baseline followed by an 84-day parallel group treatment ( 14 days titration , 70 days maintenance ) . 
 primary end points were the percent change in seizure frequency and the parent / guardian ratings of seizure severity . 
 the investigators found a significant decrease in total seizures compared with placebo ( 32.7% vs 11.7% : p = 0.0015 ) , and tonic - atonic seizures compared with placebo ( 42.5% vs + 1.4% : p < 0.0001 ) . 
 the patients on rufinamide also had a greater improvement in seizure severity and a higher 50% responder rate compared with placebo for both total seizures and tonic - atonic seizures . 
 adverse effects in this study were modest , with 24% of treated patients experiencing sedation and 21% experiencing vomiting . 
 cognitive or psychiatric adverse events were less common among rufinamide - treated patients than in the placebo group . 
 it was concluded that rufinamide is well tolerated and efficacious for seizures in lgs . a 3-year open - label followup study , published in abstract form only 
 , reports continued rufinamide effectiveness.25 that study included 124 patients treated for a median of 432 days at a dose of 10 to 45 mg / kg / day . 
 compared to the placebo group , there was a decrease in total seizures at all time points assessed up to 3 years , ranging from 42.6% to 79.3% . 
 furthermore , rufinamide was well tolerated over the long term , with only 12 patients discontinuing the drug because of adverse side effects ( most commonly vomiting , pyrexia , and somnolence ) . a recently published study from europe , using observational retrospective data from multiple centers , examined the effectiveness of rufinamide in children and adults with refractory epilepsy , including lgs.26 in the subgroup with lgs , 17 of 31 patients ( 55% ) had a response rate with greater than 50% reduction in countable seizures . 
 investigators found fatigue , vomiting , anorexia in 10% to 20% of patients but no serious adverse effects . 
 again , this study concludes that rufinamide is effective and well tolerated in patients with refractory epilepsy , including lgs . 
 although promising as a novel anticonvulsant in lgs , several caveats arise regarding rufinamide . in part , issues relate to the study populations . 
 as discussed above , patients with lgs are notoriously difficult to treat , are often treated with multiple anticonvulsant medications , and seizures are of several different types and difficult to monitor and count . therefore , the question arises as to what place rufinamide will hold in overall treatment algorithm for lgs . 
 however , a larger number of patients must be treated with this medication to establish its role in the treatment armamentarium . 
 the role of this new medication in relationship to prior drugs with some reported effectiveness in lgs needs to be further defined . 
 rufinamide is a novel , broad - spectrum anticonvulsant drug with promising potential for treatment of many seizure types . in particular 
 , the multiple seizure types seen in patients with lgs may be amenable to treatment with rufinamide . 
 rufinamide appears to have a good safety profile and is well tolerated with minimal expected side effects . 
 some of the most common side effects ( eg , somnolence , vomiting ) could perhaps be ameliorated by slow titration . 
 patient and caretaker satisfaction data need to be obtained . given the devastating nature of the seizures and cognitive impairments in lgs , any treatment with an even modest benefit is a welcome addition to the therapeutic armamentarium .","objective : this review summarizes the treatment of lennox - gastaut syndrome , an intractable epileptic encephalopathy of early childhood . in particular , the review focuses on rufinamide , a recently released anticonvulsant medication with reported effectiveness in this epilepsy syndrome.methods:a systematic literature search ( pubmed ) was performed to review the existing literature pertaining to the treatment of lennox - gastaut syndrome as well as studies involving rufinamide as an anticonvulsant medication.results:the published literature to date documents a beneficial effect of rufinamide on children over 4 years old with lennox - gastaut syndrome . 
 studies indicate a significant decrease in tonic and atonic seizure frequency as well as total seizure frequency compared to placebo - treated children . 
 rufinamide appears to be well tolerated and a safe medication , somnolence and vomiting being the most common side effects.conclusions:rufinamide is a promising adjunctive therapy for lennox - gastaut syndrome , an intractable childhood epilepsy . to ensure its optimal effectiveness 
 , clinicians must be familiar with the medication s clinical response profile and potential for adverse effects .",pubmed
"interpopulational variation in sexual chemical signals may provide the basis for premating reproductive isolation and speciation in many animals [ 1 , 2 ] . 
 phenotypic plasticity in sexual signals could play a key role in initial signal divergence , for example , as a way to maximize the efficiency of signals for communication in different environments [ 4 , 5 ] . 
 these differences can be later amplified by sexual selection leading to differences in mating preferences [ 68 ] , which could preclude mating between populations ( e.g. , [ 913 ] ) , and lead to speciation processes . in many lizards , intraspecific communication and sexual selection 
 for example , chemical compounds secreted by femoral gland of males can convey information about social status [ 1822 ] and genetic quality of a male [ 2326 ] . 
 also , differences in chemical signals may preclude interspecific mating between related sympatric species ( e.g. , [ 27 , 28 ] ) . 
 we hypothesized that interpopulational variations in femoral gland secretions within the same species might lead to reproductive isolation and thus promote speciation processes . 
 the iberian wall lizard , podarcis hispanica , is a small diurnal lizard , living in rocky habitats of the iberian peninsula . 
 molecular and morphological studies suggest that this lizard is paraphyletic and forms part of a  species complex ,  which suggests the existence of cryptic speciation within taxa previously considered to be conspecific [ 2934 ] . 
 chemical cues of males , mainly from the femoral gland secretions , are important in male - male interactions [ 19 , 20 , 40 ] and in female mate choice decisions [ 35 , 41 , 42 ] . also , chemical cues of females , in conjunction with coloration , elicit courtship by males . 
 this raises the possibility that p. hispanica lizards use chemical sexual signals to discriminate between populations , which might lead to reproductive isolation ( if variation of signals is discrete or there is a barrier to gene flow ) and explain the genetic and morphological differences observed between populations . in this study , we explored the role of chemical sexual signals in interpopulational recognition between five distinct populations of iberian wall lizards in central spain . in this area , 
 several populations inhabiting different environments live close together without geographical barriers that isolate the populations , and individuals may find each other easily [ 11 , 12 , 43 ] . however , some populations maintain clear distinct morphotypes and differ genetically [ 3234 ] , which suggests that they might be , at least partly , reproductively isolated . 
 we hypothesized that interpopulational variations in chemical signals could allow chemosensory recognition between populations and lead to premating boundaries . to test this , we first compared the morphological characteristics of these populations , and then we analyzed whether there was variation in the composition and proportions of chemical compounds in femoral gland secretions of males by using gas chromatography - mass spectrometry ( gc - ms ) . 
 we further conducted tongue - flick experiments to analyze whether males and females discriminated by chemosensory cues alone between scent of lizards from different populations . 
 we hypothesized that male and female lizards could be able to recognize by chemical cues alone , and maybe prefer , the scents of individuals of their own population , which may contribute to a reduced gene flow . 
 we expected that interpopulational differences in chemical signals of males and in population recognition abilities could suggest the probable existence of reproductive isolation and cryptic speciation between these iberian wall lizard populations . 
 during february - march 2008 , we captured by noosing male and female p. hispanica lizards at five localities within the madrid region ( central spain ) ( figure 1 ) . 
 three of these were localized in the northern mountain area (  fuenfra , 
 golondrina ,  and  pedrezuela  ) , and the other two were situated in the southern plain area (  belmonte  and  aranjuez  ) . 
 we selected these populations because lizards clearly differ in morphology and coloration [ 43 , 44 ] . in the north , we captured lizards from a population occupying different granite rock cliffs at the edge of a pine forest in the upper part of the  fuenfra  valley ( 4047n , 0403w ; 1750  m altitude ; 21 males and 26 females ) , on granite rocky outcrops inside a large oak forest  golondrina  near cercedilla village ( 4044n , 0402w ; 1250  m altitude ; 29 males and 27 females ) , and from old stone walls near crop fields close to  pedrezuela  village ( 4044n , 0336w ; 800  m altitude ; 19 males and 16 females ) . in the south , we captured lizards on human buildings and walls inside a public garden of the  belmonte del tajo  village ( 4008n , 0320w ; 735  m altitude ; 22 males and 17 females ) and on chalk and gypsum rocks in deforested bushy hills near  aranjuez  village ( 4002n , 0337w ; 494  m altitude ; 21 males and 32 females ) . 
 all lizards were individually housed at  el ventorrillo  field station ( cercedilla , madrid ) about 5  km from the capture sites of the northern populations , in indoor 60  40  cm pvc terraria containing sand substratum and rocks for cover . 
 cages were heated with 40  w spotlights during 6  h / day , and overhead lighted ( 36  w full - spectrum daylight tubes ) on a 10  h  :  14  h light / dark cycle , and were screened from each other using cardboard . every day , 
 lizards were fed mealworm larvae ( tenebrio molitor ) dusted with multivitamin powder for reptiles , and water was provided ad libitum . 
 lizards were returned to their exact capture sites with good health condition at the end of experiments . 
 we made the following morphological measurements of each individual lizard : body mass ( or weight ) ( measured with a digital balance to the nearest 0.01  g ) and body size ( snout - to - vent length , svl ; measured with a ruler to the nearest 1  mm ) . 
 we also made morphological measurements of the head using a digital caliper ( to the nearest 0.05  mm ) . 
 head length was the distance between the tip of the snout and the posterior side of the parietal scales . 
 head depth was the greatest distance from the highest portion of the head to the bottom of the lower jaw . 
 we also counted under a magnifying glass the number of femoral pores on the right and left hindlimbs of lizards and calculated an average number for both sides . 
 finally , we noted the number of small but distinctive and conspicuous blue ocelli that runs along each of the body sides on the outer margin of the belly of males and calculated an average number for both sides . these ocelli seem to have a role in sex recognition and intrasexual social relationships between males [ 39 , 45 ] . 
 all biometrical variables were log transformed prior to analysis to meet assumptions of normality and homoscedasticity . 
 we used one - way analyses of variance ( anovas ) to test for differences in morphological variables between populations . 
 immediately after capture in the field , we extracted femoral gland secretion of males by gently pressing with forceps around the femoral pores and collected secretion directly in glass vials with teflon - lined stoppers . 
 we also used the same procedure on each sampling occasion but without collecting secretion , to obtain blank control vials that were treated in the same manner to compare with the lizard samples . before the analyses we added 250  l of n - hexane ( sigma , capillary gc grade ) to each vial . 
 we analyzed lipophilic compounds in samples by using a finnigan - thermoquest trace 2000 gas chromatograph ( gc ) fitted with a poly ( 5% diphenyl/95% dimethylsiloxane ) column ( thermo fisher , trace tr-5 , 30  m length  0.25  mm i d , 0.25  mm film thickness ) and a finnigan - thermoquest trace mass spectrometer ( ms ) as detector . 
 sample injections ( 2  l of each sample dissolved in n - hexane ) were performed in splitless mode using helium as the carrier gas at 30  cm / sec , with injector temperature at 250c . 
 the oven temperature program was as follows : 50c isothermal for 5  min , then increased to 270c at a rate of 10c / min , isothermal for 1  min , then increased to 315c at rate of 15c / min , and finally isothermal ( 315c ) for 10  min . 
 initial tentative identification of secretion components was done by comparison of mass spectra in the nist / epa / nih 1998 computerized mass spectral library . 
 identifications were confirmed by comparison of spectra and retention times with those of authentic standards from sigma - aldrich chemical co. for unidentified or unconfirmed compounds we report here their characteristic ions , which we used together with retention times and characteristic m / z ratios to confirm whether these compounds were present in a given individual .   for the statistical analyses of femoral secretions , 
 the relative amount of each component was determined as the percent of the total ion current ( tic ) . 
 the relative areas of the peaks were transformed following aitchison 's formula [ 4749 ] . 
 the homogeneity of variance of these variables was tested with levene 's test , and bonferroni 's correction was applied . 
 the transformed areas were used as variables in a principal component analysis with varimax rotation . 
 the eight principal components ( pcs ) extracted ( all with eigenvalues > 1 , which explained 82.55% of variance ) were used as covariates in a discriminant analysis to test whether chemical compounds in femoral secretions could be used to predict the population of origin of a male lizard . then , we calculated the squared mahalanobis distances of individuals with all other individuals and compared them between populations . 
 lizards have been shown to react to a variety of chemical stimuli with increased and differential rates of tongue extrusions . 
 tongue - flick ( tf ) rate can , therefore , be used as a quantitative bioassay of detection of chemical cues ( e.g. , [ 11 , 38 ] ) . 
 thus , to test for differential responses to scents , we made comparisons of tf rate by lizards ( males and females ) in response to chemical stimuli presented on cotton applicators impregnated with scents of male or female p. hispanica from each of the five different populations ( aranjuez , golondrina , fuenfra , pedrezuela , and belmonte ) or with deionized water ( odorless control ) . 
 we obtained lizard scents from the femoral pores of males or from the cloacal area of females because these are the body areas most frequently and intensely investigated by tongue flicking during social encounters [ 19 , 37 , 39 ] . 
 therefore , after first dipping the cotton tip ( 1  cm ) of a wooden applicator attached to a long stick ( 50  cm ) in deionized water , we rolled the tip over those body areas ( of one population and sex per applicator ) . 
 first , males were exposed to scents from males and then to scents from females of each population tested . 
 trials were conducted in outdoor conditions during april , which coincided with the mating season of lizards in their original natural populations ( p. lpez and j. martn , unpublished data ) , and between 11:00 and 13:00   ( gmt ) when lizards were fully active .   to begin a trial , the experimenter slowly approached the terrarium and slowly moved the cotton swab to a position 1  cm anterior to the lizards ' snout . 
 lizards usually did not flee from the swab , but explore it repeatedly by tongue flicking or ignore it after the first tfs . in all cases , lizards directed tfs to the swab in all conditions . 
 the numbers of tfs directed at the swab were recorded for 60  s beginning with the first tf . 
 to examine differences among treatments , previous analyses showed that responses to the different scents differed as a function of the population of the responding lizard . 
 thus , we used separated one - way repeated measures anovas to test the effect of scent stimuli ( within factor ; fuenfra versus golondrina versus aranjuez versus pedrezuela versus belmonte versus water ) in number of tfs directed at the swab within each population of responding lizards . data were log - transformed to ensure normality . 
 tests of homogeneity of variances ( levene 's test ) showed that in all cases variances were not significantly heterogeneous after transformation . 
 there were significant differences between populations in all morphological measurements ( table 1 ) . in general , lizards from fuenfra and golondrina 
 populations were significantly heavier and longer and had greater heads than lizards from aranjuez and belmonte , which did not differ . 
 lizards from pedrezuela were intermediate in size between the other populations ( table 1 ) . however , when the effect of variation in body size between populations was removed , head size differences were significant only for head depth ( anova on residuals of head size with svl , p = 0.005 for both sexes ) , but not for head length ( p > 0.20 for both ) or width ( p > 0.05 for both ) . with respect to the number of femoral pores , both male and female lizards from aranjuez 
 had significantly less femoral pores than lizards from belmonte , fuenfra , and golondrina , which did not differ . 
 the number of femoral pores was not significantly related to body size ( p > 0.60 in all cases ) . finally , males from aranjuez , belmonte , and pedrezuela had significantly more blue ocelli than males from fuenfra and golondrina ( table 1 ) . 
 we found 53 lipophilic compounds in femoral gland secretions of male p. hispanica ( table 2 ) . 
 the lipophilic fraction of femoral gland secretions of males , all five populations pooled , is a mixture of steroids ( 83.69% of tic ) , and carboxylic acids ranged between n - c14 and n - c22 and their esters ( 10.30% ) , but we found also five alcohols between n - c16 and n - c24 ( 3.53% ) , a furanone ( 1.18% ) , four waxy esters ( 1.10% ) , squalene ( 0.60% ) , and two terpenoids ( 0.28% ) . on average , 
 the five most abundant chemicals were cholesterol ( 63.24% of tic ) , followed by cholesta-5,7-dien-3-ol ( 5.16% ) , hexadecanoic acid ( 3.73% ) , campesterol ( 3.66% ) , octadecenoic acid ( 2.46% ) , and octadecanoic acid ( 1.77% ) . 
 there were 34 chemical compounds shared by lizards from all populations , but we found differences between populations in the presence / absence of 19 compounds in femoral secretions ( table 2 ) . 
 the discriminant analysis showed that the eight pcs scores describing proportions of compounds in femoral secretions could be used to predict the population of origin of a male lizard ( wilks '  = 0.0001 , f32,355 = 607.45 , p < 0.0001 ) ( figure 2 ) . all the pairwise comparisons of the mahalanobis distances between populations , which ranged between 150.35 and 1015.13 , were significant in all cases ( 210.46 < f8,96 < 1290.20 , p < 0.0001 in all cases ) . 
 the number of tfs differed significantly between the scents presented in all cases ( table 3 ; figure 3 ) . in all populations , males discriminated between scents of any male and water ( tukey 's tests : p < 0.005 in all cases ) . 
 males from aranjuez and belmonte directed a significantly higher number of tfs to scent of males of their own population or of the other southern population than to scent of males from the three northern populations , which did not differ ( table 3 ; figures 3(a ) and 3(b ) ) . 
 the number of tfs directed by males from fuenfra was significantly higher in response to scent of males of their own population than to scent of males from any other population , which did not significantly differ ( table 3 ; figure 3(c ) ) . 
 males from golondrina directed a significantly higher number of tfs in response to scent of males of their own population than to males from aranjuez , belmonte and pedrezuela ( table 3 ; figure 3(d ) ) . 
 the number of tfs in response to scent of males of their own population and fuenfra males was not significantly different , and the latter was not significantly different from the rest of populations . 
 finally , males from pedrezuela directed significantly more tfs in response to males of their own population than to males of the two southern populations ( aranjuez and belmonte ) , which did not significantly differ ( table 3 ; figure 3(e ) ) . however 
 , responses to males of their own population did not significantly differ from responses to males of the other two northern populations . 
 the number of tfs differed between treatments in all populations ( table 3 ; figure 4 ) . in all cases , males discriminated between scents of any female and water ( tukey 's tests : 
 males from aranjuez and belmonte directed a significantly higher number of tfs to scent of females of their own population than to scent of females from all the northern populations , which did not significantly differ ( table 3 ; figures 4(a ) and 4(b ) ) . 
 the number of tfs directed by males from fuenfra was significantly higher in response to scent of females of their own population than to females from any other population ( table 3 ; figure 4(c ) ) . 
 males from golondrina directed a significantly higher number of tfs in response to scent of females from the three northern populations , including their own population , than to females from the two southern populations ( table 3 ; figure 4(d ) ) . 
 males from pedrezuela directed significantly more tfs in response to scent of females of their own population than to scent of females from any other population ( table 3 ; figure 4(e ) ) . 
 however , responses to scent of females from the two other northern populations were significantly higher than to females from the two southern populations , which did not differ . 
 the number of tfs differed between treatments in all populations ( table 3 ; figure 5 ) . 
 all females discriminated between scents of any male and water ( tukey 's tests : p < 0.005 in all cases ) . females from aranjuez and belmonte directed a significantly higher number of tfs in response to scent of males of their own population than to males from the three northern populations , which did not differ significantly ( table 3 ; figures 5(a ) and 5(b ) ) . 
 females from aranjuez and belmonte did not significantly differ in their responses to scent of males of their own population or to males from the other southern population ( belmonte or aranjuez ) . 
 the number of tfs directed by females from fuenfra was significantly higher in response to scent of males of their own population than to males from the two southern populations and from one of the northern populations ( pedrezuela ) , which did not significantly differ ( table 3 ; figure 5(c ) ) . 
 responses to scent of males of their own population and to males from golondrina were not significantly different , nor were different the responses to males from golondrina and pedrezuela . 
 females from golondrina directed a significantly higher number of tfs in response to scent of males from the three northern populations ( fuenfra , pedrezuela , and their own population ) than to scent of males from the southern populations ( aranjuez and belmonte ) ( table 3 ; figure 5(d ) ) . 
 females from pedrezuela directed significantly more tfs in response to scent of males from their own population than to males from all the other southern and northern populations , which did not differ significantly ( table 3 ; figure 5(e ) ) . 
 our study showed that different populations of iberian wall lizards p. hispanica living within a relatively small geographical area , whose environmental conditions differed between population sites , differed in morphology and in the composition and proportion of chemical compounds in femoral gland secretions of males 
 . males of each population secreted a singular and characteristic mixture of compounds used as sexual signals . 
 tongue - flick tests showed that these differences resulted in differential chemosensory recognition between some populations . 
 these results suggested that there could be premating reproductive isolation between some , but not all , populations of this lizard . 
 with respect to morphology , we could first differentiate between individuals from the south and north of the study area . 
 lizards from fuenfra and golondrina ( i.e. , northern populations ) were characterized by being larger , heavier , and with larger , more robust heads than individuals from aranjuez and belmonte ( i.e. , southern populations ) . 
 these differences could be explained by the different contrasting environments where these populations live , northern mountains ( with cold temperature , high humidity , and high altitude ) versus southern plains ( hot temperatures , dry conditions , and low altitude ) . 
 variations of body size of many animals , and in particular of vertebrates , are often explained by phenotypic plasticity or local adaptation to different climatic conditions , with individuals from colder environments being larger than those from warmer areas ( e.g. , ) . 
 lizards with a large body size have low thermal inertia ( i.e. , low cooling rates ) , and this may be an adaptation to increase effectiveness of thermoregulation in the northern populations where ambient temperatures are relatively cold , in contrast to the southern populations where temperatures are warmer and lizards are smaller . 
 moreover , iberian wall lizard populations differ in the number of femoral pores and blue spots , with males from the northern populations having more femoral pores and less blue spots than males from the southern populations . only lizards from the pedrezuela northern population had an intermediate number of femoral pores . 
 because femoral pores and blue spots are used in chemical and visual intraspecific communication , respectively ( e.g. , [ 37 , 39 ] ) , it is likely that the importance of these two sensory modes differ between populations 
 . a higher number of femoral pores may be related to a higher production of chemical secretions , whereas a larger number of blue spots may represent a higher use of visual signals . 
 the relative importance of chemical and visual signals may be explained by the effectiveness of these two types of communication in different environments [ 4 , 54 ] , which might have affected the evolution of sexual signals of different populations of p. hispanica lizards . 
 in fact , the chemical analyses showed that , similarly to other lizard species , femoral gland secretions of p. hispanica have carboxylic acids and steroids as predominant components ( reviewed in ) . 
 however , compounds found in femoral gland secretions of male p. hispanica varied in composition and proportions between populations , and these variations alone would allow a characterization of males from each population . 
 these differences could be due to local adaptation to the habitats of each population [ 4 , 54 ] . 
 selection for a better efficiency of substrate scent marks might have led to differences in composition of secretions of lizards inhabiting distinct environments , with less volatile and stable molecules being found in the southern populations where temperature and evaporation rates were higher [ 4 , 11 ] . 
 also , differences in secretions might be related to different diets or differently available food sources . 
 the question that arises is whether these differences in chemicals affect recognition systems and whether this may have consequences for speciation . 
 chemosensory recognition experiments showed that individuals of p. hispanica from each population could clearly detect scents of lizards from any population in comparison with an odorless control ( i.e. , water ) . 
 however , lizards showed different tongue - flick ( tf ) rates depending on the population of origin of the lizard 's scent presented . both females and males varied in their responses to scents from lizards from the different populations . 
 ( i.e. , a higher tf rate ) for scents from males from their own area ( i.e. , north versus south ) ; males from the northern populations made more tfs in response to scent of males from the northern populations than to scent of southern males . 
 similarly , southern males made more tfs in response to scents from southern males than to scent from northern males . 
 only males from fuenfra population showed a clearly higher response to scents from males of their own population . 
 for the rest of populations , there were not higher responses to scent of males from their own population , but there was a recognition of the area of origin ( north versus south ) of the male . 
 males from northern populations showed more interest for scents from northern females than for scents from southern females ; similarly this occurred in southern males . 
 however , we observed one interesting difference : males from the populations that are geographically located far away from the others ( i.e. , aranjuez , fuenfra , and pedrezuela ) showed a clear discrimination and interest ( i.e. , higher tf rates ) for scents of females from their own population against scent of females from any other population . 
 there was also a further secondary intermediate interest for scent of females from other populations of their own area and finally a lower interest for females from the other area . in contrast , for the populations geographically located in the middle of the madrid region ( i.e. , belmonte and golondrina ) , we did not observe a discrimination nor a higher interest of males for scent of females from their own population , but only a discrimination of females from their own area . 
 in addition , we found similar results for the males ' scents recognition by females . 
 females from northern populations made more tfs in response to scent of northern males than to southern males , and vice versa , but there were no differences between populations within each area . 
 we found only a clearly higher interest of pedrezuela females for scents of males from their own population against all the other populations . 
 these results seem concordant with the previous description of morphotypes of p. hispanica using morphological and genetic data [ 29 , 3234 ] . 
 thus , northern populations would be close to those described for the morphotype 1 , while the southern populations would be more similar to the morphotype 2 . 
 however , we observed a particular result for lizards from pedrezuela population ; these lizards live in the north , but they have chemical and morphological differences with respect to other northern populations . 
 moreover , the chemical signals in this population are singular in comparison to the other populations , and this chemical signature is effective in the chemosensory recognition of scent of males and female from their own population . 
 therefore , the assignation of this population to previously described morphotypes is not clear . in summary 
 , our results showed that male and female p. hispanica lizards from five distinct populations of the madrid region can recognize and discriminate between scents of individuals from the northern and southern populations , and have more interest for scents of lizards from their own area than for scents of lizards from the other area . 
 moreover , males from some populations discriminate and maybe prefer scents of females from their own population than from any other populations . this clear ability of males to discriminate between some female populations might suggest that there is a cryptic speciation process , probably mediated by the role of chemical signals in sexual interactions . however , we need further mating experiments to test this . 
 in addition , females also seem to discriminate male chemicals between areas ( north versus south ) , but not between populations . 
 all these results support that reproductive isolation between all the distinct populations of p. hispanica is not entirely clear , but that , at least between some populations , there could be reproductive isolation and cryptic speciation , which merits further studies .","interpopulational variation in sexual signals may lead to premating reproductive isolation and speciation . 
 genetic and morphological studies suggest that the iberian wall lizard , podarcis hispanica , forms part of a  species complex  with several cryptic species . 
 we explored the role of chemical sexual signals in interpopulational recognition between five distinct populations of iberian wall lizards in central spain . 
 results showed that these populations differed in morphology and in composition and proportion of chemical compounds in femoral gland secretions of males . 
 tongue - flick experiments indicated that male and female lizards discriminated and were more interested in scents of lizards from their own area ( i.e. , northern versus southern populations ) , but did not discriminate between all populations . 
 moreover , only males from the populations that are geographically located more far away preferred scent of females from their own population . 
 these data suggest that , at least between some populations , there may be reproductive isolation mediated by chemical signals and cryptic speciation .",pubmed
"the scientific activity of a group of researchers is manifested through published papers , which are validated and legitimized by their peers . 
 these papers can also serve as indicators of the degree to which their scientific knowledge in their area of research has been developed . 
 the formal means of releasing scientific publications , which include the results of research conducted at medical education institutions , is the collection of national and international scientific journals that have been indexed into databases representing various areas of knowledge.14 there are several major international databases that contain publications from scientific journals in the area of health science : medline / pubmed , which is organized by the national library of medicine and the national institutes of health ; the web of science , which is maintained by the institute of scientific information ( isi)/thomson reuters ; embase , which is an european database ; and current contents , which is also maintained by isi . in brazil , the main databases are lilacs ( latin american and caribbean health sciences ) and scielo ( scientific electronic library online ) , which is a database allowing free access to full texts of brazilian , latin american and caribbean scientific literature.5,6 among these databases , medline / pubmed ( http://www.pubmed.gov ) , which offers free access and has more than 5,100 titles , is the most often searched . 
 another important tool in the recovery of available indexed articles in databases is the controlled vocabulary known as medical subject headings ( mesh),7 which allows researchers to retrieve information using a more exact , standardized description of the document in question.810 international and national databases make it possible to search for an article using keywords as well as the authors last names , without considering the order of their authorship . in some cases , however , the recovery of articles and citations is compounded by the different ways that the names of authors or institutions can be entered.1113 compound last names , names with suffixes ( e.g. , neto , jr . ) or names which include  de  and  da  are often included in publications in a non - standardized way.14 in addition , different researchers from the same group frequently list their department and institution names in a non - standardized way . 
 small details like these make it difficult for researchers to find desired articles and for institutional evaluations to be made by the government or even by their peers.12,13 the journals selected for publication also reflect the level of productivity of a research group and , consequently , the institution to which they belong . 
 these values are calculated from the number of citations of the articles published by the journals over a two - year period.15,16 journals with high ifs are considered to be more relevant to , or influential on , the scientific field in question.17 few brazilian journals , even those that are indexed in databases , have ifs . 
 however , this number has recently increased , from 23 in 2006 to 30 in 2007 , despite the fact that the demand for the publication of english - language articles has favored a more visible role for them within the international community.18 in addition to the absolute number of publications , institutions are also qualitatively evaluated according to certain bibliometric indicators . 
 one such indicator is the assessment of the number of times that the scientific articles generated in a particular institution are cited by their peers.19 the evaluation of scientific production from a university is rather complex , because it involves different areas of knowledge , each one with its own peculiarities . 
 analysis of smaller units , like university departments , disciplines or services , can be made more easily . when the number of researchers is significantly high , a raffle system is often used to choose representatives from different functional categories , including professors and non - professors . in this current investigation , our purpose was to provide a panoramic view of publications from the  universidade de so paulo , faculdade de medicina 
 , during the period of 2001 to 2006 , by analyzing the scientific production of full professors ( level ms-6 ) at this institution and subsequently verifying their impact . 
 research into the number of publications in the medline / pubmed database by full professors of the faculdade de medicina da universidade de so paulo ( fmusp ) who were working during 20012006 was performed using a list of referred professors . 
 ( ataad , http://fmadm.fm.usp.br/attaad)20 provided the following data , in addition to names : job position , registration , department , discipline , kind of work and dates of admission and dismissal . 
 the inclusion criteria of the study were full professorship and employment by fmusp during the entire established period of time . 
 some full professors were excluded because they were dismissed during this period , mainly because of retirement , and others because they were hired through recruiting held during the same period . 
 the survey of the number of publications in medline / pubmed was first done by individually searching for the name of each author . 
 once published articles for that author had been identified , an excel spreadsheet was created to record relevant data : the kind of authorship ( sole author , main author , co - author ) ; the bibliographical references of each retrieved article according to the year of the search ; the if of each retrieved journal ; and the citations generated by each of the published homonyms that had been used in publication , which were determined by a search of the lattes curriculum records for each full professor . 
 in addition , complete articles that were retrieved on the internet were confirmed . after compiling the list of articles by each professor that have been indexed in the medline / pubmed database , the if of each scientific journal was determined , using the journal citation report ( jcr , 2006 edition ) . 
 finally , the number of citations by the authors peers that have been generated by these published articles was calculated using the march , 2009 update of the web of science database . 
 there were 66 full professors at the fmusp during the period between 2001 and 2006 , of whom 57 ( 86.4% ) were males and only 9 ( 13.6% ) were females . 
 we verified that 36.4% of these professors were teaching full - time ( 40 hours ) , that 60.6% of them were working intermediate shifts ( 24 hours ) , and only 3% were working partial shifts ( 12 hours ) . 
 our survey of the journal databases revealed 1,960 bibliographic records that were published in 630 international and national scientific journals . 
 the number of published articles for each year that were retrieved on medline / pubmed are provided in table 1 , with 31.3% overall being published in national journals and 68.7% in international journals . 
 there was a gradual growth in the scientific production of the researchers during these years , with an average publication rate of five articles per year by each professor increasing from 3.8 in 2001 to 5.9 in 2006 . 
 with regard to the authorship of the articles , only 2% ( 31 ) of the published articles had the professors as sole authors , and 5% ( 103 ) had them as first authors . 
 the fmusp professors were co - authors for the majority of publications ( 93% ) , which included partnerships with other researchers from the institution . 
 of the total number of articles that were published with other collaborators , 62.3% had 4 to 7 different authors . of the 630 journals in which the articles were published , only 103 ( 16.4% ) did not have ifs listed in the 2006 edition of the jcr . just considering those articles published in international journals ( 1,347 ) , 
 taking into account all of the articles , 47.7% ( 935 ) were printed in journals with ifs between 1 and 5 . 
 in addition , 6.4% of the articles were published in journals with ifs between 5 and 10 , which represented 8.2% of the journals , and 1.5% of the articles were published in journals with ifs between 10 and 52 , representing 1.7% of the journals ( table 2 ) . with regard to those articles published in brazilian journals ( 613 ) , 127 ( 20% ) were published in four journals with assigned ifs : brazilian journal medical biological research ( if = 1.075 ) , arquivos de neuropsiquiatria ( if = 0.400 ) , memrias do instituto oswaldo cruz ( if = 1.209 ) and revista de sade pblica ( if = 0.343 ) ( table 3 ) . until 2004 , 
 the journal arquivos brasileiros de cardiologia accounted for the greatest number of published articles ( 90/613 ) , followed by revista do hospital das clnicas da faculdade de medicina usp ( 65/613 ) . starting in 2005 , 
 the journal revista do hospital das clnicas da faculdade de medicina usp , which had its title changed to clinics , accounted for 21.4% of the articles published in brazil ( 66/613 , table 3 ) . 
 the qualitative analysis , which was based upon the number of times that an article was cited by peers , was performed using the web of science database . 
 almost half of the total published articles ( 45% ) were cited , which amounted to a total of 9,335 citations . 
 these citations were not only found in articles published in brazilian journals , in which 9% of the fmusp articles generated 367 citations , but also for articles published in international journals , in which 91% of the fmusp articles generated 8,968 citations . 
 we verified that 27 of the 1,960 published articles ( 1.4% ) were cited more than 50 times by their peers , as ( table 4 ) . 
 the present investigation aimed at providing an overview of the scientific production of the faculdade de medicina da universidade de so paulo , with a focus on a specific group of faculty members with high - level positions at the university , who were the full professors ( level ms-6 ) . 
 the use of only one group of professors in this study provides results that can later be confirmed for the other research professors and non - professors of the university . 
 this group was selected to be representative of the other professors , taking into consideration the level of scientific maturity achieved and especially the possibility of collaboration with other members of the same institution , as well as other national and international institutions . 
 analysis of the publications by the 66 full professors on staff during the period of 2001 to 2006 revealed a total of 1,960 published articles . 
 this number represents 0.04% of worldwide scientific publications , 2.4% of national publications , 4.3% of the all - time total publications of the fmusp and 12.33% of the total publications of the fmusp during the same period.21,22 of these articles , 68.7% were published in international journals , and 31.3% were published in brazilian journals . 
 the obligatory use of the english language in a number of the brazilian journals that published the professors articles has led to a better visibility and accessibility of research results . 
 few brazilian journals have ifs available , and only 4 of the 24 journals represented here had ifs . 
 this number , however , represents 17.39% of the total number of brazilian journals that have assigned ifs ( 2006 jcr ) . 
 there is , unfortunately , little data from the fmusp that would enable a comparison of the results of this study . in a paper published in 2005 , the scientific production that was generated by the laboratrio de investigao mdica 
 do complexo hospital das clnicas da faculdade de medicina da universidade de so paulo was evaluated . 
 the doctors , not all full professors , who represented the 62 laboratories in the hospital complex at that time published a total of 399 articles in 2004.23 a comparison of the number of publications by full professors during the same period indicates that , although the number is small ( 326 ) , it can serve as an estimate of the number of publications of the institution over a certain period . 
 although the estimation of scientific productivity was possible for full professors , it is interesting to note that these estimates can be lower than those that were actually generated by the institution of interest . 
 this type of data should be evaluated using different categories in order to make comparisons with other groups of professors . during this time period , 
 this group of full professors had an average publication rate of five published articles per year per professor . 
 records found in the  anurio estatstico da universidade de so paulo , 200722 indicate that the productivity of professors and researchers from the fmusp during the period of 2003 to 2007 was 3.1 articles per teacher per year . 
 if we consider that the number of publications per year per teacher has remained almost constant , our study shows a higher number of publications per year per professor than what was expected from the total group of teachers at the institution . 
 approximately 89% of the articles were published in international journals with assigned ifs , which provides high worldwide visibility . 
 however , the analysis of these ifs should take specific research areas into account , in addition to the kind of publication , because some areas have a greater number of researchers in comparison to others . 
 basic disciplines have a great advantage over specialized ones , in that the citation practices and publications are more focused.24 considered as a whole , the articles counted in this study were published in journals with ifs that varied from 0.063 to 51.296 . for 20.7% ( 406 ) of the articles , the ifs varied from 3.015 ( am j cardiol ) to 51.296 ( new engl j med ) . 
 for example , research in the dentistry area carried out in 2006 revealed that a large portion of dentistry publications occurred in journals with ifs varying from 0.692 to 1.569.13 with regard to citations of these published articles , 31.3% of the citations occurred in brazilian journals and 68.7% in international ones , which resulted in a total of 9,335 citations . 
 it is noteworthy that 20 of the evaluated articles received more than 70 citations . in a recent publication of 
 faculdade de medicina da universidade de so paulo ,  the collective publications of the fmusp were reported to be among the 100 most cited in the world.25,26 despite some limitations , the bibliographic record data in this study can be useful in analyzing the scientific production of a group of professors from the fmusp and for establishing a basis for comparison with other institutions in brazil . in addition , this data could possibly be used to evaluate the output of scientific production in the entire field of health .","introductionthe scientific production of institutions of higher education , as well as the dissemination and use of this published work by peer institutions , can be assessed by means of quantitative and qualitative measurements . 
 this type of analysis can also serve as the basis of further academic actions . 
 variables such as the type of evaluation , the number of faculty members and the decision to include or exclude researchers who are not professors are difficult to measure when comparing different schools and institutions.objectivesthe purpose of this study was to assess the scientific production of tenured faculty from the universidade de so paulo , faculdade de medicina performed from 2001 to 2006.methodsmedline/pubmed database was considered and the impact factors ( ifs - journal citation report , 2006 ) and the number of generated citations ( web of science / isi thomson ) were also evaluated.resultsthe analysis of the scientific production of 66 full professors ( level ms-6 ) revealed 1,960 scientific articles published in 630 scientific journals , of which 31.3% were brazilian and 68.7% were from international sources . among these , 47% of the articles were published in 62.9% of the journals with ifs above 10 , although 16.4% of the journals did not have assigned if values . 
 we verified that 45% of the published articles received 9,335 citations ( average of 11 + 17 ) , with the majority of these ( 8,968 citations ) appearing in international scientific journals.conclusionsour results indicate that it is possible to analyze the scientific production of a learning institution by the number of papers published by full professors , taking into account not only their academic position and influence , but also the fact that publication is an opportunity to stimulate joint projects with other members of the same institution .",pubmed
"according to the who experts , if rubella infection occurs during the early stages of pregnancy , the potential of passing the virus through the placenta and reaching the fetus is about 90% . 
 the most important complication of rubella is  congenital rubella syndrome ( crs ) ,  which directly relates to lack of immunity during pregnancy . on the other hand , by vaccination of all the susceptible people or women in reproductive age , or the susceptible girls at the time of marriage and induce immunity in them , it is possible to prevent the disease in mothers and its possible serious complications in their fetus . 
 so the primary purpose of rubella vaccination is to prevent congenital infection with rubella virus and crs , which affects an estimated 110,000 infants each year in developing countries . the disease is spread globally , and before vaccination era at the northern hemisphere the maximum of its attacks had been between february and april in the countries without effective policy for childhood vaccination or those countries where the vaccination policies only targeted the females , still rubella is endemic and in winter and early spring it causes some outbreaks . in the closed populations such as military camps or prisons , 
 however , in those who had suffered from this disease or vaccinated before , the possibility of infection is minimized . 
 the disease is usually rare in infants born from igg positive mothers due to antibody transfer and they will be immune for the first 46 months of their life . in general , the spread of the disease among the children has been seriously decreased , by extensive vaccination . 
 the most commonly used vaccine globally is derived from the ra27/3 virus strain , which makes more immunity in comparison with other vaccines and the immune response is more similar to the immunity after natural infection and one dose induces seroconversion in  95% of vaccinated persons . a variety of seroepidemiological studies have been done globally to evaluate the situation of protective antibodies and need assessment to vaccinate against rubella virus . 
 for example in australia , the rubella vaccination was established for the schoolgirls since 1971 and the crs was significantly decreased thereafter . in denmark , 
 the vaccination policy was optional for the schoolgirls from 1974 , but in 1987 it was replaced by extensive vaccination with two doses of mmr . 
 nine years later , the anti - rubella antibody rose to 96.5% in vaccinated people . 
 a seroepidemiological study in chahar mahal - va - bakhtiari province ( western iran ) , on 1347 urban and rural females using the hemoagglutination inhibition ( hi ) method , indicated 90.5% immunity against rubella . in another study in tabriz ( north - western iran ) , 
 the rubella level of immunity was 85.83% . in a study made on 400 high school girls in ghaem shahr ( northern iran ) in 1997 , and using the elisa method , 90.75% had immunity to rubella and 9.25% were susceptible . during the study in 1999 on 200 female university students in kordestan ( western iran ) 
 , only 14.9% of them did not have anti - rubella igg . during a study made in 1999 in kermanshahi girls at marriage , 41 out of 240 studied cases 
 did not have protective antibody and so about 20% of the referred population were susceptible , and it was then prescribed that screening for them shall be done before marriage and in susceptible cases ; they shall be vaccinated against rubella . 
 it should be mentioned that due to these studies , finally the health plans policy makers and authorities concluded to add the rubella and mumps vaccines to the iranian national vaccination plan and remove the worry resulted from the maternal rubella . so that this plan was implemented in 2003 , and almost all of the 5 to 25 age group of iranian ( 98% ) have been vaccinated , and thereafter , the mmr vaccine was contained in the country vaccination program instead of measles vaccine alone and since then the trend associated with the studies on rubella seroepidemiology was changed to compare the situation of rubella protective antibody before and after vaccination . 
 the national immunity of measles  rubella program was commenced at december 2003 , with the target of vaccination of 33,579,082 people of 5 to 25 years old iranians and 98% of the target population were vaccinated . such successful 
 proceeding resulted in a decrease in the appearance of measles and rubella for less than one case per million . 
 the main goal of this survey was to evaluate and compare the susceptibility and resistance of kermanshahi girls at marriage , before and after mass rubella vaccination . 
 the samples were randomly selected from population referred to the main marriage consultancy center in kermanshah city , west of iran . 
 about 10,000 girls in kermanshah refer to rafatieh health centre for their premarriage medical tests , annually . 
 since it was expected that the immunity rate after rubella vaccination to be increased from 80% in 1999 to more than 90% in 2006 , the sample size of 140 was calculated using the epiinfo software and 95% confidence . 
 all blood samples were taken and tested under similar conditions and by the same person at the reference laboratory . for quality assurance , about 10% of the samples 
 the results of this study were compared to those of the previous similar study conducted by the main researcher in 1999 with similar design at the same place but unvaccinated girls . 
 age distribution indicated that the majority of the study population ( 35% ) was 2024 years old . 
 their average age was 21.95 ( sd = 3.64 ) , and the youngest and oldest were 15 and 30 years old , respectively . 
 with respect to educational level , 20.14% of the samples were illiterate , 19.29% were in primary school , 20% in guidance school , 32.14% in secondary school , 5% have college degree , 11.43% were bachelor , and 2.86% were not identified . 
 a sum of 19.29% of cases was living in rural and the rest ( 80.71% ) in urban areas . 
 positive antibody , immunity against rubella , was seen in 99.3% ( 139 out of 140 vaccinated girls , compared to 79.6% ( 191 out of 240 unvaccinated girls ) [ figure 1 ] of the previous study . 
 therefore , we are faced to a significant rise of 19.7% ( 95% ci 12.7%26.7% ) of seropositivity and immunity against rubella in vaccinated versus unvaccinated girls ( z = 5.481 , p < 0.001 ) . 
 situation of anti - rubella antibody and immunity in unvaccinated ( left ) and vaccinated ( right ) kermanshahi girls at marriage , 2006 
 this study revealed that mass vaccination of rubella caused a highly significant decrease in the rate of negative antibody from 20.42% to 0.7% . 
 the finding is in line with reliable sources and extensive country study , which has compared immunity rate before and after iranian 2003 mass vaccination . 
 the result also agrees with the research conducted in mashhad ( east of iran ) . in addition , our study showed higher immunity improvement than those reported from shahre - kord and hamadan cities in the west of iran . during an extensive study made by immunology department researchers on 1217 serum samples before vaccination ( on 324 susceptible and 893 immune persons ) , and 2007 serum samples after vaccination ( 24 susceptible and 1983 immune ) in 5 to 15 years age group fulfilled in ten provinces , it was identified that the immunity rate of the prevaccination was raised from 73.4% to 98.8% of the postvaccination . in another study in mashhad , 
 the level of immunity before and after vaccination has been 70.38% and 98.5% , respectively . during a study made on 150 shahr - e - kord medical university students 
 , it was identified that 129 ( 86% ) of them were immune before vaccination , while it increased to 96% after vaccination . during a study at hamadan health center , 
 the level of anti - rubella antibody in hamadani girls at marriage had been 83.5% , while two years after such program it has increased to 98.4% [ table 1 ] . 
 the effectiveness of the ra 27/3 vaccine has been demonstrated by the elimination of rubella and crs from the western hemisphere and by the several european countries that have achieved and maintained high vaccination coverage with vaccines containing ra 27/3 . 
 mass rubella vaccination in 5 to 25 years old iranian population during 2003 , and continued thereafter as mmr in national vaccination program , has significantly resulted in increase in anti - rubella antibodies and immunity in high - risk people , as premarriage kermanshahi girls , and in comparison to many of the results specified in local and international sources , and due to the susceptibility of less than 1% of the kermanshahi girls at marriage , there is no more need to screen the vaccinated girls at marriage , which was emphasized in 1999 ; however , the screening studies is recommended every few years . 
 as the dispersed studies associated with the rubella seroepidemiology in different locations of the country resulted in awareness of lack of immunity of about 20% of the most vulnerable society members , i.e. , the pregnant women , and after that the policy makers decided to announce the rubella vaccine as the compulsory state vaccines , it is proposed such studies to be made for the immunity status against tetanus , diphtheria , etc . additionally , due to the fact that neither rubella infection nor vaccination result in life - long immunity , it is proposed that once each couple of years the studies associated with the rubella seroepidemiology to be performed in different regions of the country and if necessary , the vaccination of vulnerable society members be made , like in 2003 .","background : rubella is a mild viral infectious disease , usually occurs subclinically without a serious complication . 
 but if occurs during pregnancy , specially the first trimester , it can transmit and invade to fetus causing congenital rubella syndrome or fetal death . in 2003 , 
 nearly all iranians at the age of 5 to 25 years old were vaccinated against rubella and the vaccine was included in the compulsory immunization , mmr afterwards.methods:this study was carried out on girls attended at a marriage consultancy center in kermanshah city ( west of iran ) in 2006 . considering 95% of confidence , the sample size was estimated at 140 . 
 all samples were tested using elisa serologic method and compared with similar study conducted on 240 unvaccinated girls at the same place in 1999.results:antibody positivity was observed in 99.3% , compared to 79.58% of the previous study . a rise of 19.72% ( 95% ci 12.7%-26.7% ) of seropositivity 
 was observed in vaccinated girls versus unvaccinated ones ( p < 0.001).conclusion : in our previous study , we had recommended to screen for susceptibility to rubella before marriage , which is no longer required since more than 99% of vaccinated girls showed immunity at the time of marriage . 
 however , as sustainability of immunity after rubella vaccination is usually less than immunity due to illness , we recommend screening for rubella protective antibody every few years .",pubmed
"with the advent of cell type specific molecular markers , description and analysis of developmental morphogenesis has become possible in a number of biological systems . 
 gene - specific rna probes are now extensively used as they enable visualization of gene expression patterns and thus provide valuable information regarding the role of specific genes during development , as well as regarding positioning and movement of a particular cell type at different stages of development . 
 this technique , known as whole mount in situ hybridization ( wish ) , was initially performed using radioactive probes , but was radically simplified when nonradioactive probes were used successfully [ 24 ] . 
 it is now widely used in several biological systems , including drosophila , xenopus , quail , dictyostelium , and zebrafish . in situ hybridization ( ish ) 
 is a quite common technique among developmental research labs , yet its uses are quite diverse and include medical and prenatal diagnostics [ 916 ] . not long after its introduction 
 , protocols using two different labels for the rna probes and two color substrates were described , allowing the detection of more than one transcript simultaneously . however 
 , a major limitation of these chromogenic multilabeling techniques is that the overlapping regions of expression are very difficult to discern . 
 the use of fluorescent methods for detecting transcripts may overcome this limitation ; yet fluorophores for single- as well as double - transcript visualization   in whole mounts have only been used extensively in the fly and to some extent in the zebrafish . 
 more recently , simultaneous fluorescent detection of three transcripts was reported in the chick , and multiplexing has been used to detect up to seven transcripts in the fly [ 1921 ] . however , successful implementation of current fluorescent protocols is very rare , especially in the mouse or xenopus embryos , and even then the fluorescent images are of insufficient quality and thus can not rival the staining obtained with normal chromogenic substrates .   in addition , imaging the signal is complicated due to the strong autofluorescence of the embryos , which interferes with fluorescent detection of rna and makes the use of enzymatic amplification necessary in the mouse , xenopus , chick , and other embryos . 
 the amplification step is also necessary in the case of chromogenic protocols ; however , this step undermines the resolving ability of both methods . 
 amplification reactions create a precipitate which is deposited in the area surrounding the rna and then diffuses out . 
 it therefore does not remain localized within the particular intracellular region or compartment where the rna in question is localized . 
 in fact , it often diffuses throughout the cell and sometimes even leaks outside of the cell being labeled . 
 this problem is less pronounced in the case of tyramide amplification , where the peroxidase reaction produces tyramide radicals that react covalently with proteins at the site of the reaction , reducing the diffusion radius appreciably [ 24 , 25 ] . however , even in this case , the resolution is still limited by diffusion and depends on the time that the reaction is allowed to proceed . to combat diffusion issues , direct labeling of the probe is required , as this allows single cell , as well as , intracellular resolution of the localization of a particular mrna . 
 in addition to improved resolution , direct labeling of the probe allows simultaneous hybridization of multiple mrnas , as long as fluorophores can be spectrally resolved . 
 it is also extremely simple and can be quantitative since the fluorescence of the probe can be calibrated . despite successful implementation of direct labeling of messenger rnas in cell culture 
 [ 26 , 27 ] , this has not been possible in embryos due to the low fluorescent intensities of organic fluorophores . 
 the use of fluorescent methods for detecting transcripts is highly advantageous compared to chromogenic methods , especially because it enables higher quality three - dimensional imaging , multiplexing different rna species , and covisualization of rna with proteins . 
 as already mentioned , the major limitations preventing widespread use of fluorescence detection for wish are the high endogenous background fluorescence of many embryos , as well as the limited brightness and photostability of organic fluorophores . 
 a new type of inorganic fluorophore , namely , quantum dots ( qds ) have been used recently in several systems in vitro for detection of proteins as well as in vivo for protein labeling and lineage tracing [ 2842 ] . 
 qds were also used for in situ hybridization in clinical biopsies for the detection of multiple mrnas with successful conjugation to oligonucleotide probes . 
 qds have ideal optical properties for use in biology like strong fluorescent signal emission compared to organic and protein fluorophores [ 44 , 45 ] . 
 in addition , due to their longer excited state lifetime , their fluorescence can be separated from the background fluorescence with time - domain imaging . 
 using qds offers a number of other advantages over organic fluorophores including wide excitation spectra ( which makes the use of a single excitation filter possible ) , narrow and tunable emission spectra ( which reduces spectral overlap making the simultaneous use of more colors possible ) , large separation between the excitation and emission ( which increases the detection sensitivity ) , and resistance to photobleaching [ 47 , 48 ] . 
 their unique optical properties made qds an ideal candidate for detecting multiple mrnas in ish protocols , and their high fluorescence intensity raised the possibility of using them for rna detection in whole embryos . 
 previous efforts to do this met with aggregation issues and were time consuming since detection had to be done with rna covalently linked to hydroxylated qds . since then , we and others have reported new methods of creating hydrophilic qds and hydrophilic qd conjugates have become commercially available [ 32 , 38 , 51 ] . 
 studies have also pointed out difficulties in using qds for fish experiments [ 52 , 53 ] , including steric hindrance , degradation of qd conjugates and adherence to tubes and tips .   in this work 
 we show that the greatest limitation of these nanocrystals is penetration , since commercially available qds are quite large and therefore fail to penetrate the many cell layers of an embryo or are significantly trapped if they do . 
 however , we have determined that proteinase k can render xenopus embryos sufficiently permeable to allow qd penetration deep within embryonic tissues . 
 more specifically , we produced fluorescein- ( fitc- ) , biotin-  , and digoxigenin ( dig ) labeled rna probes and used qd - antibody and qd - streptavidin conjugates to visualize them . 
 our experiments show that this is an extremely sensitive assay that significantly improved rna detection sensitivity . 
 we then employed qds to visualize several rna probes that had been used to perform wish in xenopus embryos . 
 we demonstrate that qd detection of endogenous messenger rnas is effective and that it can be used to provide wish data of higher resolution than current techniques . 
 finally , we show that qds can be used to carry out two - color in situ hybridization simultaneously . 
 therefore , the use of qds to perform nonamplified fluorescent whole mount in situs in xenopus embryos , one of the most highly autofluorescent ( and thus demanding ) vertebrate developmental organisms , suggests that qd whole - mount in situs will find successful applications in most developmental models . 
 xenopus laevis embryos from induced spawning   were staged according to nieuwkoop and faber . 
 xenopus embryos were fertilized in vitro and dejellied using 2% cysteine - hcl , ph 7.8 , then maintained in 0.1x marc 's modified ringer 's ( mmr ) . 
 xenopus laevis embryos were fixed in 3.7% formaldehyde in memfa ( 2 hours at room temperature ) , and the vitelline envelope was manually removed with forceps . 
 permeabilization of embryos was carried out several ways : ( 1 ) overnight in 1x pbs supplemented with 0.5% triton , and 1% dmso ( pbdt ) , ( 2 ) overnight in 1x pbs supplemented with 5% triton and 1% dmso , ( 3 ) two hours in 1x pbs supplemented with 0.2% sds , and ( 4 ) for four hours in 1x pbs supplemented with 0.2% sds or ( 5 ) for 25 minutes in 10  g / ml proteinase k. embryos were then blocked for 2 hours in 1x pbs with 0.5% triton , 5% bsa , and 1% normal goat serum . 
 embryos were incubated with biotin - conjugated phosphotyrosine ( anti-4g10 , millipore ) antibody overnight at 4c at a dilution of 1  :  500 ( in block solution ) . 
 embryos were then washed ( 3  10  min ) in pbdt and incubated for 2 hours at room temperature with streptavidin conjugated cy3 or qds 655  nm at 1  :  500 dilution in fresh block solution . 
 after incubation , embryos were washed in pbdt ( 3  10  min ) . for negative control experiments , 
 clearing of embryos was performed by immersion of the embryos in two parts benzyl benzoate and one part benzyl alcohol after methanol dehydration ( murray 's clearing medium , bb  :  ba ) . 
 the refractive index of bb  :  ba closely matches the refractive index of yolk thereby rendering xenopus embryos nearly transparent . 
 the embryos were imaged on a zeiss axio imager z1 using a zeiss axiocam mr3 , the axiovision software 4.7 . 
 antisense digoxigenin - ( dig- ) , biotin-  , and fluorescein - ( fitc- ) labeled xbra ( in cs2++ ) , edd , myod ( in cs2++ ) , amylase ( in pcr4blunt - topo ) , xa-1 ( in pbsk+ ) , cardiac actin ( in psp64 ) , and ltbp1 ( in cs2++ ) probes were synthesized by in vitro transcription from linearized plasmid using rna polymerase sp6 or t3 and ribonucleotide mixture which results in rna transcripts containing bio - utp , fitc - utp , or dig - utp ( roche ) . 
 the manufacturer 's protocol was followed with a modification in the total reaction volume which was scaled down to 20  l . 
 biotin-  , fitc-  , and dig - labeled rna probes ( transcribed as described above ) were used to perform in situ hybridization using the protocol reported by harland . with some modification 
 methanol was substituted with ethanol and 4% paraformaldehyde in pbs was used to fix the embryos instead of formaldehyde . 
 after proteinase k treatment ( 5  min for chromogenic wish and 25 minutes for qd - based wish ) embryos were blocked with 0.1% bsa , 10% sheep serum in 0.1% tween in 1 x pbs(pbt ) solution , and then washes were performed in pbs . after blocking 
 , embryos were refixed for one hour in 4% paraformaldehyde followed by prehybridization at 65c . for chromogenic wish experiments , 
 the original protocol was followed . however , for qd - based fluorescent wish experiments the protocol was modified . 
 after the last 0.2x ssc wash at 65c the embryos were blocked with 1 x pbs + 0.1% bsa + 0.1% tween for one hour and then transferred to a new vial which contained 1  ml of a 1 :  500 dilution qdot - streptavidin 705  nm , qd - anti - fitc or qd - anti - dig 655  nm ( invitrogen ) conjugates in blocking solution . 
 the addition of 0.1% bsa and 0.1% tween into the incubation buffer significantly improved penetration and decreased background without appreciably affecting the qd colloidal stability or the signal intensity . 
 after the incubation , the embryos were washed in pbt ( 4  30  min ) at room temperature . 
 embryos were then cleared in bb  :  ba ( as described above ) . 
 the fluorescent signal remains localized after clearing , and this allows data acquisition from different planes within the embryo without the need for sectioning . 
 the embryos were imaged on an upright zeiss fluorescent microscope with a zeiss axiocam and the axiovision 4.7 software ( using a custom filter set ; excitation 300460  nm , emission 500  nm longpass , dichroic 475  nm ) . when detecting weak signals , the axiovision software allowed white balancing of the camera so that that the green background ( in embryos viewed with a 420 long pass filter due to the bias of the autofluorescence towards shorter wavelengths ) appears white , resulting in a major boost of the qd signal ( 605 peak emission ) , decrease of the threshold of detection and significantly better contrast . 
 if the calibration of the camera is done properly , control embryos that are not labeled with qds appear completely white under uv excitation without any traces of red . 
 this color separation method has to be performed carefully and control embryos need to appear white otherwise the risk of generating false staining increases significantly . 
 due to the fact that the embryo has several distinct regions where the background fluorescence changes not only in terms of intensity but also in terms of spectral balance the region chosen for assignment of  white  was the region in which the background had the longest average wavelength . in this manner we ensured that long - average wavelength background regions would appear white , and shorter average wavelength regions 
 we first wanted to examine whether qds could penetrate xenopus embryos to a sufficient depth so as to allow specific deep tissue staining . 
 we initially compared streptavidin - conjugated qds to streptavidin - conjugated cy3 in whole - mount immunostaining experiments for their ability to detect a biotinylated antibody against phosphotyrosine . 
 this antibody was purposely selected due to its strong and specific staining pattern of cell - cell boundaries that allows easy visual confirmation upon successful labeling . 
 not surprisingly , the staining pattern obtained using qds was very similar to the one obtained using cy3 in the superficial cell layer of the embryo ( upper area of figure 1(a ) showing superficial cells of the fin of a tadpole and data not shown ) . however , and in contrast to cy3 , qd staining could not be detected beyond the first cell layer indicating that qds encountered penetration issues , most likely due to size restrictions ( figure 1(a ) lower area showing the somites of the tadpole ) . 
 several approaches were employed to increase qd embryo permeability , including the use of harsh detergents like sds , with limited success . however , use of proteinase k treatment enabled penetration of qds into deep tissues and resulted in specific deep tissue staining ( figure 1(b ) and table 1 ) . 
 however , proteinase k treatment is not suitable for use with most whole - mount immunostaining antibodies as it may lead to degradation of the target antigen . 
 it should be noted that the best results were obtained using newly opened qd - streptavidin conjugates from invitrogen . 
 unfortunately though , the performance of these conjugates was batch dependent and significantly diminished if qd - streptavidin conjugates were stored for more than two - three months , despite a six - month shelf life stated by the manufacturer . 
 in addition , it was evident that use of qds with emissions in the nir ( 700 and 800  nm ) provided a big improvement in detection sensitivity due to a significant reduction of tissue autofluorescence in this region of the spectrum . however , commercially available qds with peak emissions in these wavelengths are quite large and suffer from even greater permeability problems . 
 consequently , there is an increased need for more reliable and smaller nir qds to become commercially available . 
 given that proteinase k treatment facilitated qd penetration in xenopus embryos and allowed specific deep tissue staining in whole - mount immunostaining experiments , we wanted to determine whether qds could be used in wish experiments in which proteinase k treatment is a standard permeabilization approach . 
 we initially tested whether anti - digoxigenin- conjugated qds could detect a dig - labeled rna probe specific for xenopus latent transforming growth factor  binding protein 1 ( ltbp1 ) . 
 as shown in figure 1 , anti - dig qds ( see figures 1(c ) and 1(d ) ) gave a similar staining pattern of xenopus ltbp1 to the published pattern obtained using the same probe but developed using a chromogenic reaction . 
 ltbp1 signal was detected in the head region including branchial arches and around the eye ( shown as inset of figure 1(c ) ) and the somites in agreement with the published expression pattern . 
 furthermore , qd - labeling of ltbp1 exhibited high signal intensity and resolution confirming that a regular fluorescence microscope is sufficient for detection ( see figures 1(c ) and 1(d ) . to further establish that the qd in situ protocol is capable of successful and specific staining , we sectioned and imaged whole - mount qd - stained embryos . 
 as shown in figure 2(a ) , qds can penetrate deep into the embryo following proteinase k treatment and stain structures independently of their proximity to the free qds in solution . 
 importantly , the use of qds for in situ staining could result in more accurate determination of transcript expression patterns . 
 figure 2(b ) shows a series of optical section images taken from a z - stack movie , using a fluorescence microscope , starting at the head region and moving posteriorly . 
 as indicated , qd labeling survives the clearing protocol used to render xenopus and other embryos transparent . 
 more importantly , however , is that the optical sections demonstrate that the staining does not originate from the notochord as originally published , but rather from the somitic mesoderm flanking the notochord . 
 therefore , the additional spatial cues provided by optical sectioning can lead to a more accurate determination of an expression domain , further emphasizing the advantages of qd in situs . 
 the above results suggest that use of anti - dig conjugated qds in wish can give highly specific staining of dig labeled mrna probes , even in deep tissues . to further determine the utility of this approach we tested whether qds could also label alternative modified oligonucleotide probes through biotin - streptavidin and fitc- anti - fitc interactions , using a similar protocol . 
 figure 3(a ) depicts in situ staining performed on a dissected xenopus tadpole gut , against a biotin labeled amylase probe using either streptavidin - conjugated qds or the chromogenic reaction . 
 it is evident , from the images , that similar staining patterns are obtained ; both the qds and the chromogenic staining were restricted to the pancreas , where amylase rna is expressed . 
 non - stained areas appear white due to background fluorescence that is present in all visible wavelengths . despite high background 
 , we obtained a good signal to noise ratio ( sufficient to allow clear visualization and delineation of the expressing region ) and excellent contrast in the most highly autofluorescent organ of the xenopus tadpole . 
 we went on to test three well - known mrnas , namely , ( a ) endodermin ( edd : a pan - endodermal marker ) , ( b ) xbra ( an early mesodermal marker ) , and ( c ) myod ( a gene encoding a dna - binding protein that can activate muscle gene expression ) , which stain the gut , the mesodermal belt , and the muscle somites , respectively . as indicated in figures 3(b ) and 3(d ) , the use of qds in in situs gives staining patterns that closely match the ones obtained using standard enzymatically amplified chromogenic reaction methods , while maintaining high resolution . 
 the degree of resolution , however , varies with the transcript of interest and its respective expression pattern . 
 for example , in the case of myod ( figure 3(b ) , fitc - labeled probe used ) , the posterior somites look fused when using the chromogenic protocol but are clearly distinct when using qds . in contrast 
 , qd staining of a biotin - labeled probe against edd , which is expressed in the highly autofluorescent gut appears weak except at the anterior , where the gene is expressed at higher levels ( figure 3(c ) ) . 
 alternatively , in the case of xbra ( figure 3(d ) ) , the staining of the chromogenic and the qd ( against biotin - labeled probe against xbra ) in situ is almost identical . 
 hoechst was used to counter - stain the nuclei blue in this experiment , and the cleared embryo was visualized from the animal pole . 
 the fact that all three common modifications of rna probes could be detected successfully with qds raised the possibility that qds could be used to detect two or more transcripts simultaneously . achieving multiple transcript labeling using chromogenic protocols is a time consuming , stepwise process which , as explained earlier , results in the inability to distinguish areas of coexpression . in order to determine whether two transcripts can be visualized simultaneously , with the use of qds , we generated two probes ; a fitc - labeled probe against xa-1 and a biotin - labeled probe against xenopus cardiac actin . 
 the two probes were hybridized at the same time and detected using spectrally distinguishable anti - fitc - conjugated and streptavidin - conjugated qds , respectively . 
 as seen in figures 4(a ) and 4(b ) both probes were visualized successfully demonstrating the ability of simultaneous detection of multiple transcripts using the qd in situ protocol . 
 in addition , due to the lack of enzymatic amplification , the resolving ability of this method is substantially better than that of amplified protocols . 
 this is evident in figures 4(c ) and 4(d ) , where we present high - magnification images of whole - mount - stained embryos showing the intracellular mrna localization for xa-1 and ltbp1 , respectively . as can be seen 
 , qd labeling of transcripts is of extremely high resolution and enables distinction of intracellular localization patterns of mrna . 
 while ltbp1 localized in the cytoplasm , the xa-1 transcript appeared to be concentrated near the plasma membrane at the cell - cell contact areas . 
 even though there is no evidence proving that the presented mrna distribution coincides with the true intracellular localization of these transcripts , the fact that there are such dramatic differences in the signal patterns from different probes suggests that this is indeed the case . 
 future work will have to focus on closely examining the resolution of this method in model systems where direct , nonamplified in situs can be performed , using traditional fluorophores for comparison . 
 nevertheless , it is clear that this level of resolution can not be achieved with existing methods for rna transcript detection in xenopus , which are mostly based on enzymatic amplification . 
 we thus propose that use of the qd approach can simultaneously give macroscopic and intracellular data regarding the distribution of mrnas in vertebrate embryos . 
 herein we describe a new application of qds in nonamplified whole - mount fluorescent in situ detection of endogenous mrnas . 
 the ideal optical properties of qds provide unprecedented resolution and strong signal intensities that have not been possible to attain using traditional fluorophores . 
 in fact , even though wish has been available for more than a decade , the requirement for an enzymatic amplification step significantly limited the resolution of this method . 
 additionally , limitations of current fluorescent protocols have prevented widespread use of fluorescent in situs in most developmental models , with the exception of drosophila , and to some extent zebrafish ; direct visualization of highly abundant transcripts is possible in drosophila , but not in most vertebrate models , in which an enzymatic amplification step is required . 
 fluorescent detection of a messenger rna opens exciting possibilities in terms of imaging and can eliminate the need for sectioning samples . 
 it can also be used to create three - dimensional maps of expression , at a previously unattainable resolution , especially if the amplification step is eliminated . 
 the study of mrna localization is limited by the current in situ methods , both fluorescent and chromogenic , due to their relatively low resolving ability . 
 chromogenic reactions have been used successfully to localize mrna transcripts in cultured cells but only for transcripts of very high abundance [ 6063 ] . 
 the method we describe is the first nonamplified fluorescent detection of mrna in situ in xenopus . 
 our protocol results in signal intensities sufficient for imaging on a regular epifluorescence microscope without the need for confocal microscopy . of great significance , in terms of xenopus and other opaque embryos like the chick , 
 is the fact that the qd in situ staining is capable of remaining localized and fluorescent for more than an hour after the embryo is cleared . 
 this has not been possible using traditional chromogenic or fluorescent methods . in the case of c. elegans , where background is not a major issue , use of fluorescent antibodies to detect labeled rna probes 
 has to be carried out in conjunction with chromogenic amplified detection of the probes in order to get a comprehensive picture of the overall expression of a gene . 
 the amplified reaction detects low expressing regions and overall expression , whereas the fluorescent antibodies are used to resolve intracellular localization . 
 the fact that our qd in situ protocol can do both in the highly autofluorescent xenopus embryo , which is highly demanding , makes us confident that the implementation of qd in situs in other less demanding model systems will be met with equal or more success . 
 more importantly , these results suggest that qds could be introduced as alternative fluorophores in other fluorescent in situ hybridization assays where their spectral properties can offer significant advantages . 
 overall , our results demonstrate that qd in situs are a viable alternative to current ish protocols , and they expand the uses of qds in biology .","the photostability and narrow emission spectra of nanometer - scale semiconductor crystallites ( qds ) make them desirable candidates for whole - mount fluorescent in situ hybridization to detect mrna transcripts in morphologically preserved intact embryos . 
 we describe a method for direct qd labeling of modified oligonucleotide probes through streptavidin - biotin and antibody - mediated interactions ( anti - fitc and anti - digoxigenin ) . to overcome permeability issues and allow qd conjugate penetration , 
 embryos were treated with proteinase k. the use of qds dramatically increased sensitivity of whole - mount in situ hybridization ( wish ) in comparison with organic fluorophores and enabled fluorescent detection of specific transcripts within cells without the use of enzymatic amplification . therefore , this method offers significant advantages both in terms of sensitivity , as well as resolution . 
 specifically , the use of qds alleviates issues of photostability and limited brightness plaguing organic fluorophores and allows fluorescent imaging of cleared embryos . 
 it also offers new imaging possibilities , including intracellular localization of mrnas , simultaneous multiple - transcript detection , and visualization of mrna expression patterns in 3d .",pubmed
"pancreatic cancer is a devastatingly lethal disease , with the highest mortality and the lowest one , three , and five - year relative survival rates of all cancers in the united states.1,2 although pancreatic cancer accounts for a relatively small number of new diagnoses at approximately 44,000 per year , with over 37,000 deaths per year,1 pancreatic cancer ranks only behind breast , colon , and lung cancer in total us cancer mortality.3 pancreatic cancer is a heterogeneous disease classified by cancer subtypes according to their cell type of origin , structure , and behavior . 
 the majority of pancreatic cancer cases are pancreatic ductal adenocarcinoma ( pdac),2 which has the most aggressive tumor biology and metastatic potential among pancreatic cancer histopathologic subtypes.4,5 the median survival time after pdac diagnosis is about four months.6 only about 19% of patients with this disease survive past one year and only about 2% survive past five years.2 however , the survival of patients diagnosed with pdac may vary significantly depending on many factors , including demographic characteristics and the pathologic nature of the tumor , as well as the type of performed treatment . to estimate the relative impact of the distinct risk factors on the survival of patients diagnosed with pdac , cox proportional regression models have been used.68 it was shown that the following demographic factors have a significant impact on the survival of pdac : gender , race , age at diagnosis,6 marital status,8 and the time period of the pdac diagnosis.6 in addition , the following clinical factors have a significant influence on the survival of pdac : tumor size , nodal status , and distant metastases.7 
 the most dramatic influence on the survival of patients diagnosed with pdac is a surgical resection of the tumor ( with our without adjuvant chemotherapy and radiation).9 unfortunately , less than 20% of patients diagnosed with pdac are suitable for surgical resection.7 for the remaining patients , the extent of disease precludes the use of surgical procedures . 
 the combination of surgery with radiation and chemotherapy has been shown by some to improve survival compared to surgery alone.9 the estimated parameters ( values of the relative impact of risk factors on the survival of cancer patients ) obtained by the cox proportional regression models are largely varied , depending on the quantity and quality of data used , the set of the considered variables , and the technical approaches utilized in developing these models . 
 the obtained estimates depend on the underlying cox models , the credibility of which can be estimated by values of their concordance indexes ( c - indexes ) , which present the probability that for any pair of randomly drawn patients , the patient with the worst outcome estimated by this model dies earlier than one with the better estimated outcome . 
 a c - index value equal to 0.5 indicates that the predictive power of the model is not better than random chance , while a level of 1.0 of this index signifies perfect discrimination . 
 the cox proportional hazards regression models for estimation of survival for patients diagnosed with pdac were used for the development of two nomograms.10,11 one nomogram estimates the survival for patients who have undergone pancreatectomy of the malignancy and accounts for the following variables ( covariates ) : tumor size , grade , stage , lymph node data , resected tissue amount , weight loss , and pancreatic subsite.10 this nomogram was constructed using data from highly selected patients treated at a single , specialized center . the other nomogram predicts the risk of perioperative mortality in pdac patients scheduled to undergo pancreatectomy and accounts for patient demographics ( age , gender , race ) , type of pancreatectomy ( distal , radical , and other ) , hospital characteristics ( size , type ) , as well as the presence of co - morbidities in pc patients ( renal failure , neurological disorders , hypothyroid , chronic heart failure ( chf ) , liver disease , uncomplicated hypertension , cardiac arrhythmia , diabetes , and chronic obstructive pulmonary disease).11 the utility of these nomograms to patients diagnosed with pdac , however , is limited to the relatively small fraction of patients with resectable pdac ( less than 20%).12,13 recently , two new cox regression models were developed to estimate the survival for patients with resectable and unresectable pdac.9 these models use the following variables : age , gender , race , tumor stage , tumor site , tumor grade , and radiotherapy . based on these models , an interactive , online clinical calculator was developed to estimate survival probability for patients diagnosed with pdac , based on an individual s unique clinicopathological profile . 
 the calculator is also capable of estimating conditional survival probabilities ( defined as the future survival probability or duration that is calculated after a given period of survival that increases over time elapsing since the diagnosis of pdac).14 because this calculator can estimate personalized survival probability , which can influence personal decision making , disease - related anxiety , and quality of life of cancer patients , it has been proposed for clinical use.9 however , this calculator does not provide the standard errors of the survival estimates and its reliability and generalizability were not checked ( or at least not disclosed in that work ) with the use of c - indexes . 
 the reliability and generalizability of a model are essential when a model s predictions are considered for clinical practice.15 in the present work , we used seer data on 18,352 pathologically confirmed cases of pdac , diagnosed during 20042009,16 and developed cox proportional hazards regression models adjusted by the following covariates : race , gender , marital status , time period , age at diagnosis , tumor site , tumor size , t - stage , m - stage , n - stage , tumor grade , type of surgery , and radiotherapy . based on these models , we developed a web - based , real - time prognostic tool for estimating survival and conditional survival probabilities ( as well as their corresponding standard errors ) for patients with pdac . 
 the reliability and generalizability of the developed models were proven by their calibration and discrimination abilities , validated by the use of both 
 in this work , we used cancer data from the surveillance epidemiology and end results ( seer ) database from 18 geographical areas ( san francisco - oakland , connecticut , detroit , hawaii , iowa , new mexico , seattle , utah , atlanta , san jose , los angeles , alaska natives , rural georgia , greater california , kentucky , louisiana , new jersey , and greater georgia).16 these data were published by seer in april 2012 and contain cases diagnosed up to 2009 . from this release , we extracted survival data for pancreatic cancer patients diagnosed with adenocarcinoma ( international classification of diseases for oncology , 3rd edition histology codes 8140 - 8149 ) . 
 we initially queried 33,252 cases of pdac diagnosed between 2004 and 2009 . from this subset 
 we excluded : 5,583 cases that did not have pancreatic cancer as the primary cancer for the patient , 999 cases that were not pathologically confirmed ; one case diagnosed at an age younger than 20 years ( ages 019 are customarily used to classify childhood cancers ) ; 2,032 cases of races that were not black or white ( cases for asian / pacific islanders , native americans , and individuals of unknown race were not available in sufficient quantities ) ; 906 cases with unknown marital status ; 271 cases with unknown radiation therapy status ; 259 cases with unknown surgery status or having non - therapeutic procedures ; 176 cases marked t0 or tis stage ; and 4,673 cases with unknown tumor extension . 
 this left 18,352 cases prior to stratification into subgroups for t1/t2 stage and t3/t4 stage . 
 data on the remaining 18,352 pdac cases were described by 13 categorical variables ( five demographic covariates , six clinical covariates , and two medical treatment covariates ) . as demographic categorical covariates ( 
 the corresponding categories are presented in brackets ) , we used : ( i ) race ( white , black ) , ( ii ) gender ( male , female ) , ( iii ) age at diagnosis ( quartile groups : 2059 , 6069 , 7077 , 78 + years ) , ( iv ) year ( time period ) of diagnosis ( january 1 , 2007 through december 31 , 2009 ; and january 1 , 2004 through december 31 , 2006 ) , and ( v ) marital status ( married , single ) . as the clinical covariates , we used : ( i ) tumor site , i.e. , pancreatic subsite ( head , body , tail , other , unknown ) , ( ii ) tumor size ( separated by the median size into 0.04.0 and > 4 cm groups ) , ( iii ) ajcc 6th edition t - stage ( t1 , t2 , t3 and t4 ) , ( iv ) ajcc 6th edition n - stage ( n0 , n1 , and nx ) , ( v ) ajcc 6th edition m - stage ( m0 , m1 , and mx ) , and ( vi ) tumor grade ( low , high , and unknown ) . 
 finally , as medical treatment covariates we used : ( i ) pancreatic resection ( pancreatoduodectomy / total pancreatectomy , partial pancreatectomy / pancreatic excision , and no surgery ) , and ( ii ) radiation therapy ( yes or no therapy ) . 
 it should be noted that comprehensive chemotherapy data are not available in the seer database , and therefore the effects of chemotherapy could not be studied in this work . 
 the prognostic effects of these variables were studied in univariate models with the kaplan - meier method and in multivariate cox proportional hazards models , in which the survival function , s , for the failure time , t , associated with a p  1 column covariate vector , z , takes the form : where s0(t ) is the baseline hazard function and  is a p  1 column vector of the regression coefficients ( the  is the transpose of the  ) . 
 the proportional hazard assumptions were assessed by visual inspection of log - log plots.17 all tests were two - sided with a significance level set at p < 0.05 . 
 , cary , nc ) was utilized to estimate the regression coefficients ,  , of the multivariate cox models . 
 the estimates ,  ( here and below the sign ,  ^  , designates an estimate ) and the estimated covariance matrix of the  parameter estimates , cv( ) , were obtained using the phreg sas procedure . 
 estimates of the baseline survival function , 0 and their standard errors , s(0 ) , were obtained by the phreg sas procedure using the baseline statement . 
 outputs of the sas phreg procedure were used to calculate estimates of the survival probability ,  , and standard error , s( ) . for a given time , 
 t , survival  is a function of the two variables:18,19 the baseline survival function , 0 , and the prognostic index , l : estimates of the standard errors of the estimates of the prognostic index , s(l ) , were obtained by the formula that is used in sas phreg procedure : estimates of the standard errors of the estimates of the survival , s( ) , were calculated by the following formula , obtained by standard rules of error propagation,19 and implemented in the phreg sas procedure : in ( 3 ) , [ / 0 ] and [ /l ] are partial derivatives , which can be easily obtained from ( 2 ) , and the covariance cv ( 0 , l ) can be evaluated by numerical experiments . 
 we found that cv ( 0 , l )  s(0 )  s(l ) ; this approximation can be further used in formula ( 5 ) . by definition , the conditional survival , s(t2 | t1 ) , represents the probability that a patient with cancer will survive an additional t2 months , given that the patient has already survived a given t1 months . in this case , the estimate of the conditional survival ,  ( t2| t1 ) , can be presented by formula : where 2 and 1 designate the corresponding estimates of the survival probabilitie  ( t2| t1 ) and  ( t1 ) . 
 survival function estimates 1 and 2 in formula ( 6 ) are dependent random variables with covariance depending on survival time t1 end t2 . neglecting this covariance while using the standard rules of error propagation 
 , we can calculate the conservative ( upper ) estimate of the standard error of the estimate of the conditional probability , s [  ( t2| t1 ) ] , by formula : wher s ( 1 ) and s ( 2 ) are determined by formula ( 5 ) . 
 the accuracy of the cox models developed in this work were assessed by the c - indexes ( to estimate the discrimination power ) and calibration plots ( to estimate an agreement between observed outcomes and predictions).20 the reproducibility and transportability of these models were also checked , as we describe below . 
 the reproducibility is defined as the performance of a model on a sample of similar patients not included in the development of the model , while transportability reflects a model s ability to predict among patients from different but  plausibly related 
 populations.21 to estimate the reproducibility of the parameters of the developed models , cross - validation with 10 iterations was done . for this purpose , 
 the systematic sample splits were performed in such a way that all patients have served once in the model evaluation dataset . 
 the end digit in the unique patient i d number , by which each case is coded in the seer database , was used as an exclusion criterion . 
 thus , the systematic sample split was done by varying the exclusion criterion , starting from the digit 0 and sequentially changing it to digits 1 , 2 , ... , and 9 . for each iteration 
 , cases not satisfying the considered exclusion criterion were used as the model training dataset , while the excluded cases were used as a testing ( validation ) dataset . the discrimination power ( c - indexes ) of the models , obtained with the use of the training dataset , was checked using the testing dataset . 
 the generalizability or transportability of the models ( i.e. , goodness of using the same variables and same coefficients ) was checked by utilizing the models for predicting the survival of the cancer patients living in different geographical areas . for this purpose , 
 the c - indexes of the models , developed by using the cases from distinct geographic areas as training datasets , were validated on testing (  external  ) datasets exclusively containing cases from the geographical areas that were not included in the training datasets . 
 estimates of observed survival , stratified by each variable , were obtained using the kaplan - meier method . for each variable , 
 the proportional hazards assumption was assessed graphically ( data not shown ) by log - log plots.17 we determined that for the following 12 categorical variables the log - log plots are nearly parallel : race , gender , marital status , time period , age at diagnosis , tumor site , tumor size , t - stage , m - stage , n - stage , tumor grade , type of surgery , and radiation therapy . for the t - stage variable , 
 however , the corresponding curves on the log - log plots were not parallel ( and , as a result , the ajcc 6th edition stage group also demonstrates non - proportional hazards ) . 
 in fact , figure 1 shows that the curves related to the t1- and t2-stages ( tumor extension localized to the pancreas ) are not parallel to the curves related to the t3- and t4-stages ( tumor extension not localized ) . 
 data on 18,352 pdac cases were stratified in two groups by degree of pdac localization . 
 the first group ( which we called localized pdac , lpdac ) contained 5,422 cases , in which the tumor is localized within the pancreas ( t1- and t2-stages ) . 
 the second group ( called extended pdac , epdac ) contained 12,930 cases , in which the tumor is extended beyond the pancreas ( t3-stage ) or is locally advanced and involves the celiac axis or the superior mesenteric artery ( t4-stage ) . 
 after separation of all pdac cases into these two groups , the proportional hazard assumptions for all categorical variables describing the cases in each of these groups were satisfied . 
 this allowed us to perform the multivariate cox regression analysis for the lpdac and epdac groups separately . in the multivariate cox regression analysis of 5,422 cases for the lpdac group 
 , we initially did not use the t - stage variable , which is not needed for this group of cases . 
 this is because the lpdac group contained only cases with t1- and t2-stages that are different from one another only by the tumor size ( cases with tumor size equal or less than 2 cm are t1-stage , and cases with tumor size larger than 2 cm are t2-stage),22 but to categorize tumor size , the tumor size variable can be used . 
 when performing the cox analysis , we found that the variable tumor subsite ( head , body , tail , and other ) does not have a significant influence on the survival of the lpdac patients , and thus we did not use this variable for the development of the final lpdac cox model . in multivariate cox regression analysis of 12,930 cases for the epdac group , we initially utilized all the variables described in materials and methods . 
 however , the cox modeling of the epdac cases showed that the variable tumor subsite ( head , body , tail , and other ) did not have a significant influence on the survival of the epdac patients . 
 analogously , the variable t - stage , which for the epdac cases varies by degree of tumor extension , also did not have a significant influence on the survival of the epdac patients . 
 therefore , we did not use the variables tumor subsite and t - stage for the development of the final epdac cox model . 
 tables 1 and 2 present the final results of multivariate cox regression analysis for the lpdac and epdac cases , correspondingly . 
 as can be seen from these tables , the vast majority of the considered covariates have significant influence on the survival of patients diagnosed with pdac . 
 there is also a good qualitative agreement in the estimated contributions of similar covariates on the survival of patients diagnosed with the localized and extended pdac . in both cases , 
 the performed treatments ( surgery and/or radiation therapy ) were the most beneficial for survival . among the clinical characteristics of a tumor , 
 the largest risk factors accelerating death from pdac include the presence of metastasis ( m1 ) , poor tumor differentiation ( high grade ) , and large ( > 4 cm ) tumor size . 
 the involvement of lymph nodes ( n1 ) makes a small ( likely due to the simultaneous effect of other , larger contributors , such as m1-stage ) , but significant contribution to survival for patients diagnosed with lpdac , but not with epdac . within the demographic characteristics of patients , age at diagnosis 
 gender , race , and marital status make modest and comparable contributions to the survival of patients diagnosed with lpdac and epdac . 
 the accuracy of the cox models developed for the lpdac and epdac cases were assessed by the c - indexes and calibration plots.20 the obtained c - indexes of these models were equal to 0.702 and 0.712 , respectively . 
 the calibration plots presented in figures 2 and 3 show how close the survival estimates are to the observed values , predicted by these models at the time point ( chosen to be equal to 12 months ) . as can be seen from these figures , 
 the reproducibility of the cox models developed for the lpdac and epdac cases were analysed by cross - validation with 10 repeats ( table 3 ) . as can be seen from table 3 , 
 the cross - validated c - indexes of the lpdac and epdac models vary within the small intervals , ( 0.6870.720 ) and ( 0.696 0.732 ) , correspondingly . 
 the averaged values of the cross - validated c - indexes were 0.700 and 0.711 , respectively , which are very close to the c - indexes ( 0.702 and 0.712 ) initially obtained for lpdac and the epdac models . 
 this suggests high reproducibility of the cox models for the lpdac and epdac cases presented in table 1 and table 2 . 
 the generalizability ( transportability ) of the lpdac and epdac models to the pdac patients living in different geographical areas were also checked . 
 specifically , the cases from one of 18 geographic areas were considered as  external  datasets and used for validation of the lpdac and epdac cox models trained by cases collected in other 17 geographic areas . 
 note that this table shows data for the geographical areas having more than 100 cases of white and black patients , which could be used as external datasets . because the utah , hawaii , alaska , and rural georgia seer registries had less than 100 cases to be used for external testing of the lpdac models 
 , we did not perform validation for these areas . as can be seen from table 4 , 
 the c - indexes of the lpdac and epdac cox models for individual geographical areas vary within the intervals , ( 0.651 0.739 ) and ( 0.6990.739 ) , respectively . 
 the averaged values of these indexes for the lpdac and epdac models were 0.700 and 0.714 , respectively . 
 these numbers are very close to the c - indexes ( 0.702 and 0.712 ) initially obtained for the lpdac and the epdac cox models . 
 this suggests that the lpdac and epdac models , the parameters of which are presented in tables 1 and 2 , are sufficiently generalized to be transportable for applications to different geographical areas of the us . 
 overall , the data presented in tables 3 and 4 confirm high reproducibility and generalizability of the lpdac and epdac cox models developed with the use of 18,352 pathologically confirmed cases of pdac diagnosed between 2004 and 2009 . 
 this allowed us to use the parameters of these models , which are presented in tables 1 and 2 , to develop a real - time prognostic estimator of survival for patients diagnosed with pdac . 
 this tool allows one to estimate the conditional survival (t2| t1 ) and its standard error se[(t2| t1 ) ] , using  , cov( ) , s0 and se(s0 ) as unchangeable datasets and z ( the personalized demographic and clinical data on patients diagnosed with pdac , as well as information on medical treatment ) , t1 ( number of months that the patient has already survived after the pdac diagnosis ) , and t2 ( number of additional months that the patient will survive ) as variable input data . depending on the value of the t - stage variable 
 , the estimator uses the lpdac ( for t1 and t2 stages ) or epdac ( for t3 and t4 stages ) models with the corresponding unchangeable datasets . 
 the conditional survival probability and its standard error have been calculated by formulas ( 6 ) and ( 7 ) , respectively . 
 the estimator is a real - time , web - based computerized tool , compatible with major browsers and user devices , including tablets and smart phones . 
 as with any other cox models , the lpdac and epdac models developed in this work have several potential limitations that may explain why predictions performed by the lpdac and epdac models may be inaccurate in up to 30% of patients ( discriminative power of these models , or c - index ~ 0.70 ) . 
 specifically , the lpdac and epdac models use variables provided by seer , while other variables , for instance , co - morbidities in pdac patients ( such as renal failure , neurological disorders , liver disease , and cardiac arrhythmia ) that may significantly contribute to mortality risk from pdac are not utilized.11 the second limitation is due to the fact that although the public - use seer data on pdac contains information on adjuvant radiation therapy , information on chemotherapy data is not provided.23,24 however , about 85% of the pdac patients who received radiation therapy also received chemotherapy.25 nevertheless , the absence of information on chemotherapy may lead to a bias in the estimation of the efficiency of radiation therapy when seer data is used.25 the third limitation is due to the use of the sum of the model variables , weighted by their coefficient values ( log hazard ratio ) as the prognostic index . in fact , 
 estimation of the prognostic index , which is at the heart of cox models , may be accurate for the majority of the observed pdac population , but it may be inaccurate for relatively small , yet very important patient subpopulations . 
 for instance , for lpdac patients who have undergone pancreatectomy for a small sized tumor , the benefits of radiotherapy , as determined by our lpdac cox model , can be overestimated . 
 nevertheless , our newly developed lpdac and epdac models and computing tool (  estimator  ) have distinct advantages over the existing models and tools , aimed at predicting survival probabilities for patients diagnosed with pdac . 
 some of these advantages are similar to those that were highlighted in the work recently published by katz et al.9 these advantages are : ( i ) the developed models are applicable for the vast majority of pdac patients ( not only for those who are qualified for pancreatectomy ) and ( ii ) the developed web - based computing tools are capable of the real - time prediction of conditional and unconditional survival probabilities for the patients diagnosed with pdac . 
 however , lpdac and epdac models have additional advantages that can be seen by exploring differences between the models and computing tools developed in our work and the katz work , as presented below . 
 the models presented in the katz work were stratified by surgery status , while our lpdac and epdac models are stratified by tumor extension . 
 our models use the following covariates ( which were not used in the katz work ) : marital status , time period , t - stage , m - stage , n - stage , and type of surgery . on the other hand , tumor subsite ( which we found did not have significant influence on survival ) and a stage covariate ( which we found did not satisfy the proportional hazards assumption ) , 
 further , our models were tested using c - indexes and cross - validated with the use of internal and external datasets , while the accuracy , reproducibility and generalizability of the corresponding models were not disclosed in the katz work . 
 finally , our models were trained using data collected in 20042009 and account for time period effects , while the models of that work were trained on data collected in 19882005 and ignore possible time period effects . 
 there are also differences in performances of the estimator , developed in our work , and the calculator , developed in the katz work . 
 in fact , the calculator assesses survival time without adjusting for time period effects and thus implicitly makes predictions anchored to the middle of the 19882005 time interval , while our estimator predicts survival , projected to the later time period ( 20072009 ) . 
 in addition , the estimator provides standard errors for the predicted survival probabilities , while the calculator does not have this capability . 
 we showed that the lpdac and epdac models developed in this work fit the observed survival data well , are well - calibrated , and have good discrimination ( c - indexes ) between cases . 
 these models were used in the development of a web - based tool ( estimator ) to assess survival probability , conditional survival probability , and their standard errors . 
 this tool uses the personalized demographic and clinical data on patients diagnosed with pdac , as well as information on ( performed , ongoing , or planned ) medical treatment as input data . 
 the estimator performs all calculations anchored to the latest time period , 2007 2009 , which contains the latest seer data available at the time of the preparation of this work . 
 in fact , clinicians mainly rely on personal experience , while the estimator is based upon the combined survival experience and clinical care of many patients and account for the effect of dozens of risk factors simultaneously . 
 the estimator removes preconceived bias and offers assessments based on the personalized demographic and clinical data of a given patient . by toggling different treatment options 
 , clinicians will be able to compare the predicted impacts of possible treatments on survival of the patient . a treatment that will not provide any significant advantage in survival , but 
 would instead be detrimental to the patient s quality of life , should not be considered as an appropriate option . using the computer - generated assessments and carefully considering the patient s co - morbidities , clinicians will be able to assist patients diagnosed with pdac in making a more educated decision regarding potential treatments .","the 18,352 pancreatic ductal adenocarcinoma ( pdac ) cases from the surveillance epidemiology and end results ( seer ) database were analyzed using the kaplan - meier method for the following variables : race , gender , marital status , year of diagnosis , age at diagnosis , pancreatic subsite , t - stage , n - stage , m - stage , tumor size , tumor grade , performed surgery , and radiation therapy . 
 because the t - stage variable did not satisfy the proportional hazards assumption , the cases were divided into cases with t1- and t2-stages ( localized tumor ) and cases with t3- and t4-stages ( extended tumor ) . for estimating survival and conditional survival probabilities in each group , a multivariate cox regression model adjusted for the remaining covariates was developed . 
 testing the reproducibility of model parameters and generalizability of these models showed that the models are well calibrated and have concordance indexes equal to 0.702 and 0.712 , respectively . 
 based on these models , a prognostic estimator of survival for patients diagnosed with pdac was developed and implemented as a computerized web - based tool .",pubmed
"we recruited japanese patients who underwent pta for cli in kansai rosai hospital , hyogo , japan , between 2003 and 2009 . 
 all patients with chronic ischemic rest pain and/or foot ulcer or gangrene were evaluated for limb ischemia by angiography . the diagnosis and management of cli were compliant with the transatlantic inter - society consensus ( tasc ) ( 10 ) or its revised consensus , tasc ii ( 1 ) . 
 once they were diagnosed as having cli , we utilized pta as the first - choice procedure for revascularization , within the recommendations in tasc ii . 
 the indication of pta was judged by consensus among vascular specialists , including vascular surgeons . 
 pta was not considered to be feasible when lesions were heavily calcified and/or with diffuse occlusions , whereas poor - risk patients , having relative contraindications for bypass surgery , those with advanced age , expected high mortality under general anesthesia , and morbidity setting , for instance , would not necessarily be contraindications for pta . 
 major amputation , namely , above - the - ankle amputation , was indicated when revascularization failed to relieve patients from rest pain or control their foot lesions . 
 the indication for amputation was judged by consensus among plastic surgeons as well as vascular specialists . 
 insufficient blood flow even after revascularization and uncontrollable limb infection were considered to be indications for amputation . in the current study , we analyzed all of the recruited patients as well as those with diabetes . 
 the preoperative variables considered in the analyses were sex , age , activities of daily living ( adls ) , fontaine stage , the presence of infection , diabetic condition , hypertension , dyslipidemia , smoking , receiving regular hemodialysis , and serum albumin level as a nutritional marker . 
 diabetic condition was determined as the presence of diabetes in the whole population and as a glycemic control in the diabetic population . 
 the diagnosis of diabetes was based on world health organization criteria , whereas glycemic control of diabetic patients was evaluated by a1c . 
 hypertension was diagnosed as systolic blood pressure 130 mmhg or diastolic blood pressure 80 mmhg or having been treated for hypertension . 
 dyslipidemia was defined as serum ldl cholesterol 100 mg / dl or hdl cholesterol < 40 mg / dl or triglycerides 150 mg / dl or having been treated for dyslipidemia . partial or total dependence in transferring ( requiring some help in moving in and out of bed / chair or a complete transfer ) were regarded as impaired adls . 
 data are given as means and sds for continuous variables or as percentages for dichotomous variables . 
 limb salvage and survival were plotted using the kaplan - meier method , and , if necessary , the differences between the two groups were assessed by a log - rank test . 
 cox proportional hazards regression model was used to determine the unadjusted association of each variable with the outcome . 
 the statistically significant variables in the univariate analyses , as well as sex and age , were entered into multivariate models to reveal the independent impact on the outcome . 
 statistical analyses were performed using spss version 15.0j ( spss , chicago , il ) . 
 data are given as means and sds for continuous variables or as percentages for dichotomous variables . 
 limb salvage and survival were plotted using the kaplan - meier method , and , if necessary , the differences between the two groups were assessed by a log - rank test . 
 cox proportional hazards regression model was used to determine the unadjusted association of each variable with the outcome . 
 the statistically significant variables in the univariate analyses , as well as sex and age , were entered into multivariate models to reveal the independent impact on the outcome . 
 statistical analyses were performed using spss version 15.0j ( spss , chicago , il ) . 
 a total of 278 cli patients undergoing primary pta procedures were recruited ; aorto - iliac lesions were revascularized in 23% of the population , femoro - popliteal in 63% , and infrapopliteal in 68% . 
 diabetic patients had a significantly higher prevalence of fontaine stage iv and hemodialysis than those without diabetes ( p = 0.006 and p = 0.001 , respectively ) . 
 baseline characteristics of the study population data are n ( % ) or means  sd 
 . differences between continuous variables of diabetic and nondiabetic groups were evaluated by unpaired t tests , whereas dichotomous variables between the two groups were compared by fisher exact tests . 
 follow - up period was 90  72 weeks ( 1.7  1.4 years ) , and 48 patients underwent major amputations and 89 died . 
 figure 1 shows the cause of death and kaplan - meier estimates of major amputation and mortality . 
 the leading cause of death was cardiac and vascular disease ( n = 40 ) ; followed by infection ( n = 31 ) , including pneumonia ( n = 16 ) , sepsis secondary to infective gangrene of the foot ( n = 8) , bed sore infection ( n = 1 ) , and catheter - related infection ( n = 1 ) ; and unspecified causes ( n = 5 ) . 
 diabetic patients had a higher rate of major amputation compared with nondiabetic patients ( p = 0.008 , log - rank test ) , whereas they had a similar life prognosis ( p = 0.717 ) . 
 the cause of death in the whole population ( a ) and in the population with diabetes ( b ) . 
 kaplan - meier estimates of major amputation ( c and d ) and survival ( e and f ) in the whole population . 
 the prognosis was compared between diabetic patients ( solid line ) and nondiabetic patients ( dotted line ) in d ( p = 0.008 , log - rank test ) and f ( p = 0.717 , log - rank test ) . 
 cv , cardiac and vascular disease ; mn , malignant neoplasm ; dm , diabetes . 
 table 2 shows the influence of preoperative variables on the prognosis in the whole population . 
 the presence of diabetes was independently associated with major amputation , and the adjusted hr was 3.101 ( 95% ci 1.2627.621 ) ( p = 0.014 ) . 
 rather , mortality was associated with age , impaired adls , hemodialysis , and serum albumin level ; the adjusted hrs were 1.036 ( 1.0101.063 ) in the 1-year increment ( p = 0.007 ) and 2.302 ( 1.4073.605 ) ( p < 0.001 ) , 2.699 ( 1.6824.332 ) ( p < 0.001 ) , and 1.788 ( 1.1502.782 ) in the 1-g / dl decrement ( p = 0.010 ) , respectively . 
 association of preoperative variables with the prognosis of cli patients undergoing pta data are hr ( 95% ci ) . 
 we also performed analyses with a focus on the patients with diabetes ( table 3 ) . here again 
 , mortality was associated with age , impaired adls , hemodialysis , and serum albumin level . 
 a1c level was , however , independently associated with major amputation , whose adjusted hr was 1.349 ( 95% ci 1.1031.650 ) in the 1% increment ( p = 0.004 ) . 
 its association was still significant in the multivariate models in which every other peripheral vascular factor available in our dataset was substituted for fontaine stage iv ( online appendix table a , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0939/dc1 ) . 
 association of preoperative variables for the prognosis with cli patients with diabetes undergoing pta data are hr ( 95% ci ) . 
 next , we divided the a1c dataset into quartiles to analyze the relationship between the increment of a1c level and the risk for major amputation in the diabetic patients . 
 5.9% was categorized into the first quartile ( q1 ) , 6.06.7% into the second quartile ( q2 ) , 6.87.6% into the third quartile ( q3 ) , and 7.7% into the fourth quartile ( q4 ) . in a stepwise multivariate model 
 , the categorized a1c level was independently associated with major amputation , with adjustment for fontaine stage iv , hemodialysis , and infection . 
 the adjusted hrs of q2 , q3 , and q4 relative to q1 were 2.030 ( 95% ci 0.6576.266 ) ( p = 0.218 ) , 3.398 ( 1.2279.412 ) ( p = 0.019 ) , and 3.983 ( 1.39811.35 ) ( p = 0.010 ) , respectively . 
 we further analyzed the influence of the presence of diabetes on major amputation according to their a1c level ; each a1c quartile of the diabetic group , defined above , was compared with the nondiabetic group in a stepwise multivariate model . as shown in fig . 
 2a , the two higher a1c quartiles of the diabetic group ( that is , the diabetic group with a1c 6.8% ) had a significantly higher risk than the nondiabetic group , whereas the two lower a1c quartiles ( that is , a1c < 6.8% ) did not . 
 based on these findings , we reanalyzed with substitution of diabetes with a1c 6.8% for the presence of diabetes in the original multivariate model shown in table 2 . 
 the result was that diabetes with a1c 6.8% , infection , and hemodialysis were significantly associated with major amputation ; their adjusted hrs were 2.907 ( 95% ci 1.6065.264 ) ( p 
 < 0.001 ) , 2.375 ( 1.1984.711 ) ( p = 0.014 ) , and 3.530 ( 1.7727.029 ) ( p 
 note that these three variables were independent of one another and therefore were expected to have additive influences on major amputation . in fact , a kaplan - meier model showed that those with the accumulation of these prognostic factors had an increased risk of major amputation ( fig . 
 when they had all of these three risk factors , their prognosis was extremely poor . 
 their estimated median time to limb loss was only 23 weeks , which would be rarely different from natural course of nonrevascularized cli patients ( 1 ) . 
 data are the adjusted hrs and 95% cis of each a1c quartile of the diabetic group relative to the nondiabetic group in the stepwise multivariate model . 
 they were adjusted for impaired activity of daily living , fontaine stage iv , infection , and receiving hemodialysis . 
 the quartiles of a1c were as follows : q1 : 5.9% , q2 : 6.06.7% , q3 : 6.87.6% , and q4 : 7.7% . 
 b : kaplan - meier estimates of major amputation according to the number of risk factors ( p < 0.001 , log - rank test ) . 
 risk factors considered here are the following three variables : diabetes with a1c 6.8% , the presence of infection , and receiving hemodialysis , all of which had independent associations in the stepwise multivariate cox proportional hazards regression model . 
 table 2 shows the influence of preoperative variables on the prognosis in the whole population . 
 the presence of diabetes was independently associated with major amputation , and the adjusted hr was 3.101 ( 95% ci 1.2627.621 ) ( p = 0.014 ) . 
 rather , mortality was associated with age , impaired adls , hemodialysis , and serum albumin level ; the adjusted hrs were 1.036 ( 1.0101.063 ) in the 1-year increment ( p = 0.007 ) and 2.302 ( 1.4073.605 ) ( p < 0.001 ) , 2.699 ( 1.6824.332 ) ( p < 0.001 ) , and 1.788 ( 1.1502.782 ) in the 1-g / dl decrement ( p = 0.010 ) , respectively . 
 association of preoperative variables with the prognosis of cli patients undergoing pta data are hr ( 95% ci ) . 
 we also performed analyses with a focus on the patients with diabetes ( table 3 ) . here again , mortality was associated with age , impaired adls , hemodialysis , and serum albumin level . 
 a1c level was , however , independently associated with major amputation , whose adjusted hr was 1.349 ( 95% ci 1.1031.650 ) in the 1% increment ( p = 0.004 ) . 
 its association was still significant in the multivariate models in which every other peripheral vascular factor available in our dataset was substituted for fontaine stage iv ( online appendix table a , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0939/dc1 ) . 
 association of preoperative variables for the prognosis with cli patients with diabetes undergoing pta data are hr ( 95% ci ) . 
 next , we divided the a1c dataset into quartiles to analyze the relationship between the increment of a1c level and the risk for major amputation in the diabetic patients . 
 5.9% was categorized into the first quartile ( q1 ) , 6.06.7% into the second quartile ( q2 ) , 6.87.6% into the third quartile ( q3 ) , and 7.7% into the fourth quartile ( q4 ) . in a stepwise multivariate model 
 , the categorized a1c level was independently associated with major amputation , with adjustment for fontaine stage iv , hemodialysis , and infection . 
 the adjusted hrs of q2 , q3 , and q4 relative to q1 were 2.030 ( 95% ci 0.6576.266 ) ( p = 0.218 ) , 3.398 ( 1.2279.412 ) ( p = 0.019 ) , and 3.983 ( 1.39811.35 ) ( p = 0.010 ) , respectively . 
 we further analyzed the influence of the presence of diabetes on major amputation according to their a1c level ; each a1c quartile of the diabetic group , defined above , was compared with the nondiabetic group in a stepwise multivariate model . as shown in fig . 
 2a , the two higher a1c quartiles of the diabetic group ( that is , the diabetic group with a1c 6.8% ) had a significantly higher risk than the nondiabetic group , whereas the two lower a1c quartiles ( that is , a1c < 6.8% ) did not . 
 based on these findings , we reanalyzed with substitution of diabetes with a1c 6.8% for the presence of diabetes in the original multivariate model shown in table 2 . 
 the result was that diabetes with a1c 6.8% , infection , and hemodialysis were significantly associated with major amputation ; their adjusted hrs were 2.907 ( 95% ci 1.6065.264 ) ( p < 0.001 ) , 2.375 ( 1.1984.711 ) ( p = 0.014 ) , and 3.530 ( 1.7727.029 ) ( p < 0.001 ) , respectively ( online appendix table b ) . 
 note that these three variables were independent of one another and therefore were expected to have additive influences on major amputation . 
 in fact , a kaplan - meier model showed that those with the accumulation of these prognostic factors had an increased risk of major amputation ( fig . 
 when they had all of these three risk factors , their prognosis was extremely poor . 
 their estimated median time to limb loss was only 23 weeks , which would be rarely different from natural course of nonrevascularized cli patients ( 1 ) . 
 data are the adjusted hrs and 95% cis of each a1c quartile of the diabetic group relative to the nondiabetic group in the stepwise multivariate model . 
 they were adjusted for impaired activity of daily living , fontaine stage iv , infection , and receiving hemodialysis . 
 the quartiles of a1c were as follows : q1 : 5.9% , q2 : 6.06.7% , q3 : 6.87.6% , and q4 : 7.7% . 
 b : kaplan - meier estimates of major amputation according to the number of risk factors ( p < 0.001 , log - rank test ) . 
 risk factors considered here are the following three variables : diabetes with a1c 6.8% , the presence of infection , and receiving hemodialysis , all of which had independent associations in the stepwise multivariate cox proportional hazards regression model . 
 as is mentioned in the tasc ii ( 1 ) , a primary outcome in cli patients would be amputation - free survival , although it remains unclear so far , especially in the population undergoing pta , which preoperative variables are associated with their prognosis and whether morality and limb loss have the same predictive factors . in the current study , a1c level 
 the multivariate model , however , showed that a1c level had a significant association with major amputation independently of infection . 
 the current study also confirmed that the presence of diabetes was significantly associated with major amputation in the whole population and that its significant association was dependent on the glycemic control . 
 some investigators already reported the association between diabetes and limb loss ( 11,12 ) , but they did not take glycemic control into consideration . 
 interestingly , when compared with nondiabetes , diabetes a1c 6.8% , but not a1c < 6.8% , was significantly associated with major amputation . 
 this finding suggests that the increased risk of major amputation for diabetic patients is mainly attributed to their poor glycemic control . 
 it is well known , however , that poor glycemic control is associated with decreased immune response and delayed wound healing , as well as the progression of microangiopathy including neuropathy , all of which play an important part of foot lesions ( 1315 ) ; the existence of poor glycemic control might reflect these underlying conditions . in discussion of limb prognoses of pad patients 
 exist several ways of assessing preoperatively their peripheral arterial severity . in the current study , we adopted fontaine stages , a widely used clinical classification , and found that it was not significantly associated with limb salvage in a multivariate model , as was poor glycemic control . to examine this paradoxical result 
 , we further analyzed with two other preoperative peripheral arterial factors available in our dataset , namely ankle - brachial index , which is physiological , and runoff below the ankle before pta , which is anatomical ( or imaging ) . 
 yet , the results were similar ; no preoperative peripheral arterial factors had independent associations with limb loss ( online appendix table a ) . 
 in contrast , a postoperative variable , the runoff below the ankle after pta , was significantly associated with major amputation . 
 one interpretation of these findings is that the limb prognosis depends on the peripheral blood flow after , rather than before , revascularization . 
 successful revascularizations would improve peripheral blood flow and could lead to avoidance of limb loss , even if the preoperative peripheral flow is severely impaired . 
 the severity of preoperative peripheral flow would not always mean the similar severity of postoperative peripheral flow . given that peripheral blood flow 
 can be changed by revascularization procedures , it is not surprising that limb prognosis after pta is associated with postoperative , rather than preoperative , peripheral arterial factors . 
 we also surveyed the influence of preoperative variables on survival , which confirmed that diabetes failed to have a significant association with morality . 
 , most studies surveyed cli patients undergoing pta together with those undergoing bypass surgery and/or nonrevascularized patients , whereas we limited our study population to those undergoing pta . in addition , we revealed that a1c level in diabetic patients had no significant association with mortality . 
 the prognostic factors for mortality were , rather , age , impaired adls , hemodialysis , and serum albumin level . 
 these significant associations were affirmed even with additional adjustment for cardiac function or ejection fraction evaluated with echocardiography ( online appendix table c ) . 
 previous reports ( 9,18,19 ) investigated some of these prognostic variables , but they did not mention nutritional status in the population . as is well known , malnutrition affects life prognosis in various clinical situations ( 20,21 ) . 
 we adopted serum albumin level as a nutritional marker in the current study because of its great advantages in ease and cost and its wide use in clinical practice . 
 it is possible that serum albumin level is affected by clinical factors other than malnutrition . 
 but we confirmed the relationship between malnutrition and mortality by the analyses with other nutrition - related markers , such as lymphocyte count and serum cholinesterase level ( online appendix table d ) . in conclusion , we investigated the association of preoperative variables with the prognosis of cli patients undergoing pta , which suggests that prognostic indicators are somewhat different between survival and limb salvage . 
 diabetes and poor glycemic control , for instance , was significantly associated with limb loss but not mortality . 
 diabetes with poor glycemic control , infection , and hemodialysis was independently associated with limb loss , and the accumulation of these prognostic factors increased the amputation risk . 
 especially , those with all of the three risk factors had such poor limb prognosis that they might fail to receive a prognostic benefit of pta . 
 it is possible , however , that correction of poor glycemic control by adequate interventions leads to prognostic improvement . 
 a further prospective investigation is required to determine whether the intervention on glycemic control subsequently improves their prognosis . 
 ","objectiveto reveal the influence of preoperative factors on the prognosis of patients undergoing percutaneous transluminal angioplasty ( pta ) for critical limb ischemia ( cli).reseach design and methodswe recruited 278 japanese patients who underwent pta for cli between 2003 and 2009 . 
 the outcome measures were mortality and major amputation . 
 cox proportional hazards regression analyses were performed.resultsthe prevalence of diabetes was 71% , and a1c was 7.0  1.4% . 
 the follow - up period was 90  72 weeks , and 48 patients underwent major amputations and 89 died . 
 the presence of diabetes in the whole population and a1c level in the diabetic population had no influence on morality ; rather , mortality was associated with age ( p = 0.007 ) , impaired activities of daily living ( p < 0.001 ) , hemodialysis ( p < 0.001 ) , and albumin level ( p = 0.010 ) . 
 in contrast , the presence of diabetes and a1c level had significant association with major amputation ( p = 0.012 and p = 0.007 , respectively ) . 
 the quartile analysis showed that diabetic subjects with an a1c 6.8% , but not < 6.8% , had a significantly higher risk of major amputation than nondiabetic subjects . 
 the adjusted hazard ratio of diabetes with a1c 6.8% was 2.907 ( 95% ci 1.6065.264 ) ( p < 0.001).conclusionsdiabetes with poor glycemic control is associated with major amputation , but not mortality , in cli patients undergoing pta . 
 prognostic indicators seem somewhat different between survival and limb salvage in the population .",pubmed
"human cystic echinococcosis ( ce ) , caused by echinococcus granulosus , is one of the most important and widespread parasitic zoonoses ( 1 ) . 
 humans acquire infection by accidental ingestion of e. granulosus eggs voided in the faeces of infected dogs and the disease is common in parts of the world where there is close contact between the intermediate and definitive hosts , usually sheep and dogs , respectively ( 2 ) . 
 cystic echinococcosis in humans usually presents with symptoms associated with the presence of fluid - filled cysts in the liver , lungs , or other viscera and diagnosis is usually established by a combination of radiology and serology ( 3 ) . 
 one of the problems that can be encountered after treating ce patients is the risk of postsurgical relapses or treatment failure due to nonradical surgical procedures or perisurgical spillage of parasite material , especially protoscoleces . 
 relapses in the form of newly developing cysts have been reported and may affect between 2 and 25% of cases after therapy , according to previous studies ( 46 ) . 
 therefore , postsurgical follow - up of ce patients for years is necessary , with the aim of detecting newly growing cysts as soon as possible . 
 a posttreatment follow - up method to prognostically determine the efficacy of treatment should therefore include markers that allow the detection of newly growing or relapsing cysts and tracking of previously undetected but still viable cysts . 
 serology has been one of the methods selected for the post - operative control of hydatidosis . however , the long persistence of anti - e . 
 granulosus antibodies after recovery makes difficult the diagnosis of relapse by serology ( 7 ) . in this sense , many serologic techniques have been evaluated ( latex agglutination , passive hemagglutination , immunoelectrophoresis and specific ige , igm , igg enzyme - linked immunosorbent assay ) in the post - operative monitoring of hydatid disease patients . therefore , there is still a need to develop or improve immunodiagnostic tools in order to meet the requirements expressed by clinicians . 
 one approach consists of searching for and identifying new antigens that specifically allow the classification of patients into cured and noncured categories . 
 this study examined the pattern of antigenic bands essential for the serologic diagnosis of ce , revealed by immunoblotting analysis . 
 we also report on the post - operative evolution of patients treated for this disease and also determined the diagnostic performance of western blot . 
 blood samples were obtained from 50 patients ( 16 males and 34 females ; mean sd age was 31.111.2 years , range 969 years ) with clinically / radiologically diagnosed ce , 40 non - ce patients with different parasitic infections and malignancy and from 20 sex and age - matched healthy controls . 
 samples from all subjects were centrifuged at 2000g for 10 minutes at 4 c to obtain the serum . 
 the sera was divided in to 3 tubes for each subject and stored immediately at 70 c until analysis . 
 all ce patients were sampled 1 week before treatment and received antihelminthic treatment of albendazole , 400 mg twice a day for 3 months , plus praziquantel , 40 mg / kg / day for two weeks as per standard guidelines ( 8) and underwent surgical procedures . 
 all procedures were approved by the local ethical committee and all subjects gave their informed consent to the study . 
 the results were compared between preoperative and post - operative group and were evaluated statistically using paired  t test . 
 hydatid cyst fluid antigen ( hcf ) was prepared according to standard procedure as described earlier ( 9 ) . 
 briefly , the hydatid cyst fluid ( hcf ) was aseptically aspirated from fertile hydatid cysts obtained from livers of naturally infected sheep slaughtered at the local abattoir . 
 the aspirated fluid was centrifuged at 2000g for 20 min at 4 c to remove the protoscolices . 
 the supernatant was then filtered through a whatman wcn type membrane filter ( cellulose nitrate , 47 mm diameter , 0.45 m pore size ) and dialyzed against distilled water overnight at 4c using dialysis tubing ( sigma aldrich , usa ) with molecular weight cut off 2000 da ( dalton ) . 
 the antigen protein concentration was estimated by the lowry method ( 10 ) with bovine serum albumin ( bsa ) as a reference standard . 
 hydatid sheep antigen ( 40 g ) was subjected to discontinuous sds - page , using 12.5% gel in a bio - rad apparatus at 50 ma / gel for 1 hour . 
 the proteins separated were transferred from un - stained gels to nitrocellulose membranes ( schleicher & schuell , inc . , keene , u.s.a . ) by means of the phasttransfer ( pharmacia lkb ) following standardized procedures ( 11 ) . 
 efficacy of transfer was checked by staining the membrane with ponceau s stain ( 0.001 g / ml in 3% trichloro acetic acid ) . 
 the membranes with blotted antigen were cut into strips and blocked with 5% ( w / v ) of skimmed milk in washing buffer ( 10 mm tris , 150 mm nacl , and 0.05% tween 20 ; ph 7.4 ) for 2 hours . 
 the strips were incubated with test sera ( 1/100 dilution in washing buffer with 1% bovine serum albumin ( bsa ) for 2 hours at room temperature . 
 after 3 washes ( each 15 min ) , the strips were incubated with horseradish peroxidase conjugated anti - human igg ( sigma ) at a dilution of 1/2000 ( in washing buffer+1% bsa ) for 2 h at room temperature . 
 after 3 washes as before , bound antigens was developed using diaminobanzidine ( dab ) substrate ( 0.1% h2o2 + 6 mg / ml dab in 50 mm tris - hcl , ph 7.6 ) . 
 molecular weight estimates were made by comparing the mobility of the different antigen fractions with these of prestained protein marker , broad range ( 8250 kda ) , ( new england biolabs , usa ) which were separated in the same gel of a hydatid fluid sample and electrotransferred to the same sheet . 
 hydatid cyst fluid antigen ( hcf ) was prepared according to standard procedure as described earlier ( 9 ) . 
 briefly , the hydatid cyst fluid ( hcf ) was aseptically aspirated from fertile hydatid cysts obtained from livers of naturally infected sheep slaughtered at the local abattoir . 
 the aspirated fluid was centrifuged at 2000g for 20 min at 4 c to remove the protoscolices . 
 the supernatant was then filtered through a whatman wcn type membrane filter ( cellulose nitrate , 47 mm diameter , 0.45 m pore size ) and dialyzed against distilled water overnight at 4c using dialysis tubing ( sigma aldrich , usa ) with molecular weight cut off 2000 da ( dalton ) . 
 the antigen protein concentration was estimated by the lowry method ( 10 ) with bovine serum albumin ( bsa ) as a reference standard . 
 hydatid sheep antigen ( 40 g ) was subjected to discontinuous sds - page , using 12.5% gel in a bio - rad apparatus at 50 ma / gel for 1 hour . 
 the proteins separated were transferred from un - stained gels to nitrocellulose membranes ( schleicher & schuell , inc . , keene , u.s.a . ) by means of the phasttransfer ( pharmacia lkb ) following standardized procedures ( 11 ) . 
 efficacy of transfer was checked by staining the membrane with ponceau s stain ( 0.001 g / ml in 3% trichloro acetic acid ) . 
 the membranes with blotted antigen were cut into strips and blocked with 5% ( w / v ) of skimmed milk in washing buffer ( 10 mm tris , 150 mm nacl , and 0.05% tween 20 ; ph 7.4 ) for 2 hours . 
 the strips were incubated with test sera ( 1/100 dilution in washing buffer with 1% bovine serum albumin ( bsa ) for 2 hours at room temperature . 
 after 3 washes ( each 15 min ) , the strips were incubated with horseradish peroxidase conjugated anti - human igg ( sigma ) at a dilution of 1/2000 ( in washing buffer+1% bsa ) for 2 h at room temperature . 
 after 3 washes as before , bound antigens was developed using diaminobanzidine ( dab ) substrate ( 0.1% h2o2 + 6 mg / ml dab in 50 mm tris - hcl , ph 7.6 ) . 
 molecular weight estimates were made by comparing the mobility of the different antigen fractions with these of prestained protein marker , broad range ( 8250 kda ) , ( new england biolabs , usa ) which were separated in the same gel of a hydatid fluid sample and electrotransferred to the same sheet . 
 in the present study the age of patients varied between 969 years . the mean sd age of the patients was 31.1211.24 years . 
 the highest incidence of disease was recorded among patients between 20 to 49 years age . 
 the predominance of hydatidosis was in females ( 68% ) than in males ( 32% ) . 
 thirty four ( 34 ) of the radiologically and surgically confirmed cases had hepatic cysts while 16 had extrahepatic cysts ( 12-lung cysts , 3-liver and lung and 1-thigh cyst ) . 
 all patients responded to pharmacological and surgical treatment except for two women ( 32 and 36 years old ) in whom multiple cysts ( 12 and 7 cysts ) were detected in liver and lung two years after the first operation . 
 igg reactivity from patients with cystic echinococcosis revealed multiple immunoreactive bands ranging from 18239 kda ( fig . 
 igg immunoreactivity of hydatid sera against different antigenic fractions in pre - operative samples the major immunoreactive bands were 24 , 39 , 46 , 52 , 57 , 6061 and 63 kda ( table 1 ) . 
 sera from patients with other parasitic infections and malignancy showed cross - reactivity with the cluster of 5459 kda bands . 
 comparative evaluation of igg immunoreactivity with antigenic fractions in pre and post - operative samples antigenic fractions of 24 kda and 39 kda decreased significantly in six month ; one year and two year follow up samples the antigenic fractions of 46 , 52 , 57 , 6061 and 63 kda present before surgery in higher number of samples persisted significantly in post surgical six month , one year and two year follow up samples the healthy control sera were not reactive to any antigenic fraction ( table 2 ) . 
 therefore the antigenic bands with molecular weight of 52 , 24 , 39 , 46 , 63 and 6061 kda which were reactive to 72.09% , 62.79% , 60.46% , 48.83% , 34.88% and 30.23% samples of pre - operative patients were specific for hydatid disease , suggesting thereby that theses antigenic fractions have higher diagnostic value . similarly the 39 kda band reactivity decreased significantly from 60.46% in pre - operative samples to 35.71% with six month , 21.42% with one year and 7.5% with two year follow - up samples ( table 1 , fig . 
 however 24 kda and 39 kda persist in 2 patients who had a relapse at two year follow - up . 
 igg antibody immunoreactivity with antigenic fractions in control serum samples igg immunoreactivity of hydatid sera against different antigenic fractions in post - operative samples the antigenic fractions of 52 , 46 , 39 and 24 kda in combination may serve as useful diagnostic markers . 
 immunoreactive bands of 52 , 46 and 57 kda present in pre - operative samples persisted in higher number of post - operative samples . 
 the percentage immunoreactivity of 24 and 39 kda decreased significantly within two year post - operative samples . 
 the 24 kda immunoreactivity decreased from 62.79% in pre - operative samples to 30.95% with six month , 19.04% with one year and 7.5% with two year follow - up samples . 
 igg reactivity from patients with cystic echinococcosis revealed multiple immunoreactive bands ranging from 18239 kda ( fig . 
 igg immunoreactivity of hydatid sera against different antigenic fractions in pre - operative samples the major immunoreactive bands were 24 , 39 , 46 , 52 , 57 , 6061 and 63 kda ( table 1 ) . 
 sera from patients with other parasitic infections and malignancy showed cross - reactivity with the cluster of 5459 kda bands . 
 comparative evaluation of igg immunoreactivity with antigenic fractions in pre and post - operative samples antigenic fractions of 24 kda and 39 kda decreased significantly in six month ; one year and two year follow up samples the antigenic fractions of 46 , 52 , 57 , 6061 and 63 kda present before surgery in higher number of samples persisted significantly in post surgical six month , one year and two year follow up samples the healthy control sera were not reactive to any antigenic fraction ( table 2 ) . 
 therefore the antigenic bands with molecular weight of 52 , 24 , 39 , 46 , 63 and 6061 kda which were reactive to 72.09% , 62.79% , 60.46% , 48.83% , 34.88% and 30.23% samples of pre - operative patients were specific for hydatid disease , suggesting thereby that theses antigenic fractions have higher diagnostic value . similarly 
 the 39 kda band reactivity decreased significantly from 60.46% in pre - operative samples to 35.71% with six month , 21.42% with one year and 7.5% with two year follow - up samples ( table 1 , fig . 
 2 ) . however 24 kda and 39 kda persist in 2 patients who had a relapse at two year follow - up . 
 igg antibody immunoreactivity with antigenic fractions in control serum samples igg immunoreactivity of hydatid sera against different antigenic fractions in post - operative samples the antigenic fractions of 52 , 46 , 39 and 24 kda in combination may serve as useful diagnostic markers . 
 immunoreactive bands of 52 , 46 and 57 kda present in pre - operative samples persisted in higher number of post - operative samples . 
 the percentage immunoreactivity of 24 and 39 kda decreased significantly within two year post - operative samples . 
 the 24 kda immunoreactivity decreased from 62.79% in pre - operative samples to 30.95% with six month , 19.04% with one year and 7.5% with two year follow - up samples . 
 different conventional serological techniques for the diagnosis of human cystic echinococcosis have been developed , but their diagnostic value is limited because of the variable sensitivity and specificity ( 12 ) . in recent years , western 
 blotting has created a new era in immunodiagnosis , which greatly reduces cross - reactions . 
 this technique has been reported to give specific results in human studies ( 13 , 14 ) . 
 therefore , the present study was designed to use this technique for the diagnosis and post - operative follow - up of ce patients . 
 the igg reactivity in the sera of pre - operative patients identified multiple immunoreactive bands with the molecular weight ranging from 18239 kda . 
 our results are in agreement with previous study , where the predominance of 24 , 3234 , 4446 and 5254 kda was observed in sera of patients with hepatic and pulmonary cystic echinococcosis . 
 kanwer et al . , ( 16 ) also noticed the antigenic bands of high molecular weight ranging from 8116 kda with the frequent occurrence of 8 kda , 16 , 24 , 38 , 45 , 58 kda in ce patients . 
 the antigenic bands of 1214 , 16 , 20 , 2426 , 34 , 39 and 42 kda in molecular weight enabled the detection of antibodies in the pre - surgical samples and antibodies specific to 39 and 42 kda disappeared in less than one year in the patients cured after surgery in ce patients ( 17 ) . 
 al - olayan and helmy , ( 18 ) observed the antigenic bands of 22 , 24 , 35 , 38 , 40 , 45 , 50 , 55 , 6065 , 80 , 97 and 110 kda were recognised by sera from hydatidosis patients . 
 there are not enough studies about the immunoreactivity of igg with high molecular mass antigenic bands in patients with ce ( 16 , 19 , 20 ) . 
 the results revealed that 57 kda band cross - reacts with patient sera containing antibodies of other parasitic infections such as ascariasis , amoebiasis , toxoplasmosis and malignancy . 
 the non - specificity may be caused mainly by a sharing of hydatid antigen with those of other parasites ( 21 ) but other possible reasons are interaction with some blood group antigen ( 22 ) or with non - specific host proteins in hydatid fluid ( 23 ) . 
 the variability in the results could be due to many reasons , the source of antigen used as sheep hydatid fluid ( 15 ) , camel hydatid fluid ( 18 ) , human hydatid fluid ( 24 ) , the differences in purification protocols may also change the ability of antigens to recognized the specific antibodies ( 25 ) , differences in experimental condition , differences in echinococcus strains and geographical / clinical characteristics of patients ( 26 ) . 
 the results verified all the purified antigenic bands except 57 kda were specific to hydatid antibodies as they did not cross - react with the antibodies of other parasitic infections . 
 the application of western blotting for monitoring the surgical treatment is a subject that is not available in the literature . with the use of different antigenic bands 
 , it was verified that western blotting technique shows a disappearance of some bands in the post - operative case , as well as the persistence of some bands . 
 this latter situation agrees with the differences in the reactivity of the different isotypes of immunoglobulins with the hydatid antigens , observed with the western blotting technique applied to sera of patients suffering hepatic hydatidosis ( 20 ) . 
 the western blotting appears to be a useful method for diagnosing hydatidosis and for post - surgical monitoring , incapable at the moment of being detected by any other immunologic technique , given that the other techniques react with all the antigens without the ability of observing the appearance or disappearance of the bands observed only with the western blotting . in this sense , the antibodies against proteins of 24 , and 39 kda constitute a good marker for post - surgical monitoring , since in the cases of surgically cured patients these disappear in post - operative samples , while they remain as long as cysts persist . 
 further studies are also required to analyze the behaviour of these antibodies after medical treatment . 
 the western blotting appears to be a useful method for post - surgical monitoring because the antibodies against proteins of 24 and 39 kda constitute a good marker for post - surgical monitoring , since in the cases of cured patients these disappear in postoperative samples , while they remain as long as cysts persist . 
 ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .","background : cystic echinococcosis ( ce ) is one of the most important parasitic zoonosis worldwide . 
 due to the high recurrence rate of the disease after surgery , follow up of the patient is necessary . 
 the aim of current research was to assess the performance of western blotting ( wb ) , using sheep hydatid fluid , for serological diagnosis and post - treatment monitoring of human ce.methods:serum samples obtained from 50 clinically / radiologically proven cases of ce along with serum samples from non - ce patients and healthy persons were tested by wb , using sheep hydatid fluid as antigen.results:the wb test enabled the detection of antibodies in the pre - operative samples for proteins of 18239 kda in molecular weight . from 50 sera of ce patients , 31 cases ( 72.09% ) 
 detected 52 kda subunit , 27 cases ( 62.79% ) detected 24 kda band , 26 cases ( 60.46% ) recognised 39 kda band and 21 cases ( 48.83% ) identified 46 kda component of sheep hydatid antigen . 
 sera from patients with other parasitic infections and malignancy showed cross - reactivity with the cluster of 5459 kda bands . 
 the healthy control sera were not reactive to any antigenic fraction . 
 the antigenic bands with molecular weight of 52 , 24 , 39 and 46 kda were specific for ce , and may serve as useful diagnostic markers . 
 the antibodies specific to proteins 24 and 39 kda significantly decreased in the patients cured after surgery , while in patients with recurrent parasitism the bands present before surgery persisted.conclusion:the wb with sheep hydatid antigen might be useful in the diagnosis and post - surgical monitoring of ce patients .",pubmed
"obesity is a common metabolic disease world - wide and dyslipidemias among the most common metabolic disorders associated with obesity [ 17 ] . 
 obesity is often described by classical parameters such as body mass index ( bmi ) , waist circumference ( wc ) , and waist - to - hip ratio ( whr ) [ 13,7,8 ] . 
 results of meta - regression analysis of prospective , randomized studies showed that whr and wc may play important roles in assessment of increased risk of cardiovascular ( cv ) events . in recent years 
 , attention has been drawn to new obesity indices ( e.g. , waist - to - height ratio ( whtr ) , visceral adiposity index ( vai ) , and body adiposity index ( bai ) , which also take into account adipose tissue distribution [ 1316 ] . 
 results of the some recently published studies suggest that whtr , the newer obesity index , may be helpful for assessing risk of metabolic complications , including dyslipidemia , and it may also be a predictor of cardiovascular events in obese patients [ 1,8,1620 ] . nevertheless , there is little data about the associations between other newer obesity indices ( e.g. , vai and bai ) and lipid profile in obese patients . 
 therefore , the aim of this study was to examine the relationship between obesity parameters or indices ( bmi , wc , whr , whtr , bai , vai ) and serum lipid levels of total cholesterol ( tc ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides ( tg ) , and lipid indices such as tc / hdl - c , ldl - c / hdl - c , and tg / hdl - c ratios in obese non - diabetic patients . 
 this cross - sectional study was conducted in a population of 66 consecutively enrolled obese non - diabetic patients ( bmi 30 kg / m ) with mean age of 55.811.6 years , including 40 women ( mean age 57.510.8 years ) and 26 men ( mean age 53.312.3 years ) treated in the outpatient department of hypertension and lipid disorders of wam university hospital in lodz , poland . patients with fasting hyperglycemia 5.6 
 mmol / l ( 100 mg / dl ) underwent a glucose tolerance test ( 75 g ) to exclude diabetes mellitus ( dm ) . 
 patients with dm , pregnancy , cancer , acute stroke , acute hepatic or renal diseases and acute cardiovascular events or with history of abdominal surgery , which could have an impact on abdominal fat distribution , were excluded from the study . 
 patients with psychiatric disorders receiving antipsychotic agents that might impact appetite and lead to weight gain were also excluded . based on calculated bmi values , patients were divided into 3 groups according to their obesity level : group i consisted of patients with obesity class i with bmi of 30.034.9 kg / m ; group ii consisted of patients with obesity class ii with bmi of 35.039.9 kg / m ; and group iii consisted of patients with obesity class iii with bmi 40 kg / m . 
 the study was conducted after receiving approval from the bioethics committee of the medical university of lodz , resolution no . 
 all the patients underwent a complete blood count , lipid and liver profile , tsh , glucose , renal function indices ( bun and creatinine ) , and general urinalysis . 
 the serum ldl - c level was calculated based on friedewald s formula ( in mmol / l ) : measurements accurate to within 0.1 kg were made of body mass , and measurements accurate to within 0.5 cm were made of height , wc , and hc . wc was measured at mid - distance between the last rib and the iliac crest , while hc was measured at the level of the greater trochanter . 
 based on these measurements , the following body mass - related indices were calculated using the following formulas : bmi = body mass ( kg ) / [ height ( m ) ] whr = wc ( cm ) / hc ( cm ) whtr = wc ( cm ) / height ( cm ) men vai = [ wc / 39.68 + ( 1.88  bmi ) ]  ( tg / 1.03 )  ( 1.31 / hdl ) ; women vai = [ wc / 36.58 + ( 1.89  bmi ) ]  ( tg / 0.81 )  ( 1.52 / hdl ) . 
 bai = [ hip circumference ( cm ) / height ( m ) ]  18 [ 1315,18 ] . 
 in addition , based on the lipid profile results obtained , the following lipid metabolism indices were calculated : tc / hdl - c ratio 
 values > 5 indicate increased cardiovascular risk ; ldl - c / hdl - c ratio  increased cardiovascular risk with values > 3 ; tg / hdl - c ratio  increased cardiovascular risk with values 3 . 
 the results are presented as mean values with standard deviation ( normality of distribution was tested using the shapiro - wilk test ) . 
 significance of differences between groups was assessed using the t - test for independent samples , and in the case of multiple comparisons ( more than 2 study groups ) , univariate analysis of variance ( anova ) was applied with subsequent use of post - hoc multiple comparison tests ( tukey s test ) . for statistical analysis of discontinuous results , 
 values of p<0.05 were assumed to be statistically significant . to assess the relationship and correlation between obesity indices and lipid metabolism parameters , pearson s linear correlation analysis was used , along with a linear regression model with a 95% confidence interval . 
 this cross - sectional study was conducted in a population of 66 consecutively enrolled obese non - diabetic patients ( bmi 30 kg / m ) with mean age of 55.811.6 years , including 40 women ( mean age 57.510.8 years ) and 26 men ( mean age 53.312.3 years ) treated in the outpatient department of hypertension and lipid disorders of wam university hospital in lodz , poland . patients with fasting hyperglycemia 5.6 
 mmol / l ( 100 mg / dl ) underwent a glucose tolerance test ( 75 g ) to exclude diabetes mellitus ( dm ) . 
 patients with dm , pregnancy , cancer , acute stroke , acute hepatic or renal diseases and acute cardiovascular events or with history of abdominal surgery , which could have an impact on abdominal fat distribution , were excluded from the study . 
 patients with psychiatric disorders receiving antipsychotic agents that might impact appetite and lead to weight gain were also excluded . based on calculated bmi values , patients were divided into 3 groups according to their obesity level : group i consisted of patients with obesity class i with bmi of 30.034.9 kg / m ; group ii consisted of patients with obesity class ii with bmi of 35.039.9 kg / m ; and group iii consisted of patients with obesity class iii with bmi 40 kg / m . 
 the study was conducted after receiving approval from the bioethics committee of the medical university of lodz , resolution no . 
 all the patients underwent a complete blood count , lipid and liver profile , tsh , glucose , renal function indices ( bun and creatinine ) , and general urinalysis . 
 the serum ldl - c level was calculated based on friedewald s formula ( in mmol / l ) : measurements accurate to within 0.1 kg were made of body mass , and measurements accurate to within 0.5 cm were made of height , wc , and hc . wc was measured at mid - distance between the last rib and the iliac crest , while hc was measured at the level of the greater trochanter . based on these measurements , 
 the following body mass - related indices were calculated using the following formulas : bmi = body mass ( kg ) / [ height ( m ) ] whr = wc ( cm ) / hc ( cm ) whtr = wc ( cm ) / height ( cm ) men vai = [ wc / 39.68 + ( 1.88  bmi ) ]  ( tg / 1.03 )  ( 1.31 / hdl ) ; women vai = [ wc / 36.58 + ( 1.89  bmi ) ]  ( tg / 0.81 )  ( 1.52 / hdl ) . 
 bai = [ hip circumference ( cm ) / height ( m ) ]  18 [ 1315,18 ] . 
 in addition , based on the lipid profile results obtained , the following lipid metabolism indices were calculated : tc / hdl - c ratio  values 
 > 5 indicate increased cardiovascular risk ; ldl - c / hdl - c ratio  increased cardiovascular risk with values > 3 ; tg / hdl - c ratio  increased cardiovascular risk with values 3 . 
 the results are presented as mean values with standard deviation ( normality of distribution was tested using the shapiro - wilk test ) . 
 significance of differences between groups was assessed using the t - test for independent samples , and in the case of multiple comparisons ( more than 2 study groups ) , univariate analysis of variance ( anova ) was applied with subsequent use of post - hoc multiple comparison tests ( tukey s test ) . for statistical analysis of discontinuous results , 
 values of p<0.05 were assumed to be statistically significant . to assess the relationship and correlation between obesity indices and lipid metabolism parameters , pearson s linear correlation analysis was used , along with a linear regression model with a 95% confidence interval . 
 there were no statistically significant differences in clinical characteristic between compared groups ( table 1 ) . among persons enrolled in the study , 
 the largest group consisted of dyslipidemic patients , accounting for 90.9% of the general study population , while hypercholesterolemia was present in 43.9% , mixed hyperlipidemia in 36.4% , and hypertriglyceridemia in 10.6% of subjects ( table 1 ) . 
 hypertension was diagnosed in 83.3% patients from the whole study population ( table 1 ) . 
 patients from group iii had greater mean wc compared to group ii and i ( table 2 ) . 
 patients in obesity class ii also had greater mean wc than in group i. mean whtr , bmi , and bai values were higher in group iii compared to group ii and group i ( table 2 ) . 
 higher mean whtr and bmi were observed in group ii compared to group i ( table 2 ) . 
 mean serum tc and ldl - c in group iii were significantly lower than in group i ( table 2 ) . 
 negative significant correlations were found in the whole study population between : bmi and tc , ldl - c , hdl - c ; wc , whr , vai and hdl - c ( table 3 ) . 
 by contrast , positive significant correlations were found between wc , whr , and tg / hdl - c , as well as between vai and : tc , tg , tc / hdl - c , ldl / hdl - c , and tg / hdl - c ( table 3 ) . 
 lipid disorders were the most frequent concomitant disorders in our study population . the high incidence of dyslipidemia in obese patients was also shown in 2 large polish epidemiological studies 
 interestingly , the group of patients with class iii obesity had lower mean serum levels of tc and ldl - c compared with patients from the other 2 groups , and this difference was statistically significant when compared to the patients with class i obesity . 
 this result was not related to lipid - lowering treatment because of lack of statistically significant differences between compared groups . 
 similar results were obtained in the study lipidogram 2004 , where higher serum levels of tc , ldl - c , and tg were found in overweight people compared with the obese . on the other hand 
 , the negative correlations observed in our study between bmi , wc , and ldl - c might have resulted from a lower ldl - c level in patients with morbid obesity as previously observed by others [ 2628 ] . 
 found a 2-phase relationship between bmi , wc , and ldl - c levels , especially in women . 
 increase in bmi and wc values initially resulted in increases in ldl - c levels , subsequently leading to reductions in the most obese persons . in native americans , 
 increase in ldl - c levels was positively correlated with bmi only for the first 3 quartiles , then this relationship was reversed . 
 the authors suggest that increase in adipose tissue in this population may decrease ldl - c levels in 2 ways : through increased activity of ldl - c receptors in adipose tissue or because of diluting the circulating pool of ldl - c through the increased circulating blood volume in more obese people . 
 it must also be noted that levels of small , dense ldl ( sdldl ) fractions , as in our study , were not measured and a preponderance of sdldl induced by insulin resistance can lead to significant reductions in ldl - c . 
 howard et al . concluded , based on results of clinical studies , that the relationship between bmi and ldl - c serum levels is complex and depends on numerous factors such as age or sex . 
 it was demonstrated that higher ldl - c levels were seen in young ( 2044 years of age ) obese women , and were positively correlated with bmi . 
 the authors did not observe significant differences between patients from different obesity classes with regards to levels of hdl - c and tg . in the strong heart study conducted in 773 women and 739 men of native american origin , 
 hu et al . found significant negative correlation between bmi and hdl - c in women . 
 wc values were positively correlated with serum tg levels and negatively correlated with hdl - c levels , and the relation between wc and assessed lipid fraction levels in women was much stronger than in the case of bmi . in men , 
 positive correlations were observed between bmi , wc , and tg levels , as well as a negative correlation between bmi , wc , and hdl - c levels , although , in contrast with women , the strength of correlation was comparable for both obesity parameters . 
 data obtained from the available literature suggest that increasing bmi results in decreasing hdl - c levels and increasing tg levels . 
 these studies , however , were conducted in general populations rather than selected groups of patients with obesity . 
 these observations are also supported by the results of a study conducted in 1518 adult peruvians , where the authors found a very strong negative correlation between bmi , vai , and the hdl - c fractions , as well as a positive correlation with tg ; however , this was irrespective of gender . 
 we also found a negative correlation between bmi , wc , whr , and hdl - c , but not with tg . 
 similar findings were observed in the prospective epidemiological study of myocardial infarction ( prime ) and by mojiminiyi et al . . 
 the negative correlation between wc and hdl - c levels observed in our study is supported by results presented by hu et al . 
 ; however , unlike the present authors , no statistically significant correlation between bmi , wc , and tg was found , although a similar tendency was present . 
 the above - mentioned negative correlation between wc and hdl - c was also reported by chehrei et al . in 750 non - obese people . 
 the results of our study suggest that obesity , especially abdominal obesity , is significantly associated with dyslipidemia , which manifests itself mainly with reductions in hdl - c . 
 it is especially evident with the vai index , which demonstrated the strongest correlation with tg and hdl - c levels and with the lipid indices tc / hdl - c , ldl - c / hdl - c , and tg / hdl - c . 
 this is not surprising , however , given the fact that the vai formula includes both the hdl - c and tg levels . 
 the positive correlation observed in our study between whr and tg / hdl - c ratio is supported by results obtained by marrotta et al . 
 results of our study demonstrate a relationship between some selected obesity indices , especially those pertaining to visceral obesity , and lipid parameters . 
 this is especially the case with new , recently proposed obesity indices , such as vai , whtr , and bai , which have been investigated in only a few studies to date [ 1,1316 ] . 
 one strength of this study is that there is relatively little information on obese patients grouped according to obesity level , especially among the morbidly obese ( bmi 40 kg / m ) . 
 most studies have assessed the relationship between obesity indices and lipid metabolism parameters in the general population , and sometimes in non - overweight or healthy people . 
 obesity is associated with lipid disturbances , especially with hdl - c reduction , in obese non - diabetic patients . 
 vai is strongly related with lipid profile and thus may be the most valuable obesity index in obese patients with dyslipidemias . 
 ","backgroundthe aim of this cross - sectional study was to examine the relationship between obesity and lipid markers.material/methodswe divided 66 non - diabetic adult obese patients ( mean age : 55.811.6 years ) into 3 groups according to body mass index ( bmi ) . 
 all patients were measured for waist circumference ( wc ) , hip circumference ( hc ) , body mass index ( bmi ) , waist - to - hip ratio ( whr ) , waist - to - height ratio ( whtr ) , body adiposity index ( bai ) , and visceral adiposity index ( vai ) . 
 serum levels of total cholesterol ( tc ) , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , and triglycerides ( tg ) were determined , and lipid indices tc / hdl , ldl / hdl , and tg / hdl were also estimated.resultstc and ldl - c in group iii were lower than in group i ( 5.01.0 vs. 6.01.0 mmol / l , and 2.90.9 vs. 3.81.2 
 mmol / l ; p<0.05 for both ) . 
 negative correlations were found between : bmi and tc , ldl , and hdl ( r=0.291 ; r=0.310 , r=0.240 , respectively ) ; and wc , whr , vai , and hdl ( r=0.371 , r=0.296 , r=0.376 , respectively ) . 
 positive correlations were found between wc , whr , and tg / hdl ( r=0.279 , r=0.244 , respectively ) and between vai and : tc ( r=0.327 ) , tg ( r=0.885 ) , tc / hdl ( r=0.618 ) , ldl / hdl ( r=0.480 ) , and tg / hdl ( r=0.927).conclusionsobesity is associated with lipid disturbances , especially with hdl - c reduction , in obese non - diabetic patients . 
 vai is strongly related to lipid profile and thus may be the most valuable obesity index in obese patients with dyslipidemias .",pubmed
"there has been an explosive growth in the use of internet not only in india but also worldwide in the last decade . 
 there were about 42 million active internet users in urban india in 2008 as compared to 5 million in 2000 . 
 the internet is used by some to facilitate research , to seek information , for interpersonal communication , and for business transactions . on the other hand 
 , it can be used by some to indulge in pornography , excessive gaming , chatting for long hours , and even gambling . 
 there have been growing concerns worldwide for what has been labeled as  internet addiction . 
 griffith considered it a subset of behavior addiction and any behavior that meets the 6  core components  of addiction , i.e. , salience , mood modification , tolerance , withdrawal , conflict , and relapse . while davis avoided the term internet addiction , referring it as a dependency on psychoactive substances , he instead preferred the term  pathological internet use  ( piu ) . 
 young linked excessive internet use most closely to pathological gambling , a disorder of impulse control in dsm iv and adapted the dsm iv criteria to relate to internet use in the internet addiction test developed by her . according to her , 
 various types of internet addiction are cyber - sexual addiction , cyber - relationship addiction , net compulsions , information overload , and computer addiction . 
 his findings indicated that social isolation plays a greater role in behavioral symptoms of piu than does the presence of psychopathology . 
 hence , caplan suggested replacing the term  pathological internet use  with  problematic internet use . 
 the prevalence found by greenfield was about 6% among the general population , while scherer found it to be 14% among the college - based population . 
 surveys conducted online revealed that 4 - 10% of the users meet the criteria for internet addiction . 
 the addicted averaged 38.5 h / week on a computer , whereas the non - addicted averaged 4.9 h / week . measuring 
 goldberg developed the internet addictive disorder ( iad ) scale by adapting the dsm iv . 
 brenner developed the internet - related addictive behavior inventory ( irabi ) comprising of 32 true and false questions . 
 young initially developed 8-question internet addiction diagnostic questionnaire ( dq ) based on dsm iv . 
 later , she included 12 new items in addition to the 8 items to formulate an internet addiction test ( iat ) . 
 young 's iat is the only available test whose psychometric properties have been tested by widyanto and mcmurran . in india , 
 hence , it was found necessary to study pattern of internet usage in young adults in indian setting and its relationship with their mental and physical health . with this background 
 the aim of the study was to study the prevalence of internet addiction and association of any psychopathology in college going student population in the city of mumbai . 
 the aim of the study was to study the prevalence of internet addiction and association of any psychopathology in college going student population in the city of mumbai . 
 the cross - sectional survey was carried out in 3 different colleges in the city of mumbai during the period of august 
 it covered about 1000 college students ( aged 16 - 18 years ) having access to the internet from the past 6 months . 
 the semi - structured proforma along with the scales were distributed in classes , each with roughly 40 students , and necessary instructions were given . 
 the study was conducted after obtaining the approval from the institutional review board and permission was sought from the college authorities of all the colleges . of the total 1000 students , 13 could not be included in the study as they were not using internet . 
 the tools used in this study were as follows : 
 semi - structured proforma that contained details of demographics , educational qualification and status , purpose of using the internet ( by choosing among the options like education , entertainment , business transactions , or social networking ) , money spent per month , place of access ( home , cybercaf , or workplace if working part - time ) , the time of day when the internet is accessed the most ( by choosing between morning , afternoon , evening , or night ) , and the average duration of use per day . data was collected from those using internet for at least since last 6 months.the internet addiction test ( iat ; young , 1998 ) is a 20-item 5-point likert scale that measures the severity of self - reported compulsive use of the internet . 
 total internet addiction scores are calculated , with possible scores for the sum of 20 items ranging from 20 to 100 . 
 the scale showed very good internal consistency , with an alpha coefficient of 0.93 in the present study.according to young 's criteria , total iat scores 20 - 39 represent average users with complete control of their internet use , scores 40 - 69 represent over - users with frequent problems caused by their internet use , and scores 70 - 100 represent internet addicts with significant problems caused by their internet use.the duke health profile is a 17-item generic questionnaire instrument designed to measure adult self - reported functional health status quantitatively during a 1-week time window . 
 there are 11 scales with maximum score for each scale being 100 and minimum being 0 . 
 six scales ( i.e. , physical health , mental health , social health , general health , perceived health , and self - esteem ) measure function , with high scores indicating better health . five scales ( i.e. , anxiety , depression , anxiety - depression , pain disability ) measure dysfunction , with high scores indicating greater dysfunction . 
 most extensive use was in family practice patients with the broadest spectrum of diagnoses , but it was used in patient populations with specific diagnoses such as insulin - dependent diabetes mellitus , end - stage renal disease , ischemic disease , and impotence . 
 both internal consistency ( cronbach 's alpha ) and temporal stability ( test - retest ) testing have supported reliability of the duke . 
 validity has been supported for the duke scales by ( a ) comparison of the duke scores with scores of other health measures for the same patients , ( b ) comparison of duke scores between patient groups having different clinical diagnostic profiles and severity of illness , and ( c ) prediction of health - related outcomes by duke scores . 
 semi - structured proforma that contained details of demographics , educational qualification and status , purpose of using the internet ( by choosing among the options like education , entertainment , business transactions , or social networking ) , money spent per month , place of access ( home , cybercaf , or workplace if working part - time ) , the time of day when the internet is accessed the most ( by choosing between morning , afternoon , evening , or night ) , and the average duration of use per day . 
 the internet addiction test ( iat ; young , 1998 ) is a 20-item 5-point likert scale that measures the severity of self - reported compulsive use of the internet . 
 total internet addiction scores are calculated , with possible scores for the sum of 20 items ranging from 20 to 100 . 
 the scale showed very good internal consistency , with an alpha coefficient of 0.93 in the present study . 
 according to young 's criteria , total iat scores 20 - 39 represent average users with complete control of their internet use , scores 40 - 69 represent over - users with frequent problems caused by their internet use , and scores 70 - 100 represent internet addicts with significant problems caused by their internet use . 
 the duke health profile is a 17-item generic questionnaire instrument designed to measure adult self - reported functional health status quantitatively during a 1-week time window . 
 there are 11 scales with maximum score for each scale being 100 and minimum being 0 . 
 six scales ( i.e. , physical health , mental health , social health , general health , perceived health , and self - esteem ) measure function , with high scores indicating better health . five scales ( i.e. , anxiety , depression , anxiety - depression , pain disability ) measure dysfunction , with high scores indicating greater dysfunction . 
 most extensive use was in family practice patients with the broadest spectrum of diagnoses , but it was used in patient populations with specific diagnoses such as insulin - dependent diabetes mellitus , end - stage renal disease , ischemic disease , and impotence . 
 both internal consistency ( cronbach 's alpha ) and temporal stability ( test - retest ) testing have supported reliability of the duke . 
 validity has been supported for the duke scales by ( a ) comparison of the duke scores with scores of other health measures for the same patients , ( b ) comparison of duke scores between patient groups having different clinical diagnostic profiles and severity of illness , and ( c ) prediction of health - related outcomes by duke scores . 
 the cross - sectional survey was carried out in 3 different colleges in the city of mumbai during the period of august 
 it covered about 1000 college students ( aged 16 - 18 years ) having access to the internet from the past 6 months . 
 the semi - structured proforma along with the scales were distributed in classes , each with roughly 40 students , and necessary instructions were given . 
 the study was conducted after obtaining the approval from the institutional review board and permission was sought from the college authorities of all the colleges . of the total 1000 students , 13 could not be included in the study as they were not using internet . 
 the tools used in this study were as follows : 
 semi - structured proforma that contained details of demographics , educational qualification and status , purpose of using the internet ( by choosing among the options like education , entertainment , business transactions , or social networking ) , money spent per month , place of access ( home , cybercaf , or workplace if working part - time ) , the time of day when the internet is accessed the most ( by choosing between morning , afternoon , evening , or night ) , and the average duration of use per day . 
 data was collected from those using internet for at least since last 6 months.the internet addiction test ( iat ; young , 1998 ) is a 20-item 5-point likert scale that measures the severity of self - reported compulsive use of the internet . 
 total internet addiction scores are calculated , with possible scores for the sum of 20 items ranging from 20 to 100 . 
 the scale showed very good internal consistency , with an alpha coefficient of 0.93 in the present study.according to young 's criteria , total iat scores 20 - 39 represent average users with complete control of their internet use , scores 40 - 69 represent over - users with frequent problems caused by their internet use , and scores 70 - 100 represent internet addicts with significant problems caused by their internet use.the duke health profile is a 17-item generic questionnaire instrument designed to measure adult self - reported functional health status quantitatively during a 1-week time window . 
 there are 11 scales with maximum score for each scale being 100 and minimum being 0 . 
 six scales ( i.e. , physical health , mental health , social health , general health , perceived health , and self - esteem ) measure function , with high scores indicating better health . 
 five scales ( i.e. , anxiety , depression , anxiety - depression , pain disability ) measure dysfunction , with high scores indicating greater dysfunction . 
 most extensive use was in family practice patients with the broadest spectrum of diagnoses , but it was used in patient populations with specific diagnoses such as insulin - dependent diabetes mellitus , end - stage renal disease , ischemic disease , and impotence . 
 both internal consistency ( cronbach 's alpha ) and temporal stability ( test - retest ) testing have supported reliability of the duke . 
 validity has been supported for the duke scales by ( a ) comparison of the duke scores with scores of other health measures for the same patients , ( b ) comparison of duke scores between patient groups having different clinical diagnostic profiles and severity of illness , and ( c ) prediction of health - related outcomes by duke scores . 
 semi - structured proforma that contained details of demographics , educational qualification and status , purpose of using the internet ( by choosing among the options like education , entertainment , business transactions , or social networking ) , money spent per month , place of access ( home , cybercaf , or workplace if working part - time ) , the time of day when the internet is accessed the most ( by choosing between morning , afternoon , evening , or night ) , and the average duration of use per day . 
 the internet addiction test ( iat ; young , 1998 ) is a 20-item 5-point likert scale that measures the severity of self - reported compulsive use of the internet . 
 total internet addiction scores are calculated , with possible scores for the sum of 20 items ranging from 20 to 100 . 
 the scale showed very good internal consistency , with an alpha coefficient of 0.93 in the present study . according to young 's criteria , total iat scores 20 - 39 represent average users with complete control of their internet use 
 , scores 40 - 69 represent over - users with frequent problems caused by their internet use , and scores 70 - 100 represent internet addicts with significant problems caused by their internet use . 
 the duke health profile is a 17-item generic questionnaire instrument designed to measure adult self - reported functional health status quantitatively during a 1-week time window . 
 there are 11 scales with maximum score for each scale being 100 and minimum being 0 . 
 six scales ( i.e. , physical health , mental health , social health , general health , perceived health , and self - esteem ) measure function , with high scores indicating better health . five scales ( i.e. , anxiety , depression , anxiety - depression , pain disability ) measure dysfunction , with high scores indicating greater dysfunction . 
 most extensive use was in family practice patients with the broadest spectrum of diagnoses , but it was used in patient populations with specific diagnoses such as insulin - dependent diabetes mellitus , end - stage renal disease , ischemic disease , and impotence . 
 both internal consistency ( cronbach 's alpha ) and temporal stability ( test - retest ) testing have supported reliability of the duke . 
 validity has been supported for the duke scales by ( a ) comparison of the duke scores with scores of other health measures for the same patients , ( b ) comparison of duke scores between patient groups having different clinical diagnostic profiles and severity of illness , and ( c ) prediction of health - related outcomes by duke scores . 
 in the present study , out of 1000 students , 13 were excluded and the remaining 987 adolescents who participates included 681 ( 68.9% ) females and 306 ( 31.1% ) males . 
 subjects had more girls than boys as the data was collected during routine lectures and the attendance of girls might have been more 
 the subjects belonged to different streams : 33.5% to science , 30.3% to commerce , and 36.2% to arts . 
 using young 's original criteria , the users were divided into groups : 74.5% as moderate users , 24.8% as possible addicts , and 0.7% as addicts . 
 males in comparison to females were significantly more likely to be addicted ( x2=10.2 , p=0.006 ) . 
 moderate users and the possible addicts used the internet mostly for social networking , academic purposes , chatting , emailing , gaming , and downloading media files and pornography . 
 they indulged more in social networking , chatting , and downloading media files ( x2=76 , p<0.001 ) . 
 significantly , most of the addicts used the internet mostly in the evening and nights as compared to other users who used it in the mornings and afternoons as well ( x2=26.4 , p=00019 ) . 
 interesting findings were noted with respect to the place of accessing internet . about half of the adolescents with addiction were also working part - time and 
 this finding too was statistically significant ( x2=144 , p<0.001 ) . in this study , no significant relationship was found between internet addiction and the hours of use per day . 
 moreover , the criteria used in iat does not take into consideration the exact duration of use . 
 losing track of time while being online and staying online longer than intended are more likely to be seen in addicts . 
 using duke 's health profile , it was found that addicts have poor mental , physical , and mental health score [ figure 1 ] . 
 addicts had high anxiety , depression , and anxiety depression score ( x2=12.26 , p<0.0022 ) [ table 1 ] . internet use and psychopathology 
 a number of studies have been conducted across the world , especially among adolescents with respect to internet addiction . 
 this study is a preliminary step toward understanding the extent of internet addiction among college students in india . 
 the findings of the present study corroborates with previous studies stating that addiction is more common in males than in females(anderson et al . ) . also , the finding that addicts are more likely to use their workplace to access internet is comparable to  internet abuse at workplace  reported by young and case . 
 this could be a confounding factor for comparison as our study had students as subjects . 
 employers have found that employees with access to the internet at their desks spend a considerable amount of their working day engaging in non - work - related internet use ( beard , 2002 ) . in a study done previously , young concluded that depression assessment should be done in suspected cases of piu . 
 although it is not clear whether depression precedes the development of internet abuse or it is a consequence , yet assessment of the same is imperative . in 2005 , a study by nemiz et al 
 . dealt with british university students showed that those students who were pathological internet users had low self - esteem and were socially inhibited online . 
 young showed that withdrawal from significant real - life relationships is a consequence of pathological internet users . 
 in the last one decade , internet has become an integral part of our life . 
 it can put into three groups using young 's original criteria : 74.5% as moderate users , 24.8% as possible addicts , and 0.7% as addicts . 
 those towards the addict part of spectrum reported had high anxiety , depression , and anxiety depression score . in the emerging era , where young people have been more exposed to the internet and use online activity as an important form of social interaction . 
 however , it may still remain a matter of debate whether to call internet addiction a distinct disorder by itself or a behavioral problem secondary to another disorder . at present 
 whether or not we will have any such diagnosis included in the future is yet to be seen . in future , 
 if it is added , it is more likely to be classified as an impulse control disorders not elsewhere classified rather than in the diagnostic criteria for substance dependence . by studying the association of internet usage and its effects on human behavior 
 , we can formulate interventions like setting boundaries and detecting early warning signs of underlying psychopathology at the earliest . 
 in the emerging era , where young people have been more exposed to the internet and use online activity as an important form of social interaction . 
 however , it may still remain a matter of debate whether to call internet addiction a distinct disorder by itself or a behavioral problem secondary to another disorder . at present 
 whether or not we will have any such diagnosis included in the future is yet to be seen . in future , 
 if it is added , it is more likely to be classified as an impulse control disorders not elsewhere classified rather than in the diagnostic criteria for substance dependence . by studying the association of internet usage and its effects on human behavior 
 , we can formulate interventions like setting boundaries and detecting early warning signs of underlying psychopathology at the earliest .","background : there has been an explosive growth of internet use not only in india but also worldwide in the last decade . 
 there is a growing concern about whether this is excessive and , if so , whether it amounts to an addiction.aim:to study the prevalence of internet addiction and associated existing psychopathology in adolescent age group.materials and methods : a cross - sectional study sample comprising of 987 students of various faculties across the city of mumbai was conducted after obtaining institutional ethics committee approval and permission from the concerned colleges . 
 students were assessed with a specially constructed semi - structured proforma and the internet addiction test ( iat ; young , 1998 ) which was self - administered by the students after giving them brief instructions . 
 dukes health profile was used to study physical and psychosocial quality of life of students . 
 subjects were classified into moderate users , possible addicts , and addicts for comparison.results:of the 987 adolescents who took part in the study , 681 ( 68.9% ) were female and 306 ( 31.1% ) were males . 
 the mean age of adolescents was 16.82 years . 
 of the total , about 74.5% were moderate ( average ) users . 
 using young 's original criteria , 0.7% were found to be addicts . those with excessive use internet had high scores on anxiety , depression , and anxiety depression.conclusions:in the emerging era of internet use , we must learn to differentiate excessive internet use from addiction and be vigilant about psychopathology .",pubmed
"the frequency response of the resonators was measured using a downmixing method , described in detail in . 
 the device was electrostatically actuated , and the driving voltage was applied to a side - gate parallel to the resonator . to reduce parasitic signals , 
 the drive signal was set to half the actuation frequency 2 , thus the amplitude of motion of the resonator was proportional to the square of the actuation voltage . 
 a bias voltage at (  +  ) through the gauges was used to down - mix their resistance change ( occurring at the actuation frequency  ) , and the low - frequency readout signal at  was detected using a lock - in amplifier . 
 typical measurement values were 1.5 v for the bias voltage at a measurement frequency of 500 khz . 
 all measurements were performed in a vacuum chamber at a pressure of 10 mbar and at room temperature . 
 thermomechanical noise was measured using the same set - up , with the drive electrode disconnected . 
 measurements were taken with a lock - in amplifier , which also generated the drive and bias signals . 
 the allan deviation was measured in open - loop configuration , and the frequency stability was extracted from the response of the resonator actuated at resonance frequency with a fixed driving frequency . 
 the phase of the measured signal , (t ) , was monitored for a certain amount of time , and then transformed into frequency fluctuations using the phase response of the resonator . 
 close to the resonance frequency , this phase response was linear , f2qf0 . using the complete phase response of the resonator instead of this linearization does not significantly alter the allan deviation . 
 harmonics appearing at the frequency of the electricity supply ( multiples of 50 hz ) were filtered out of data during post - processing . using this method , we obtained n samples of the resonance frequency of the resonator f1fn , each averaged over an integration time , 0 . the allan deviation for this integration time could then be defined as : 
 ( 2)a(0)=12(n1)1n1(fi+1fif0)2 to obtain the frequency stability for higher integration times from the same set of frequency samples , we followed the standard method . 
 initial samples were averaged in groups of n samples , and the allan deviation for the new array was calculated using equation ( 2 ) to determine a(n0 ) . 
 this process was repeated multiple times until the number of samples was too low to provide a statistically significant result . 
 correlation measurements were performed by simultaneously measuring the response of the resonator at different frequencies within the resonator s bandwidth . 
 the measurement set - up was based on the one described in supplementary section 3 , but here each signal was doubled , using two drive signals at different frequencies , two bias signals , and two measurement signals ( supplementary figure s9 shows a detailed measurement scheme ) . 
 particular care was taken when choosing the drive signal amplitudes so that the resonator remained in the linear regime . 
 moreover , the two measurement frequencies were chosen to avoid cross - talk ( e.g. 302 khz and 367 khz ) . 
 although here we used a down - mixing set - up , correlation could also be measured with a homodyne method . 
 the phase traces were converted to frequency traces corresponding to the different integration times , as described above . here , the complete phase response of the resonator was used rather than the linear approximation , as the frequencies for phase samples can be quite different from the resonance frequency . with this method we obtained two frequency sample arrays with an integration time 0 . 
 the graph in figure 4b shows the correlation of these frequency traces versus the integration time . we processed the signals so that the correlation for a given  only depends on frequency variations with characteristic time close to . for each  of the plot , we filtered the two frequency traces with a band - pass filter centered on . for a consistent correspondence between allan deviation and correlation integration times , we chose the allan deviation transfer function as the band - pass filter , defined as : 
 ( 3)ha(f)2=2sin4f(f)2 finally , the correlation coefficient of the filtered frequency traces f1 and f2 , each of length n , was defined by : 
 ( 4)corrf1f2=i=1n(f1,if1)(f2,if2)nsf1sf2 
 where f1 and f2 are the sample means of f1 and f2 , respectively , and sf1 and sf2 are their standard deviations . 
 the frequency response of the resonators was measured using a downmixing method , described in detail in . 
 the device was electrostatically actuated , and the driving voltage was applied to a side - gate parallel to the resonator . to reduce parasitic signals , 
 the drive signal was set to half the actuation frequency 2 , thus the amplitude of motion of the resonator was proportional to the square of the actuation voltage . 
 a bias voltage at (  +  ) through the gauges was used to down - mix their resistance change ( occurring at the actuation frequency  ) , and the low - frequency readout signal at  was detected using a lock - in amplifier . 
 typical measurement values were 1.5 v for the bias voltage at a measurement frequency of 500 khz . 
 all measurements were performed in a vacuum chamber at a pressure of 10 mbar and at room temperature . 
 thermomechanical noise was measured using the same set - up , with the drive electrode disconnected . 
 measurements were taken with a lock - in amplifier , which also generated the drive and bias signals . 
 the allan deviation was measured in open - loop configuration , and the frequency stability was extracted from the response of the resonator actuated at resonance frequency with a fixed driving frequency . 
 the phase of the measured signal , (t ) , was monitored for a certain amount of time , and then transformed into frequency fluctuations using the phase response of the resonator . 
 close to the resonance frequency , this phase response was linear , f2qf0 . using the complete phase response of the resonator instead of this linearization does not significantly alter the allan deviation . 
 harmonics appearing at the frequency of the electricity supply ( multiples of 50 hz ) were filtered out of data during post - processing . using this method , we obtained n samples of the resonance frequency of the resonator f1fn , each averaged over an integration time , 0 . the allan deviation for this integration time could then be defined as : 
 ( 2)a(0)=12(n1)1n1(fi+1fif0)2 to obtain the frequency stability for higher integration times from the same set of frequency samples , we followed the standard method . 
 initial samples were averaged in groups of n samples , and the allan deviation for the new array was calculated using equation ( 2 ) to determine a(n0 ) . 
 this process was repeated multiple times until the number of samples was too low to provide a statistically significant result . 
 correlation measurements were performed by simultaneously measuring the response of the resonator at different frequencies within the resonator s bandwidth . the measurement set - up was based on the one described in supplementary section 3 , but here each signal was doubled , using two drive signals at different frequencies , two bias signals , and two measurement signals ( supplementary figure s9 shows a detailed measurement scheme ) . 
 particular care was taken when choosing the drive signal amplitudes so that the resonator remained in the linear regime . 
 moreover , the two measurement frequencies were chosen to avoid cross - talk ( e.g. 302 khz and 367 khz ) . 
 although here we used a down - mixing set - up , correlation could also be measured with a homodyne method . 
 the phase traces were converted to frequency traces corresponding to the different integration times , as described above . here , the complete phase response of the resonator was used rather than the linear approximation , as the frequencies for phase samples can be quite different from the resonance frequency . with this method we obtained two frequency sample arrays with an integration time 0 . 
 the graph in figure 4b shows the correlation of these frequency traces versus the integration time . we processed the signals so that the correlation for a given  only depends on frequency variations with characteristic time close to . for each  of the plot , we filtered the two frequency traces with a band - pass filter centered on . for a consistent correspondence between allan deviation and correlation integration times , we chose the allan deviation transfer function as the band - pass filter , defined as : 
 ( 3)ha(f)2=2sin4f(f)2 finally , the correlation coefficient of the filtered frequency traces f1 and f2 , each of length n , was defined by : 
 ( 4)corrf1f2=i=1n(f1,if1)(f2,if2)nsf1sf2 
 where f1 and f2 are the sample means of f1 and f2 , respectively , and sf1 and sf2 are their standard deviations . 
 ","frequency stability is key to performance of nanoresonators . this stability is thought to reach a limit with the resonator s ability to resolve thermally - induced vibrations . 
 although measurements and predictions of resonator stability usually disregard fluctuations in the mechanical frequency response , these fluctuations have recently attracted considerable theoretical interest . 
 however , their existence is very difficult to demonstrate experimentally . here , through a literature review , 
 we show that all studies of frequency stability report values several orders of magnitude larger than the limit imposed by thermomechanical noise . 
 we studied a monocrystalline silicon nanoresonator at room temperature , and found a similar discrepancy . 
 we propose a new method to show this was due to the presence of frequency fluctuations , of unexpected level . 
 the fluctuations were not due to the instrumentation system , or to any other of the known sources investigated . 
 these results challenge our current understanding of frequency fluctuations and call for a change in practices .",pubmed
"cardiovascular disease ( cvd ) is the leading cause of death in the united states . in 2010 , cvds killed over 700 000 americans , and accounted for 29.4% of total deaths . 
 moreover , cvd accounts for a significant portion of the financial burden of medical care , costing > $ 312.6 billion in healthcare spending and lost productivity in 2010 . 
 aspirin therapy is a prevention measure that can reduce the risk of major cardiovascular events such as heart attack and stroke , and is recommended by the us preventive services task force to prevent heart attack and ischemic stroke . 
 aspirin is used as a primary prevention measure to aid in the prevention of a first occurrence of cvd . 
 it can also be used as a secondary prevention measure among individuals who have experienced a heart attack or stroke to prevent additional cardiovascular events . 
 the american heart association recommends the use of lowdose aspirin daily for people at high risk of a heart attack and regular use of lowdose aspirin for heart attack survivors . 
 a variety of studies have examined whether individuals at risk of having a heart attack in the united states were taking aspirin . however , many of these studies failed to ascertain whether a person was recommended to take aspirin by their physician or whether they were taking it on their own initiative . 
 thus , these studies may be not an accurate assessment of physician recommendations for aspirin use . 
 further , much of the past research has used various risk factors to determine whether people were at high risk of cvd , rather than using the framingham risk score ( frs ) as recommended by the us preventive services task force in 2009 , the 2010 american diabetes association / american heart association / american college of cardiology recommendations , and the 2008 american college of chest physician guidelines . 
 prevention of cvd events is particularly important , and understanding physician recommendations for aspirin therapy is critical for delivering quality health care . 
 our current understanding of who is being encouraged to take aspirin for cvd prevention is limited . 
 we sought to evaluate patient use of aspirin and reported physician recommendations of aspirin therapy for cvd prevention , in a nationally representative sample . 
 we analyzed the national health and nutrition examination survey ( nhanes ) for the years 20112012 . 
 the nhanes samples the noninstitutionalized population of the united states by using a stratified multistage probability sample design . 
 the national center for health statistics uses a multilevel weighting system to account for survey design and nonresponse . 
 the current study is focused on adults 40 years of age and older , because the nhanes asked this sequence of questions about aspirin use only to adults aged 40 and older . 
 the respondents were asked if their doctor recommended that they take lowdose aspirin for prevention of cvd , stroke , or cancer . 
 individuals who reported a recommendation to take aspirin were also asked if they were following this advice . 
 individuals who did not report a recommendation to take aspirin were asked if they were taking lowdose aspirin on their own . for the purposes of this analysis , 
 these individuals were combined with those who were taking aspirin on their physician 's recommendations , in order to get a better understanding of who is taking aspirin . 
 individuals who had never been told by a physician that they had a stroke or heart attack were considered candidates for primary prevention . 
 their eligibility for guidelinerecommended use of aspirin was based on the assessment of their risk for coronary heart disease ( chd ) . 
 high risk for chd was classified as having a 10year chd risk of > 10% according to the frs with no history of heart attack or stroke . 
 low risk for chd was considered a risk of 10% according to the frs with no history of heart attack or stroke as stated in the 2010 american diabetes association / american heart association / american college of cardiology guidelines . 
 the frs utilizes gender , age , total cholesterol , highdensity lipoprotein cholesterol , systolic blood pressure , use of blood pressure medications , and smoking status . for nhanes participants , 
 systolic blood pressure was calculated by averaging 3 readings taken during the medical examination portion of the nhanes . 
 the scores were grouped into 2 categories for this analysis : low risk ( having a 10 year risk of heart attack be < 10% on the framingham risk calculator ) and high risk ( 10% or above ) . for people with diabetes ( defined as having been told by a physician that the respondent had diabetes [ excluding gestational diabetes ] ) , as per the american diabetes association / american heart association / american college of cardiology guidelines , individuals scoring < 5% were considered low risk , and individuals scoring > 10% were considered high risk . 
 individuals with diabetes who had an intermediate risk score between 5% and 10% were removed from the analysis because there is no firm guideline for this group regarding aspirin use from the american diabetes association / american heart association / american college of cardiology guidelines . 
 we did , however , examine whether there were differences in being told to take aspirin and whether they were taking it , and how they compared to low and highrisk diabetes patients . 
 individuals who reported that a doctor had previously told them that they had been diagnosed with a stroke or a heart attack were candidates for secondary prevention . although obesity is not a specific characteristic that would justify a recommendation of aspirin for primary prevention , it is possible that both patients and physicians may interpret the cvd risk inherent in obesity as justifying aspirin . 
 the nhanes 20112012 does not ask questions regarding history of peptic ulcer or other gastrointestinal conditions that could increase risk of bleeding . 
 it does , however , collect information about stomach cancer , a condition for which aspirin use is contraindicated . to control for this , we removed individuals with a history of stomach cancer from the analysis . 
 age was selfreported and divided into 2 categories : 40 to 64 and 65 and older . race 
 / ethnicity was also selfreported and was categorized as nonhispanic whites , nonhispanic blacks , hispanics , and asians / other . 
 education was examined because of its potential impact on selfinitiation of aspirin use . as having access to care 
 may affect whether patients receive appropriate care for ambulatorysensitive conditions including diabetes and cvd , we examined both whether the respondent had health insurance and a regular source of health care . 
 insurance status was defined as whether or not the individual reported having health insurance , of any form , at the time of the interview . 
 regular access to care was defined by whether the respondent reported having a place to regularly go to receive health care . 
 the nhanes uses a stratified multistage probability sample design . to account for this complex design 
 the analysis utilized the nesting capability in sudaan with variables provided in nhanes ( that account for stratum level differences and primary sampling unit differences ) for all analysis , along with the appropriate weight identified by national center for health statistics for the type of data being used in our analysis . 
 utilizing these weights and sampling design variables allows us to account for the complexity of the sampling design in performing univariate analyses ,  tests , and logistic regression models and make population estimates for the noninstitutionalized adult population of the united states . 
 we examined obesity status , demographics , and access to care characteristics of the respondents . 
 we also examined the bivariate relationship between being told to take aspirin / taking aspirin on doctor 's orders / taking aspirin on their own with the individual 's high / low risk for chd based on frs / previous diagnosis of diabetes using  tests to test for significant differences . 
 we examined both primary prevention ( those who have not been told by their physician that they have had either a stroke and/or heart attack ) and secondary prevention ( have been told by their physician that they have had either a heart attack or stroke ) . 
 we examined the bivariate relationship between being told to take aspirin and taking aspirin with risk level using  tests to test for significant differences . 
 we also conducted separate analyses stratified by race using  tests to examine the impact of race on other independent variables as well as on the outcome variables . 
 bivariate estimates for stratifying secondary prevention by heart attack or stroke were examined , but the sample size was too small for stratified estimates to produce reliable estimates . 
 all bivariate calculations used the standard method of pairwise deletion to account for missing data . 
 we computed forcedinclusion logistic regression to examine the relationship of risk status , age , gender , race , education level , obesity status , having insurance , and having a regular source of care on the likelihood of whether patients were told to take aspirin by their physicians . 
 we conducted the regression for the primary prevention sample and again separately for individuals at high and low risk . 
 we were unable to conduct logistic regressions for individual racial / ethnic groups because the sample size was too small to generate reliable estimates . 
 we also conducted tests for multicollinearity of risk status ( based on the frs ) with obesity , age , and gender . 
 specifically , we computed spearman 's correlations as well as examined the tolerance and variance inflation factors of independent variables in the regressions 
 . the frs uses gender and age as variables in its construction , and obesity is a known risk factor for cvd . 
 the respondents were asked if their doctor recommended that they take lowdose aspirin for prevention of cvd , stroke , or cancer . 
 individuals who reported a recommendation to take aspirin were also asked if they were following this advice . 
 individuals who did not report a recommendation to take aspirin were asked if they were taking lowdose aspirin on their own . for the purposes of this analysis , 
 these individuals were combined with those who were taking aspirin on their physician 's recommendations , in order to get a better understanding of who is taking aspirin . 
 individuals who had never been told by a physician that they had a stroke or heart attack were considered candidates for primary prevention . 
 their eligibility for guidelinerecommended use of aspirin was based on the assessment of their risk for coronary heart disease ( chd ) . 
 high risk for chd was classified as having a 10year chd risk of > 10% according to the frs with no history of heart attack or stroke . 
 low risk for chd was considered a risk of 10% according to the frs with no history of heart attack or stroke as stated in the 2010 american diabetes association / american heart association / american college of cardiology guidelines . 
 the frs utilizes gender , age , total cholesterol , highdensity lipoprotein cholesterol , systolic blood pressure , use of blood pressure medications , and smoking status . for nhanes participants , 
 systolic blood pressure was calculated by averaging 3 readings taken during the medical examination portion of the nhanes . 
 the scores were grouped into 2 categories for this analysis : low risk ( having a 10 year risk of heart attack be < 10% on the framingham risk calculator ) and high risk ( 10% or above ) . 
 for people with diabetes ( defined as having been told by a physician that the respondent had diabetes [ excluding gestational diabetes ] ) , as per the american diabetes association / american heart association / american college of cardiology guidelines , individuals scoring < 5% were considered low risk , and individuals scoring > 10% were considered high risk . 
 individuals with diabetes who had an intermediate risk score between 5% and 10% were removed from the analysis because there is no firm guideline for this group regarding aspirin use from the american diabetes association / american heart association / american college of cardiology guidelines . 
 we did , however , examine whether there were differences in being told to take aspirin and whether they were taking it , and how they compared to low and highrisk diabetes patients . 
 individuals who reported that a doctor had previously told them that they had been diagnosed with a stroke or a heart attack were candidates for secondary prevention . 
 although obesity is not a specific characteristic that would justify a recommendation of aspirin for primary prevention , it is possible that both patients and physicians may interpret the cvd risk inherent in obesity as justifying aspirin . 
 the nhanes 20112012 does not ask questions regarding history of peptic ulcer or other gastrointestinal conditions that could increase risk of bleeding . 
 it does , however , collect information about stomach cancer , a condition for which aspirin use is contraindicated . to control for this , we removed individuals with a history of stomach cancer from the analysis . 
 age was selfreported and divided into 2 categories : 40 to 64 and 65 and older . race 
 / ethnicity was also selfreported and was categorized as nonhispanic whites , nonhispanic blacks , hispanics , and asians / other . 
 education was examined because of its potential impact on selfinitiation of aspirin use . as having access to care 
 may affect whether patients receive appropriate care for ambulatorysensitive conditions including diabetes and cvd , we examined both whether the respondent had health insurance and a regular source of health care . 
 insurance status was defined as whether or not the individual reported having health insurance , of any form , at the time of the interview . 
 regular access to care was defined by whether the respondent reported having a place to regularly go to receive health care . 
 the nhanes uses a stratified multistage probability sample design . to account for this complex design 
 the analysis utilized the nesting capability in sudaan with variables provided in nhanes ( that account for stratum level differences and primary sampling unit differences ) for all analysis , along with the appropriate weight identified by national center for health statistics for the type of data being used in our analysis . 
 utilizing these weights and sampling design variables allows us to account for the complexity of the sampling design in performing univariate analyses ,  tests , and logistic regression models and make population estimates for the noninstitutionalized adult population of the united states . 
 we examined obesity status , demographics , and access to care characteristics of the respondents . 
 we also examined the bivariate relationship between being told to take aspirin / taking aspirin on doctor 's orders / taking aspirin on their own with the individual 's high / low risk for chd based on frs / previous diagnosis of diabetes using  tests to test for significant differences . 
 we examined both primary prevention ( those who have not been told by their physician that they have had either a stroke and/or heart attack ) and secondary prevention ( have been told by their physician that they have had either a heart attack or stroke ) . 
 we examined the bivariate relationship between being told to take aspirin and taking aspirin with risk level using  tests to test for significant differences . 
 we also conducted separate analyses stratified by race using  tests to examine the impact of race on other independent variables as well as on the outcome variables . 
 bivariate estimates for stratifying secondary prevention by heart attack or stroke were examined , but the sample size was too small for stratified estimates to produce reliable estimates . 
 all bivariate calculations used the standard method of pairwise deletion to account for missing data . 
 we computed forcedinclusion logistic regression to examine the relationship of risk status , age , gender , race , education level , obesity status , having insurance , and having a regular source of care on the likelihood of whether patients were told to take aspirin by their physicians . 
 we conducted the regression for the primary prevention sample and again separately for individuals at high and low risk . 
 we were unable to conduct logistic regressions for individual racial / ethnic groups because the sample size was too small to generate reliable estimates . 
 we also conducted tests for multicollinearity of risk status ( based on the frs ) with obesity , age , and gender . 
 specifically , we computed spearman 's correlations as well as examined the tolerance and variance inflation factors of independent variables in the regressions 
 . the frs uses gender and age as variables in its construction , and obesity is a known risk factor for cvd . 
 a total of 3435 individuals answered questions about aspirin use , representing 142 677 272 americans . 
 demographic characteristics of the sample , primary prevention group , lowrisk primaryprevention group , and highrisk primaryprevention group are shown in table 1 . 
 characteristics of individuals eligible for primary prevention , individuals at high risk , and individuals at low risk na indicates not applicable . 
 table 1 shows that among the total sample eligible for aspirin as primary prevention , less than a third reported being recommended by their doctor to take aspirin . 
 table 1 also indicates that over three fourths of primaryprevention patients were classified as low risk . 
 less than half of the primaryprevention patients who had a high chd risk level received a recommendation to take aspirin while > one fourth of lowrisk patients were also recommended to take aspirin ( table 1 ) . 
 of those told to take aspirin by their physicians , the vast majority complied with the recommendation . specifically , 77.4% of the total sample , 79.0% of the highrisk patients , and 76.5% of the lowrisk patients reported taking aspirin ( table 1 ) . 
 table 2 shows the bivariate relationships between the demographic variables , health insurance , having a regular place to go for health care , and having a diabetes diagnosis and being told by the physician to take aspirin as primary prevention . 
 analysis examining differences between low , intermediate , and highrisk diabetes showed that among lowrisk diabetes patients , 54.6% were told to take aspirin , compared to 72.8% of intermediaterisk diabetes patients and 56.2% of highrisk diabetes patients ( p = 0.17 ) . 
 bivariate results for patients told to take aspirin , for individuals eligible for primary prevention , individuals at high risk , and individuals at low risk table 3 presents bivariate relationships among respondents who were taking aspirin after having received a recommendation to do so by their physician . in an additional subanalysis 
 , there were no statistically significant differences for taking aspirin for low , intermediate , and highrisk diabetes patients , with 51.5% of lowrisk diabetes patients taking aspirin , 42.2% of intermediaterisk diabetes patients taking aspirin , and 47.1% highrisk diabetes patients taking aspirin ( p = 0.53 ) . 
 bivariate results for taking aspirin among respondents told by their physicians to take aspirin , for individuals eligible for primary prevention there were a number of significant differences in demographic and accesstocare variables by race / ethnic group among primaryprevention patients , as shown in table 4 . 
 bivariate results for demographic differences by race , for individuals eligible for primary prevention of the 8.3% of the total sample eligible for secondary prevention , 75.9% were told to take aspirin by their physician . of those who were told by their physicians to take aspirin , 
 table 5 shows the results of 3 logistic regression models for being told to take aspirin among individuals eligible for primary prevention , only individuals at high risk , and only individuals at low risk . 
 the logistic regression examining all patients without a history of heart attack or stroke that included risk level as an independent variable indicated that the frs was not predictive of receiving a physician recommendation for aspirin . among all primaryprevention patients , education level , insurance status , and having a regular source of care were predictive of receiving a recommendation for aspirin for cvd prevention . among highrisk individuals , age , health insurance , and being of asian / other descent were predictive of receiving a recommendation for aspirin for cvd prevention . among lowrisk individuals , age , health insurance status , education level , and obesity status were all predictive of receiving a recommendation for aspirin therapy . 
 logistic regression results predicting aspirin recommendation for individuals eligible for primary prevention , individuals at high risk , and individuals at low risk or indicates odds ratio . 
 table 1 shows that among the total sample eligible for aspirin as primary prevention , less than a third reported being recommended by their doctor to take aspirin . 
 table 1 also indicates that over three fourths of primaryprevention patients were classified as low risk . 
 less than half of the primaryprevention patients who had a high chd risk level received a recommendation to take aspirin while > one fourth of lowrisk patients were also recommended to take aspirin ( table 1 ) . of those told to take aspirin by their physicians , the vast majority complied with the recommendation . 
 specifically , 77.4% of the total sample , 79.0% of the highrisk patients , and 76.5% of the lowrisk patients reported taking aspirin ( table 1 ) . 
 table 2 shows the bivariate relationships between the demographic variables , health insurance , having a regular place to go for health care , and having a diabetes diagnosis and being told by the physician to take aspirin as primary prevention . 
 analysis examining differences between low , intermediate , and highrisk diabetes showed that among lowrisk diabetes patients , 54.6% were told to take aspirin , compared to 72.8% of intermediaterisk diabetes patients and 56.2% of highrisk diabetes patients ( p = 0.17 ) . 
 bivariate results for patients told to take aspirin , for individuals eligible for primary prevention , individuals at high risk , and individuals at low risk 
 table 3 presents bivariate relationships among respondents who were taking aspirin after having received a recommendation to do so by their physician . in an additional subanalysis 
 , there were no statistically significant differences for taking aspirin for low , intermediate , and highrisk diabetes patients , with 51.5% of lowrisk diabetes patients taking aspirin , 42.2% of intermediaterisk diabetes patients taking aspirin , and 47.1% highrisk diabetes patients taking aspirin ( p = 0.53 ) . 
 bivariate results for taking aspirin among respondents told by their physicians to take aspirin , for individuals eligible for primary prevention 
 there were a number of significant differences in demographic and accesstocare variables by race / ethnic group among primaryprevention patients , as shown in table 4 . 
 of the 8.3% of the total sample eligible for secondary prevention , 75.9% were told to take aspirin by their physician . 
 of those who were told by their physicians to take aspirin , 89.9% were taking aspirin . 
 table 5 shows the results of 3 logistic regression models for being told to take aspirin among individuals eligible for primary prevention , only individuals at high risk , and only individuals at low risk . 
 the logistic regression examining all patients without a history of heart attack or stroke that included risk level as an independent variable indicated that the frs was not predictive of receiving a physician recommendation for aspirin . among all primaryprevention patients , education level , insurance status , and having a regular source of care were predictive of receiving a recommendation for aspirin for cvd prevention . among highrisk individuals , age , health insurance , and being of asian / other descent were predictive of receiving a recommendation for aspirin for cvd prevention . among lowrisk individuals , age , health insurance status , education level , and obesity status were all predictive of receiving a recommendation for aspirin therapy . 
 logistic regression results predicting aspirin recommendation for individuals eligible for primary prevention , individuals at high risk , and individuals at low risk or indicates odds ratio . 
 the results of this study indicate that recommendations for aspirin for cvd prevention do not match up to the objectively computed future risk . for individuals at high risk , fewer than half report receiving recommendations for aspirin . 
 moreover , in a multivariate analysis , objective risk level was not associated with an increased risk of recommendation for aspirin . 
 this suggests that decisions for treatment recommendations seem to be being made on criteria other than objectively calculated future chd risk . 
 only 40.9% of patients at high risk reported having been told by their physician to take aspirin . 
 patient reports of recommendations for aspirin use among secondary prevention patients was 75.9% , but still suboptimal . 
 respondents who were at low risk of developing cvd and had body mass indexes over 30 were more likely to be told to take aspirin for primary prevention than normalweight patients . 
 the benefit of using aspirin in lowrisk patients who do not have clinical cvd is negated by the risk of harms from gastrointestinal bleeding . 
 it is possible that the seemingly suboptimal rates of recommendations to take lowdose aspirin are the result of the provider weighing the benefits of aspirin against the very real risks of upper gastrointestinal bleeding . 
 lin et al have shown that the risk of upper gastrointestinal bleeding is 90% higher among lowdose aspirin users who are using aspirin for primary prevention compared to nonusers and 40% higher for secondaryprevention patients . 
 the number needed to harm per 1 year of lowdose aspirin use was 601 for primaryprevention patients and 391 for secondaryprevention patients . 
 patients with a history of upper gastrointestinal bleeding and peptic ulcers also have a higher incidence of upper gastrointestinal bleeding , as do patients with concomitant use of clopidogrel or nonsteroidal antiinflammatory drugs . 
 all of these factors may have contributed to the lack of recommendations for aspirin use to highrisk patients . 
 first , nhanes does not account for hemorrhagic stroke ( 13% of all strokes ) and aspirin use would be inappropriate for these patients . 
 second , secondaryprevention patients could be taking other blood thinners that would obviate the need for lowdose aspirin . in order to better understand physician prescribing practices of aspirin and patient usage of aspirin , additional research of aspirin use in the context of other antiplatelet and anticoagulant use 
 the apparent disconnect between risk for future cvd and aspirin recommendations may benefit from improved clinical decision support . helping to compute 
 the actual risk rather than simply identifying general risk factors should aid in making accurate decisions . 
 actual risk could be computed in electronic health records , which could also provide the physician with the recommendation for treatment . 
 in addition , such tools could provide other affiliated treatment recommendations such as use of gastricprotective agents in select patient populations . 
 in addition to a significant disconnect between who needs and who receives recommendations for aspirin use , the analysis indicated a persistent problem with access to care and receipt of recommendations for aspirin use . 
 lack of health insurance can be a significant barrier to accessing health care , especially for preventive services . 
 previous research has found no relationship between insurance status and aspirin use among patients who accessed clinical preventive services . 
 these results suggest that access to care may play a role in recommendations for the advice of aspirin for cvd prevention , and the use of it by patients 
 . the present results may differ from previous studies in that the previous studies did not assess whether the provider recommended aspirin for cvd prevention . 
 race and ethnicity did have an impact among primaryprevention patients , with nonhispanic whites more likely to receive a recommendation for aspirin than were asians / others . 
 additional analyses indicated that among primaryprevention patients , a greater percentage of nonhispanic whites had health insurance compared with other racial / ethnic groups . 
 as with curative medical care , the ability to access preventive medical care is in large part influenced by insurance status . 
 therefore , part of the racial / ethnic disparities in primary prevention that we observed are possibly attributable to lack of health insurance and a usual source of care . that a higher level of education for primaryprevention and lowrisk patients was predictive of lower likelihood of receiving a recommendation for aspirin was somewhat surprising , particularly that the finding emerged after controlling for race , age , gender , obesity , insurance status , and access to a regular source of care . 
 it is possible that physicians may perceive more highly educated people as having a lower risk of heart disease , based on the known connection between educational attainment and health outcomes . 
 this investigation relies on selfreport of whether or not an individual 's doctor recommended aspirin as prevention of heart attack , stroke , and cancer , as well as selfreport of a number of variables for analysis . 
 selfreported data are very common in studies of health behaviors , but there are questions regarding the reliability of the data . 
 patient selfreport can be unreliable , especially regarding behaviors and risk factors relevant to cvd and cancer . 
 the national center for health statistics conducts rigorous testing of its survey instruments to ensure validity through the questionnaire design research laboratory , ensuring that all questions utilized are of high reliability and validity . 
 in addition , lab and physical examination values were used when possible to minimize bias introduced by selfreport . 
 an additional limitation is that the study only examines adults age 40 and over , as this is the only group that nhanes asks about aspirin use 
 . there could be important patterns of use among younger individuals that this analysis can not look for . 
 another limitation of this study is that we do not know how many respondents were recommended to take aspirin for heart attack or stroke prevention versus for cancer prevention . 
 thus , it is possible that some proportion of aspirin recommendations may be for cancer prevention , rather than heart attack or stroke prevention . 
 however , the us preventive services task force currently recommends against the use aspirin for the prevention of cancer , thereby suggesting that recommendations to take aspirin based on cancer prevention should be unlikely . 
 in addition , this study did not control for use of warfarin or other anticoagulant / antiplatelet therapy among respondents . 
 anticoagulant use could explain some of the deficit in the use and recommendations to take aspirin among primaryprevention patients . finally , 
 because of limitations in what information is collected by the nhanes , we do not know what role the risk of gastrointestinal bleeding played in the decision of physicians to recommend aspirin . 
 gastrointestinal bleeding is a significant issue that we would expect to have an impact on the likelihood of a physician recommending aspirin use for patients who may otherwise benefit from it . 
 the available data from nhanes do not allow us to elucidate who is at greater risk for gastrointestinal bleeding and for whom a recommendation to take aspirin would be inappropriate . 
 highrisk patients who are not receiving the recommendation are put at increased risk of cvd events . 
 lowrisk patients are exposed to adverse risk with unnecessary use of aspirin based upon a physician 's recommendation . 
 it is clear that quality of care for primary prevention falls far short of ideal . 
 further research on clinical decision support systems and interventions designed to reinforce the use of appropriate risk calculation is necessary to ensure that patients receive appropriate preventive care .","backgroundaspirin use has been shown to be an effective tool in cardiovascular disease ( cvd ) prevention among highrisk patients . 
 the patientreported physician recommendation for aspirin as preventive therapy among high and lowrisk patients is unknown.methods and resultswe conducted an analysis of the national health and nutrition examination survey 20112012 to examine the use of aspirin for cvd prevention . 
 patients without previously diagnosed cvd were classified into high and low risk based on their framingham risk score ( 10year coronary heart disease risk ) . among patients without previously diagnosed cvd , 
 22.5% were classified as high risk . of the highrisk individuals , 
 40.9% reported being told by their physician to take aspirin , with 79.0% complying . among those who were at low risk , 
 26.0% were told by their physician to take aspirin , with 76.5% complying . 
 logistic regression analysis indicated that age , access to a regular source of care , education , and insurance status were significant predictors of patientreported physician recommendations for aspirin use for primary prevention . among highrisk patients , age , race , and insurance status were significant predictors of reported recommendations for aspirin use . among lowrisk patients , age , education , obesity , and insurance status were significant predictors of reported recommendations for aspirin use.conclusionspatient reports indicate nonideal rates of being told to take aspirin , for both high and lowrisk patients for primary prevention . 
 clinical decision support tools that could assist physicians in identifying patients at risk may increase patient reports of physician recommendations for aspirin use .",pubmed
"mitochondria produce reactive oxygen species ( ros ) during oxidative metabolism through the single - electron reduction of molecular oxygen ( o2 ) . 
 the resulting superoxide anion ( o2 ) , is converted to hydrogen peroxide ( h2o2 ) by cellular superoxide dismutases ( sods ) . 
 similar to phosphorylation , ubiquitination , and acetylation , oxidation of protein cysteine residues represents a form of reversible , posttranslational protein modification which can regulate signaling . 
 oxidation of cysteine thiol groups ( sh ) by h2o2 results in formation of sulphenic acid ( soh ) which quickly forms disulfide ( ss ) or sulfenyl amide ( sn ) bonds , changing protein function . 
 the cellular thioredoxin and glutathione reductase systems restore these oxidatively modified residues back to their reduced forms in a manner analogous to phosphatases , deubiquitinating enzymes , or histone deacetylases . 
 mitochondrial ros ( mros ) generation is required for the propagation of numerous cellular signaling pathways including those regulating tumorgenesis , immune responses , and cellular adaptation to stresses such as hypoxia . in most cases 
 it is unknown exactly what oxidative events are required for the effects of ros on these pathways and it is likely that multiple oxidative targets potentiate transduction through a given pathway . 
 increasing evidence suggests that mitochondria and mros generation play key roles in cellular differentiation programs . 
 cellular mitochondrial content and oxidative capacity are increased when mouse embryonic stem cells ( escs ) , induced pluripotent stem cells ( ipscs ) , or mesenchymal stem cells are induced to differentiate . 
 furthermore , increased cellular ros levels and oxidation products correlate with the differentiation of escs and ipscs as well as mesenchymal , neural , and epithelial stem cells . 
 hematopoietic stem cells ( hscs ) contain significantly lower levels of ros than do the more - differentiated common myeloid progenitor cells . 
 thus increased mitochondrial and oxidant content appears to correlate with stem cell differentiation while low mitochondrial mass and oxidant content correlates with stem cell maintenance . in our recent report 
 , we used the mammalian epidermis as a model system to test the hypothesis that mitochondrial metabolism and ros production promote signaling through pathways required for differentiation of mammalian stem cell populations in vivo . 
 the epidermis is a self - renewing stratified squamous epithelium and is thus regulated by stem cell populations . to maintain epidermal homeostasis , cells within the proliferative basal layer 
 withdraw from the cell cycle and differentiate as they process outward through the suprabasal layers , compensating for cellular loss via desquamanation from the outermost epidermal layer . 
 the epidermis also elaborates appendages such as hair follicles ( hfs ) which are themselves regulated by stem cell populations . 
 multiple transcriptional networks are associated with differentiation within the epidermis including notch , p63 , c / ebp , and ap2 ( interfollicular epidermis ) and -catenin ( hf ) . 
 it is incompletely understood how these various factors are regulated to promote epidermal homeostasis and what role cellular metabolism might play in this regulation . to test the hypothesis that mitochondria play an active role in the regulation of epidermal homeostasis , 
 we conditionally deleted tfam ( transcription factor a , mitochondrial ) in basal , undifferentiated epidermal keratinocytes . tfam is required for the replication and transcription of the mitochondrial genome , and cells lacking tfam are unable to conduct oxidative phosphorylation or produce mros . 
 mice conditionally lacking tfam in epidermis ( tfam cko mice ) lacked hair and developed an epidermal barrier defect which contributed to their perinatal mortality . 
 similar to in vivo , primary keratinocytes derived from tfam cko mice displayed impaired differentiation in vitro . 
 differentiation of wild - type keratinocytes was inhibited by antioxidant treatment , and differentiation marker expression in tfam cko cells could be partly restored by treatment with exogenously applied h2o2 , clearly demonstrating that oxidative signaling promotes keratinocyte differentiation . 
 we went on to demonstrate that mros generation is required for activation of the notch and -catenin transcriptional programs that promote epidermal differentiation and hair growth respectively . 
 together , our results show that mros act as pro - differentiation signals and are key upstream regulators of stem cell fate decisions . 
 while the role for mros in signal transduction is becoming increasingly accepted , the identities of the exact targets of oxidation which promote signaling remain unknown in most situations . 
 our work identified nucleoredoxin ( nxn ) as a putative target of mros - mediated wnt--catenin signaling . 
 nxn is a member of the thioredoxin family which has previously been demonstrated to regulate -catenin - dependent transcription in a redox - sensitive manner . 
 our results show that nxn becomes oxidized when wild - type keratinocytes are treated with the -catenin activator wnt-3a . 
 this did not occur in tfam cko keratinocytes , suggesting that mros - mediated nxn oxidation plays a causal role in the transduction of signals between wnt receptors and -catenin . 
 further studies will be required to determine how mros regulate notch transcriptional activity during keratinocyte differentiation . 
 this raises the interesting possibility that aberrant keratinocyte metabolism may be associated with , and presents targets for , the treatment of epidermal disease . 
 while the genetic causes of these diseases are increasingly studied , little is known about how disease - causing mutations might affect cellular metabolism . 
 more generally , the interactions between cellular differentiation programs and cellular metabolism will be increasingly studied in years to come . 
 the development of regenerative therapies depends on the ability to maximize the renewal capacity of stem cells , and subsequently , to promote complete differentiation into desired cell types . 
 cellular metabolism represents an intriguing  rheostat  which may be used to modulate these ends . 
 a further understanding of the mechanisms by which differentiation programs both regulate , and are regulated by metabolism will prove useful in this regard . 
 proper regulation is likely dependent on cell type - specific factors as well as environmental influences such as hypoxia . in keratinocytes , 
 as an epidermal calcium gradient exists in vivo , it is hypothesized that calcium concentrations regulate epidermal differentiation in vivo as well . 
 calcium uptake into mitochondria is known to stimulate the activity of tca cycle enzymes ( pyruvate- , isocitrate- , and oxoglutarate - dehydrogenases ) , promoting mitochondrial respiration . 
 we found that inhibition of mitochondrial calcium uptake prevented keratinocyte differentiation in vitro , suggesting that mitochondrial calcium uptake might promote the mitochondrial metabolism and ros production required for epidermal differentiation . 
 a recent study suggests that mitochondrial uncoupling proteins may play a role in regulating differentiation - dependent metabolism . 
 uncoupling protein 2 ( ucp2 ) is highly expressed in ipscs , with reduced expression in differentiated cells . 
 ectopic ucp2 expression inhibited oxidative metabolism of pyruvate , reduced cellular ros content , and prevented differentiation suggesting that this protein could act as a metabolic switch that controls stem cell differentiation . 
 how ucp2 expression is repressed during differentiation remains to be determined and it will also be of interest to determine if ucp2 regulates the metabolism of less - pluripotent , tissue - specific stem cell populations . beyond promotion of oxidant - dependent 
 signaling , the mechanisms by which mitochondrial metabolism promotes cellular differentiation remain poorly understood . some differentiated lineages may require the efficient atp production provided by oxidative phosphorylation ; however , another intriguing possibility involves the nutrient - dependent regulation of chromatin ( fig . 
 acetyl - coa , a key fuel for oxidative metabolism , is also a substrate used by histone acetyltransferases to acetylate histones and other cellular proteins . 
 global increases in histone acetylation are associated with differentiation of escs , 3t3-l1 preadipocytes , and keratinocytes . 
 treatment of primary human keratinocytes with histone deacetylase inhibitors is sufficient to induce cell cycle arrest and expression of several differentiation markers . 
 although differentiation programs likely regulate histone acetylation in a gene and time - dependent manner ; these reports suggest that cellular differentiation may depend on availability of acetyl - coa to carry out acetylation reactions . in mammalian cells , 
 most cytosolic acetyl - coa is produced through the cleavage of tca - cycle - derived citrate by the enzyme atp - citrate lyase ( acl ) . 
 inhibition of acl expression prevents global increases in histone acetylation and differentiation of 3t3-l1 cells . 
 electrons are transferred from tca cycle substrates to complexes i and ii of the electron transport chain via nadh and fadh2 respectively . 
 these electrons are transferred to complex iii ( via coenzyme q ) and subsequently to complex iv ( via cytochrome c ) where they are used to reduce molecular oxygen to water . 
 complexes i , ii , and iii of the respiratory chain contain sites in which electrons can prematurely react with oxygen , producing ros . 
 our recent report demonstrates that mitochondrial ros production is required for activation of notch and -catenin transcriptional programs which promote epidermal differentiation and hair development respectively . 
 mitochondrial metabolism may also promote keratinocyte differentiation by providing substrates for histone acetyltransferases ( hats ) and histone demethylases ( such as jmjd3 ) . 
 histone methylation status is regulated by histone methyltransferases and demethylases with various methylation  codes  associated with either transcriptional activation or repression . one particular methyl modification 
 trimethylation of histone 3 on lysine 27 ( h3k27me3)is associated with transcriptional repression of epidermal differentiation markers . 
 genes such as keratin 1 and involucrin display high levels of h3k27me3 in undifferentiated keratinocytes , and lose this mark as cells differentiate . 
 demethylation of h3k27me3 requires the demethylase jmjd3 and cells lacking jmjd3 expression are unable to differentiate in culture . 
 jmjd3 is a member of the jumanji - c domain containing family of demethylases which use oxygen , fe(ii ) , and the tca cycle intermediate -ketoglutarate as cofactors and are thus potentially sensitive to cellular metabolic state . 
 many other nutrient - sensitive mechanisms are likely to regulate stem cell maintenance and differentiation in both the epidermis and in other compartments . 
 cancer researchers are currently striving to identify cancer - specific metabolic signatures which can be therapeutically targeted . 
 metabolic study of cellular differentiation is an emerging area which , in concert with genetic approaches , has the potential to lead to strategies for targeted control of cell fate decisions and therapies for diseases associated with aging .","mitochondrial metabolism has traditionally been thought of as a source of cellular energy in the form of atp . 
 the recent renaissance in the study of cellular metabolism , particularly in the cancer field , has highlighted the fact that mitochondria are also critical biosynthetic and signaling hubs , making these organelles key governors of cellular outcomes.1 - 5 using the epidermis as a model system , our recent study looked into the role that mitochondrial metabolism and ros production play in cellular differentiation in vivo.6 we showed that conditional deletion of the mitochondrial transcription factor , tfam within the basal cells of the epidermis results in loss of mitochondrial ros production and impairs epidermal differentiation and hair growth . 
 we demonstrated that mitochondrial ros generation is required for the propagation of notch and -catenin signals which promote epidermal differentiation and hair follicle development respectively . 
 this study bolsters accumulating evidence that oxidative mitochondrial metabolism plays a causal role in cellular differentiation programs . 
 it also provides insights into the role that mitochondrial oxidative signaling plays in a cell type - dependent manner .",pubmed
"hepatitis c virus ( hcv ) is a single - stranded rna virus , a member of the flaviviridae family . since the first identification of the hcv genome by choo et al . 
 , hcv study has progressed mainly in the field of hcv functional analysis and therapeutic implications . 
 because of low viral levels in the serum , the diagnosis of hcv infection has been made with a branched chain dna signal amplification assay and reverse - transcription polymerase chain reaction ( rt - pcr ) [ 24 ] . 
 pathologists are often requested to detect viral pathogens within the routinely prepared biopsy tissue samples , and therefore simple and reliable histochemical techniques are needed . 
 reports have described the detection of hcv in human liver tissue by using immunohistochemistry and in situ hybridization ( ish ) [ 414 ] , as well as by in situ rt - pcr [ 4 , 6 , 1520 ] . at the moment , hcv detection with immunohistochemistry and ish is not yet reproducible enough , we believe . in situ rt - pcr 
 reliable histochemical techniques for detecting hcv in formalin - fixed , paraffin - embedded ( ffpe ) liver tissues are needed , particularly for routine diagnostic purpose . in the present study 
 , we established an ish technique using a locked nucleic acid ( lna ) probe and biotin - free tyramide amplification system ( csaii ) for detecting hcv - rna in ffpe tissue sections of the liver . 
 as positive controls , we sampled the humanized liver of hcv - infected chimeric mice [ 21 , 22 ] , and ffpe tissue sections were prepared . 
 the chimeric mice were maintained by m. kohara , department of microbiology and cell biology , tokyo metropolitan institute of medical science , tokyo . 
 a total of 13 humanized liver specimens from hcv - infected chimeric mice were used ( noninfected 3 , genotype 1a - infected 3 , genotype 1b - infected 4 , and genotype 2a - infected 3 ) . 
 a total of 11 needle biopsy liver specimens of chronic hepatitis c were culled from the computer file at the diagnostic pathology division of fujita health university hospital , toyoake , japan , and ffpe sections were cut . 
 needle biopsy specimens of hepatitis - b - virus- ( hbv-)infected chronic hepatitis , virus - negative fatty liver and autoimmune hepatitis ( n = 1 , resp . ) , as well as two surgically removed hcc lesions caused by hcv or hbv infection , were also examined . 
 the nonneoplastic part of the liver tissue ( chronic hepatitis b ) in the hbv - related hcc case was also evaluated . 
 non - neoplastic liver tissue was scarcely included in the hcv - related hcc case . 
 the activity of inflammation and the degree of fibrosis were evaluated according to the new inuyama classification ( 1996 ) . 
 serum hcv - rna levels in the patients were measured using an rt - pcr assay , the amplicor hcv monitor assay ( roche diagnostics ) , and serum hcv subtypes were determined with nested rt - pcr ( roche diagnostics ) . the sequence of an hcv - common probe was designed according to the previous description . 
 the target was within the hcv 5-untranslated region ( hcv-5utr ) , which is a highly conserved portion in the hcv genome . 
 an lna - modified oligonucleotide probe labeled with digoxygenin at the 3-end was prepared in gene design inc . , 
 the nucleotide sequence of the 45-mer probe was 5-altttlggglcgtlgcclccclgcglagalctglctalgcclgagltagltgtltgglgt-3 , in which la , lt , lc , and lg represent lna monomers corresponding to the bases a , t , c , and g , respectively . 
 endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol for 60 minutes at room temperature ( rt ) . 
 after rinsing thrice in diethyl - pyrocarbonate- ( depc-)treated water , sections were digested with 40  g / ml proteinase k ( roche diagnostics , tokyo , japan ) at 37c for 15 minutes , washed thrice in depc - treated water , submerged in 95% ethanol for 1 minute , and air - dried completely . 
 then , sections were heat - treated for 5 minutes at 95c on a hot plate and hybridized with 0.01 
 mol / l lna - modified oligonucleotide probe diluted with in situ hybridization buffer ( enzo life science , bulter pike , pa , usa ) in an incubation chamber overnight at 37c . after rinsing in 1x saline sodium citrate ( ssc ) for 30 minutes at 50c 
 , sections were rinsed twice in 50  mm tris - hcl - buffered saline ( tbs ) , ph 7.6 , at rt . a horseradish - peroxidase - labeled antidigoxygenin antibody ( roche diagnostics ) diluted at 1  :  100 with 1% bovine serum albumin in tbs was applied for 60 minutes at rt . 
 for amplifying signals , fluorescein - isothiocyanate- ( fitc-)conjugated tyramide enclosed in csaii ( dako , glostrup , denmark ) was applied to the sections for 15 minutes at rt . 
 finally , a horseradish - peroxidase - labeled anti - fitc antibody equipped in the csaii kit was reacted for 30 minutes at rt . 
 reaction products were visualized in 50  mm tris - hcl buffer , ph 7.6 , containing 20  mg / dl diaminobenzidine tetrahydrochloride and 0.006% hydrogen peroxidase . 
 the nuclei were lightly counterstained with mayer 's hematoxylin . in order to verify the specificity of the ish technique for hcv - rna detection 
 , we carried out the treatment with dnase and rnase before the hybridization with the lna probe on consecutive sections . 
 namely , either 10  iu / ml dnase i ( wako , osaka , japan ) or 100  g / ml rnase ( sigma - aldrich , st . louis , mo , usa ) was incubated for 30 minutes at 37c . in order to distinguish mouse hepatocytes from chimeric human hepatocytes in hcv - infected chimeric mice 
 , we utilized a human - specific ck8/18 monoclonal antibody ( clone : ncl 5d3 , mp biochemicals , santa ana , ca , usa ) . after blocking endogenous peroxidase activity , 
 hydrated heat - assisted epitope retrieval was employed using a pressure pan cooker ( delicio 6l , t - fal , clichy , france ) for 10 minutes . 
 preliminary study revealed that as a soaking solution , 1  mm ethylenediamine tetraacetic acid solution , ph 8.0 , was optimal to retrieve the antigenicity . 
 after pressure cooking , sections were left for 30 minutes at rt for cooling . in order for avoiding nonspecific signals in mouse hepatocytes , a mouse stain kit ( nichirei bioscience , tokyo ) 
 was applied before and after the primary antibody incubation . the human - specific ck8/18 monoclonal antibody at a 1  :  100 dilution was incubated overnight at rt . 
 after rinsing in 10  mm phosphate - buffered saline ( pbs ) , ph 7.2 , the sections were reacted with a secondary polymer reagent , novolink ( novocastra , newcastle , uk ) . 
 total rna was extracted from dewaxed sections using recoverall total nucleic acid isolation kit ( applied biosytems , austin , tx , usa ) , according to the manufacturer 's protocol , and stored at 80c until use . 
 the present study was approved by the institutional ethical review board for clinical and epidemiological investigations at fujita health university , toyoake . 
 in he - stained sections , mouse hepatocytes showed densely eosinophilic cytoplasm , while chimeric human hepatocytes were weakly eosinophilic with frequent deposition of fat droplets . 
 immunoreactivity of human ck8/18 was seen only in human hepatocytes . in all the 10 hcv - infected livers but not the livers of three hcv noninfected mice , ish demonstrated diffuse cytoplasmic signals in the human hepatocytes . in figure 1 employing consecutive sections , 
 hcv - rna was visualized with the ish technique in human ck8/18-positive chimeric hepatocytes from chimeric mice infected hcv genotypes 1a , 1b , and 2a ( in figure 1 , dotted lines encircle the mouse hepatocyte area ) . 
 it is of note that hcv - rna was demonstrated in the cytoplasm of almost all chimeric human hepatocytes . 
 the positive signals remained after dnase treatment but were completely abolished after rnase treatment . 
 figure 2 illustrates hcv genotype 1b - infected chimeric mouse liver , showing he histology and positive cytoplasmic signals , which were resistant to dnase but sensitive to rnase . 
 most of the hcv - positive patients showed high levels of hcv - rna in the serum . 
 exceptionally , a surgical case of hcv - related hcc showed a low level of hcv - rna in the serum . 
 nested rt - pcr genotyping for hcv using total rna extracted from ffpe sections was successful in 8 ( 73% ) of 11 biopsied chronic hepatitis c lesions , and in all the 8 cases , the genotypes were comparable with the serum analysis . 
 lna - based ish detected signals in 9 ( 82% ) of 11 chronic hepatitis c lesions , including three showing diffuse cytoplasmic positivity , and six showing focal or partial positivity . in three nested rt - pcr negative lesions , ish revealed negativity ( 1 lesion ) or focal / partial positivity ( 2 lesions ) . 
 no hcv - rna was detected in hbv - related lesions , fatty liver , autoimmune hepatitis , and in hcc lesions . 
 nonspecific binding of the csaii reagent to kupffer cells or lymphoid cells was occasionally observed in both the hcv - infected and hcv - unrelated lesions , as arrows indicate . 
 we showed herein that lna - based ish for hcv - rna yielded clear cytoplasmic reactivity in infected hepatocytes in ffpe sections . 
 the lna - modified oligonucleotide probe we employed represents one of the most sensitive probes for ish [ 2529 ] . 
 lna - based ish has been applied to detecting cellular microrna in ffpe tumor tissues [ 3032 ] . 
 the 45-mer lna probe was targeted at the hcv-5utr containing a sequence common to the hcv subtypes . 
 positive signals for hcv - rna were clearly demonstrated in hcv 1a- , 1b- , and 2a - infected humanized livers of chimeric mice . 
 hcv - infected chimeric mice with humanized liver thus functioned as a useful model for histochemically demonstrating the hcv genome , particularly when immunostaining for human - specific ck8/18 was combined . in needle - biopsied human liver specimens , 
 hcv - rna was detected in 8 of 11 hcv - infected samples with the nested rt - pcr analysis and in 9 of 11 with the ish technique . in nested rt - pcr negative cases , 
 ish was negative or partially positive , suggesting that the detection threshold may depend on the virus load . 
 hcv - rna was undetectable in the hepatocytes in hbv - infected livers , nonviral liver disorders ( fatty liver and autoimmune hepatitis ) , and in hcv - related hcc . 
 with ish using a digoxygenin - labeled cdna probe , tang et al . reported positivity in 8 ( 67% ) of 12 hcv - related hcc . 
 it has been documented with an in situ rt - pcr technique that hcv - rna decreases along with the progression from liver cirrhosis to hcc . 
 more cases of hcc need to be analyzed , in relation to the distribution of the hcv genome in cancerous lesions . 
 revie and salahuddin reviewed that hcv replicated in macrophages , b and t lymphocytes , and other nonhepatocellular components . 
 however , we judged the nonhepatocellular bindings as nonspecific reactions , primarily because of the staining in non - hcv cases . under our present condition , nonspecific binding of the reagents , particularly csaii , to kupffer cells and lymphoid cells 
 further technical improvement is requested for reliable and reproducible hcv - rna detection in ffpe specimens . in conclusion , 
 the ish technique using the lna - modified oligonucleotide probe and csaii can be applied to detecting hcv - rna in liver biopsy specimens , the sensitivity probably being comparable to in situ rt - pcr . for detecting the hcv genome in routinely prepared liver biopsy specimens , 
 our ish sequence is relatively simple and requires no special equipment to perform . at the moment 
 , this method seems to be suitable for demonstrating hcv genome in ffpe sections and will become a valuable tool for routine histopathological diagnosis of hcv infection using ffpe liver biopsy specimens .","background . in situ hybridization ( ish ) with high sensitivity has been requested to demonstrate hepatitis c virus ( hcv ) rna in formalin - fixed , paraffin - embedded ( ffpe ) sections of the liver . 
 methods . 
 ish employing a locked - nucleic - acid- ( lna-)modified oligonucleotide probe and biotin - free catalyzed signal amplification system ( csaii ) was applied to hcv - rna detection in the liver tissue . 
 nested reverse - transcription polymerase chain reaction ( rt - pcr ) was performed for hcv genotyping using total rna extracted from ffpe sections . 
 the target tissues included ffpe tissue sections of humanized livers in hcv - infected chimeric mice ( hcv genotypes 1a , 1b , and 2a and noninfected ) and of needle - biopsied livers from hcv - infected patients . results . 
 hcv - rna was demonstrated with the ish technique in hcv - infected liver tissues from both chimeric mice and 9 ( 82% ) of 11 patients with hcv infection . 
 the hcv signals were sensitive to rnase . 
 nested rt - pcr confirmed the genotype in 8 ( 73% ) of 11 livers ( type 1b : 6 lesions and type 2a : 2 lesions ) . 
 hcv - rna was not identified in chronic hepatitis b lesions , fatty liver , autoimmune hepatitis , and hepatocellular carcinoma . 
 conclusion . 
 ish using the lna - modified oligonucleotide probe and csaii was applicable to detecting hcv - rna in routinely prepared ffpe liver specimens .",pubmed
"there are many concerns about endocrine - disrupting chemical effects on wild animals as well as humans 
 . considerable numbers of chemicals have been shown to alter the thyroid system in vertebrates , and disruption of the thyroid axis has been identified as an important consideration for the regulation of chemicals . 
 thus , focusing on thyroid toxicity , the oecd and epa established guidelines for investigation of frog metamorphosis . 
 because amphibian tadpoles have a thin , permeable skin and inhabit aquatic environments , they are exposed to toxicants through both dermal and dietary routes and appear to be particularly susceptible to a number of contaminants in wastewater discharged from agricultural fields and from industrial and household areas . in addition , the fact that frog metamorphosis is regulated by thyroid hormones ( ths ) , promoting the remodeling of the aquatic larvae into an adult tetrapod , means that the dramatic structural and functional changes of larval tissues can be readily applied as parameters reflecting endocrine disruption . 
 1 ) , show various morphological changes during metamorphosis and under conditions of exposure to certain compounds . here 
 , the regulation by ths , focusing on affecting factors in addition to the metamorphosis assay , is reviewed . 
 the thyroid gland secretes thyroxine ( t4 ) , which is converted to a more biologically active form , 3,3,5-triiodothyronine ( t3 ) , mainly in the peripheral target tissues . 
 transport of ths in the blood is accomplished through binding to transport proteins , predominantly transthyretin ( ttr ) , with conjugation and degradation processes occurring in the liver and subsequent secretion in bile . 
 some ths are coupled with bacterial deconjugation and undergo reabsorption in the intestine . th structures are conserved between vertebrates , and their production and secretion are essentially the same in amphibians and mammals . 
 there are several processes that influence the thyroid hormone balance , such as sulfation , deiodination and glucuronidation ( see fig . 
 2 ) . both inhibition of thyroidal iodide uptake and suppression of thyroidal peroxidase activity can disrupt th synthesis and secretion . 
 in addition , inhibition of 5-deiodinase in the peripheral tissue is related to reduction of conversion from t4 to t3 , while induction of hepatic microsomal enzymes such as phenol sulfotransferase and udp - glucuronyl transferase leads to increase in excretion of both t4 and t3 into bile . 
 furthermore , competitive binding to thyroid transport proteins results in reduced levels of total and free ths in serum . 
 therefore , there are many potential targets of environmental contaminants that could be involved in disruption of th metabolism . rahman and yamauchi found th sulfating activity to be present in the liver cytosol in frog tadpoles and that the t3:t4 sulfating activity ratio varies during developmental stages , sulfation being inhibited by chemical compounds such as halogenated phenol and phenolic compounds , p - nitrophenol , dopamine , 17beta - estradiol ( e2 ) and dihydroxyepiandrosterone . compared with sulfation 
 , glucuronidation has been poorly studied in amphibians , in line with its character as a relatively minor pathway . however , a subset of the responsible enzymes , glucuronidases ( ugts ) , can reduce circulating levels of ths through biliary elimination as in mammals . regarding phase i species 
 , hepatic microsomal cytochrome p450 2b1 was found to be induced by pentobarbital in adults of the semiaquatic frog , rana pipiens , but not in frog tadpoles and adults of the aquatic frog xenopus laevis . 
 deiodinase enzymes , type 2 and 3 iodothyronine deiodinase , are found in several peripheral target tissues in amphibians , and the level of expression is closely connected with metamorphosis . 
 the most characteristic feature of ttr from nonmammalian vertebrates , such as amphibians , is their higher affinity for t3 than for t4 . 
 binding of chemicals to ttr decreases their effective free concentrations in plasma and changes the apparent affinity for ths , which can diminish cellular uptake and biological responses and would alter plasma th homeostasis . 
 therefore , chemicals interfering with t3 binding to ttr may directly affect the free concentration of plasma t3 and its plasma clearance rate . 
 chemical compounds including diethylstilbestrol ( des ) , phenolic and phenol compounds and pentachlorophenol and ioxynil have the potential to bind to ttr , and yamauchi et al . reported that with amphibian ttr , des possesses similar affinity to t3 , which is the natural ligand . 
 the premetamorphic stage is the period until the appearance of the hind limbs , and the prometamorphic period is from their appearance to that of the forelimbs . during the period of metamorphic climax , resorption of the tail and gills and development of lungs 
 amphibian metamorphosis is divided roughly into 3 stages , premetamorphosis , prometamorphosis and climax ( see fig . 
 the premetamorphic stage is the period until the appearance of the hind limbs , and the prometamorphic period is from their appearance to that of the forelimbs . 
 subsequently , during the period of metamorphic climax , resorption of tail and gills and development of lungs occur . 
 for example , treatment of tadpoles with synthetic thyroid hormone receptor antagonists ( e.g. , nh-3 ) leads to retardation or even complete blockade of xenopus laevis ( x. laevis ) metamorphic development . in turn 
 , exposure of x. laevis tadpoles to low nanomolar concentrations of t3 or synthetic thyroid hormone receptor agonists ( e.g. , gc-1 ) causes precocious induction of metamorphosis . 
 the sensitivity to th varies according to the developmental stage , and the amount of spontaneously released thyroid stimulating hormone ( tsh ) is higher in late prometamorphic and climactic tadpoles than in early prometamorphic larvae and juvenile and adult frogs . 
 low levels of circulating thyroglobulin are found in tadpoles at the early stages , but extremely high levels are evident at the metamorphic climax . in amphibians , both corticotrophin - releasing hormone ( crh ) and gonadotropin - releasing hormone ( gnrh ) , in addition to trh , have been considered as candidates for the tsh - releasing factor . in the bullfrog , crh was found to be the most potent tsh - releasing substance in both adult and larval pituitary cells , while both trh and gnrh moderately stimulated the release of tsh from the adult pituitary but not larval cells . 
 kaneko et al . demonstrated that th suppressed the crh - induced release of tsh , but not the basal release , from larval , juvenile and adult bullfrog pituitaries in vitro . 
 in addition , jacobs et al . reported that intravenously injected mammalian luteinizing hormone - releasing hormone ( lhrh ) was able to raise circulating levels of t4 as well as testosterone ( t ) in three frog species , rana ridibunda , rana temporaria and rana esculenta . 
 regarding prolactin ( prl ) , inhibitory effects may be exerted on metamorphosis , but this is controversial . 
 huang and brown concluded that prl does not play a role as a juvenile hormone in x. laevis , but overexpression of prl does specifically inhibit some but not all programs of tail resorption . 
 research has indicated concomitant elevation of ths and corticoids as metamorphosis progresses . according to kikuyama et al . , the aldosterone plasma level is low prior to the onset of climax , but then there is a sharp rise , and combined adrenocorticotropin ( acth ) and t treatment causes a marked increase in concentration . 
 gray and janssens observed that testosterone and e2 inhibited t3-induced metamorphosis in x. laevis tadpoles , while hogan et al . demonstrated a delay in the time for rana pipiens tadpoles to undergo metamorphosis when exposed to ethinylestradiol during either mid - metamorphosis or throughout the entire larval period . 
 in the past few decades , several thyroid disrupting substances have been tested for toxicity using amphibians . 
 xenopus and other anurans have been generally applied to assess the developmental effects of a variety of xenobiotics . 
 there have been a considerable number of reports regarding inhibitors of t4 synthesis that inactivate peroxidases . after exposure to propylthiouracil ( ptu ) , 
 also known to inhibit deiodinase , x. tropicalis tadpoles showed no signs of decline in body length or body weight but a considerable reduction in the developmental stage and hind limb length . 
 reported effects of exposure to ptu and another peroxidase inhibitor ethylenthiourea ( etu ) , in x. laevis . 
 metamorphic retardation caused by etu was associated with concentration - dependent histological changes in the thyroid gland and increased mrna expression of tsh - beta in the pituitary . 
 exposed pre- and pro - metamorphic larvae to methimazole , ptu and t4 and induced changes in a concentration - dependent manner . 
 methimazole and ptu caused a delay in larval development and morphological changes in the thyroid gland , which were characterized as reduced colloid , glandular hypertrophy and cellular hyperplasia and hypertrophy . 
 on the other hand , t4 treatment resulted in a concentration - dependent increase in the developmental rate . 
 one other potent inhibitor of t4 synthesis , perchlorate , disturbs iodide uptake by the follicular cells of the thyroid gland . 
 ammonium perchlorate was found to inhibit forelimb emergence , hindlimb development and tail resorption , linked to significant hypertrophy of the thyroid follicular epithelium at concentrations below those reported in contaminated surface waters . 
 showed that sodium perchlorate inhibits th synthesis via effects on the sodium - iodide symporter , resulting in retarded metamorphosis and histological effects on thyroid as the most sensitive endpoint . 
 hu et al . reported the results of thyroid immunohistochemistry for t4 in x. laevis exposed to perchlorate ; t4 immunoreactivity was concentrated in a ring of colloid adjacent to follicle cells , independent of the developmental stage . 
 theirs is the only report regarding this point of view , but they suggested the utility of this immunohistochemical biomarker , because the intensity of the colloidal t4 ring is more sensitive than any other morphological changes ( such as hind limb length , forelimb emergence , tail resorption , thyrocyte hypertrophy or colloid depletion ) . 
 found that time until metamorphic transformation was prolonged , the body weights of froglets were increased after exposure to polychlorinated biphenyls ( pcb ) and t4 levels were lowered , although not significantly . 
 established that bisphenol a ( bpa ) induced deceleration of both spontaneous and t4-induced metamorphic changes , with suppression of thyroid hormone receptor ( tr ) beta gene expression . 
 polybrominated diphenyl ether ( bde-47 ) and a commercial pentabromodiphenyl ether mixture ( de-71 ) caused delay in tail regression in x. laevis . according to carlsson et al . 
 , autoradiograms revealed high concentrations and long - term retention of 2,2,4,4,5-pentabromodiphenyl ether ( bde-99 ) in adipose tissue and melanin in frogs exposed as both tadpoles and juveniles , with lower developmental stages suggesting possible thyroid hormone disruption . 
 recorded delayed metamorphosis and enlarged thyroid glands with follicular hyperplasia in x. tropicalis and x. laevis tadpoles exposed to the insecticide methoxychlor . regarding actual environmental contamination , sowers et al . 
 reported that municipal wastewater effluent containing measurable amounts of a variety of pharmaceuticals , personal care products , industrial compounds and natural and synthetic hormones could alter the timing of the metamorphic process in frogs . in their latest report of a field study of the native wood frog in the athabasca oil sands region of northern alberta , canada , 
 metamorphosis was delayed or not complete at the end of the 75 day study period . 
 showed that the herbicide acetochlor accelerated forelimb emergence and the beginning of metamorphic climax , apparently by interacting with t3 , but also possibly by weakly opposing the inhibitory actions of corticosterone on development of premetamorphic tadpoles . 
 the doses used in most of these studies were greater than the levels to which anurans are exposed in the natural environment , so further investigations are required to determine whether exposure to chemicals at environmentally relevant concentrations can affect frog metamorphosis . 
 in contrast to the data showing a clear effect on metamorphosis of chemical compounds , some negative data have been reported . 
 uv filters such as 4-methylbenzylidene camphor ( 4-mbc ) and 3-benzylidene camphor ( 3-bc ) have been found to not affect the thyroid system and the rate of metamorphosis at environmental concentrations , and thus , they seem not to exhibit thyroid - mimicking or antithyroid activity . 
 carr et al . reported exposure to one herbicide , atrazine , did not exert any effects on metamorphosis . 
 there are several reports concerning effects on amphibian larvae of triclosan , which is a bacteriocide used in personal care and consumer products such as shampoo , soaps , deodorants , toothpaste and plastic ware , and so on . 
 the release of triclosan into the environment is of particular concern as it is structurally similar to th . 
 conducted a glp study in compliance with the guidelines mentioned below , and indicated that environmentally relevant concentrations do not alter the normal course of thyroid - mediated metamorphosis in this standard anuran model . 
 the amphibian hypothalamic - pituitary - thyroid ( hpt ) axis controls th levels in the same way as it does in mammals , although crh also plays a role . 
 therefore , the amphibian metamorphosis assay ( ama ) represents a generalized vertebrate model to the extent that it is based on the functions of the hpt axis . on september 7 , 2009 , 
 the organization for economic co - operation and development ( oecd ) adopted the ama using x. laevis as a guideline for testing of chemicals . 
 the us environmental protection agency ( epa ) office of prevention , pesticides , and toxic substances ( oppts ) also includes the ama for detection of thyroid - active chemicals in tier 1 testing of their endocrine screening program . 
 the epa developed the oecd test guidelines through a process of harmonization in october 2009 . 
 previously , in 1998 , the oecd initiated a high - priority activity to revise existing guidelines and develop new guidelines for the screening and testing of potential endocrine disrupters . 
 one element was a test guideline for the screening of substances active on the thyroid system of vertebrate species . 
 the 1st oect expert consultation on endocrine disrupter testing in amphibians was held in 2001 . 
 subsequently , the ama underwent extensive validation programs consisting of 3 phases , which included intra- and interlaboratory studies demonstrating the relevance and reliability of the assay . mainly the u.s . 
 the primary objective was a comparative evaluation of the utility and sensitivity of two proposed exposure scenarios for detection of stimulating and inhibiting effects of thyroid system - disrupting substances . 
 the united kingdom and switzerland also participated in this phase in addition to germany , japan and the united states . 
 three model substances representing different modes of action on the thyroid system were used : sodium perchlorate , t4 and iopanoic acid ( iop ) . 
 phase 3 studies were conducted to assess the utility of the ama protocol to detect weakly active thyroid system - disrupting substances and to distinguish thyroid system - related changes from activity resulting from mechanisms not directly related to thyroid system function . 
 benzophenone-2 ( bp-2 ) and e2 were selected as weakly active and non- or indirectly related substances . as a result , 
 the effect of a weakly antithyroid substance , bp-2 , was detected , while the endpoints with exposure to e2 did not indicate any effect on the thyroid system . 
 subsequently , validation of the assay was subjected to peer review by a panel of independent experts . 
 the primary endpoints and observation time points are mortality daily , developmental stage , hind limb length , snout - vent length and wet body weight on days 7 and 21 and thyroid gland histology on day 21 . 
 the most prominent morphological staging landmark is hind limb morphology , which is positively associated with agonistic effects . regarding the histopathology of the thyroid gland , the guidelines mention core criteria and additional qualitative criteria along with grading , as shown in table 3 . 
 for a more detailed understanding , information is available in  amphibian metamorphosis assay : part 1  technical guidance for morphologic sampling and histological preparation  and  amphibian metamorphosis assay : part 2  approach to reading studies , diagnostic criteria , severity grading and atlas  . 
 in addition , in support of the oecd ama test guideline , a document was developed that provides a standardized approach for evaluating the histology / histopathology of thyroid glands , including an atlas of the normal architecture of amphibian thyroid glands over the course of metamorphosis . 
 one article regarding an examination conducted in compliance with these guidelines supported the use of the ama as a tier 1 endocrine screen for detection of potential thyroid pathway activity , but the lack of a true negative response ( no effect ) during the validation process prevents full evaluation of this assay s specificity at this time . 
 thyroid glands after exposure for 21 days beginning at stage 51 . left : control . 
 each of the mechanisms mentioned above can alter concentrations of circulating ths . under conditions of th decrease 
 , a result of feedback of circulating ths on the hpt axis , there is increased secretion of tsh from the pituitary gland . 
 such excessive stimulation would be expected to result in an increased thyroid gland size , increased degree of follicular cell hyperplasia and/or increased degree of follicular cell hypertrophy , as is well documented for the rodent thyroid gland . 
 stated that histopathological change of the thyroid gland is the most valuable information regarding thyroid toxicants , and histological examination of the amphibian thyroid gland has been shown to be a very sensitive approach in several studies . 
 although the developmental stage and hind limb length are important endpoints of antithyroid activity , developmental delay can not , by itself , be considered a diagnostic indicator of antithyroidal activity . 
 a series of validation assays verified that thyroid histopathology is sensitive and reliable for antithyroidal activity resulting from either inhibition of thyroidal iodide uptake or iodide organification . on the other hand , according to the phase 1 validation study , histopathological analyses of thyroid glands of t4-treated tadpoles were less consistent between the laboratories and more difficult to interpret compared with the effects seen in the ptu studies . 
 furthermore , the phase 2 validation report mentioned that while thyroid histopathology was sensitive for antithyroidal activities , weak agonistic activity could not be reliably detected . 
 depletion of colloid stores and increases in epithelial cell height are known to occur at climax stages during normal development when tsh synthesis and release by the pituitary and t4 synthesis and secretion by the thyroid gland reach maximum levels . 
 therefore , there may be difficulty in distinguishing whether histological changes occur in response to alterations of the hpt axis or alternatively merely reflect advanced stages in tadpoles . 
 in fact , the guidelines insist that the most appropriate sampling approach for histological analyses is to use stage - matched individuals whenever possible . in order to select stage - matched individuals 
 , all larvae should first be staged prior to selection and subsequent processing for data collection and preservation . 
 this is necessary because normal divergence in development will result in differential stage distributions within each replicate tank . 
 there is one example showing that amphibian thyroid histopathology is extremely complex , and when misinterpretation occurs , it leads to the opposite conclusion . in a controversial issue regarding the effect of triclosan , fort and pawlowski reported in regard to the histopathological changes that an increase in the occurrence of minimal thyroid gland hypertrophy was not accompanied by follicular hypertrophy or hyperplasia . 
 the lack of follicular hypertrophy or hyperplasia was suggested to be the result of both the minimal nature of the response and the increased body size of the treated specimens . 
 because disruption of the thyroid axis has been identified as an important consideration for regulation of chemicals , the oecd and epa have established guidelines that make for use of larval african clawed frogs ( x. laevis ) and frog metamorphosis for screening and testing of potential endocrine disrupters . in the test guidelines , 
 thyroid gland histology is one of the primary endpoints , along with mortality , developmental stage , hind limb length , snout - vent length and wet body weight . 
 a series of validation studies and many reports have revealed that the histopathology of the thyroid gland is very sensitive and reliable for detection of antithyroidal activity but , on the other hand , is less useful for weak agonistic effects . 
 since the morphology of the amphibian thyroid gland changes continuously throughout metamorphosis with the fluctuation in th levels , the guidelines stress the need for well experienced toxicologic pathologists who are familiar with normal x. laevis thyroid histology , thyroid gland physiology and general responses of the thyroid gland to agonists or antagonists .","there are continued concerns about endocrine - disrupting chemical effects , and appropriate vertebrate models for assessment of risk are a high priority . 
 frog tadpoles are very sensitive to environmental substances because of their habitat and the complex processes of metamorphosis regulated by the endocrine system , mainly thyroid hormones . during metamorphosis , 
 marked alteration in hormonal factors occurs , as well as dramatic structural and functional changes in larval tissues . 
 there are a variety of mechanisms determining thyroid hormone balance or disruption directly or indirectly . 
 direct - acting agents can cause changes in thyroxine synthesis and/or secretion in thyroid through effects on peroxidases , thyroidal iodide uptake , deiodinase , and proteolysis . at the same time 
 , indirect action may result from biochemical processes such as sulfation , deiodination and glucuronidation . because their potential to disrupt thyroid hormones has been identified as an important consideration for the regulation of chemicals , the oecd and the epa have each established guidelines that make use of larval african clawed frogs ( xenopus laevis ) and frog metamorphosis for screening and testing of potential endocrine disrupters . 
 the guidelines are based on evaluation of alteration in the hypothalamic - pituitary - thyroid axis . 
 one of the primary endpoints is thyroid gland histopathology . 
 others are mortality , developmental stage , hind limb length , snout - vent length and wet body weight . regarding histopathological features , 
 the guidelines include core criteria and additional qualitative parameters along with grading . 
 taking into account the difficulties in evaluating amphibian thyroid glands , which change continuously throughout metamorphosis , histopathological examination has been shown to be a very sensitive approach .",pubmed
"cesarean sections ( cs ) performed following a medical indication is necessary for life - saving . 
 it is a surgical intervention to prevent maternal or perinatal complications and the appropriate rate of use should be one associated with the lowest rate of maternal and perinatal morbidity and mortality . 
 however , women are increasingly undergoing cs without any medical indication , which may contribute to the world - wide higher rates of cs . 
 there has been a debate about the appropriateness of cs performed due to maternal request or what physicians suggest to their patients . 
 result of the world health organization ( who ) global survey in a large cross - sectional study demonstrated severe maternal morbidity in planned cesarean delivery are higher than planned vaginal delivery ( vd ) . who recommended 10 - 15% rate of cesarean section . 
 this recommendation was based on the cesarean section rates of the countries with the lowest maternal and neonatal mortality rates in the world at that moment . 
 many studies have shown that the actual rate of cs in numerous countries is far higher than the recommended range . 
 the rate of cesarean section out of all deliveries in iran increased by six - fold over the past three decades . 
 these results are in accordance with previous reports that confirm the growing rate of cs in iran . 
 however , the debate on the best practices ( vd vs. cs ) to minimize postnatal morbidity still is a matter of controversy both from professionals perspectives and from women 's perceptions of the childbirth experience . 
 the postpartum period are critical life events for women leading to physiological , emotional , and social changes . 
 postpartum mothers experience certain physical health conditions that may affect their quality - of - life ( qol ) , future health , and health of their children . 
 several studies confirm that socio - economic deficiencies and medical problem are risk factors for decreased qol and depressive symptoms in women during the postnatal period . 
 mode of delivery and childbirth experience may have a long - term effect on self - rated health . 
 several studies have investigated the association between type of delivery and health related quality - of - life ( hrqol ) , but there is conflicting , with some studies reporting decreased hrqol in cesarean delivery . whereas , other studies did not confirm any relations between cs and hrqol . in a study 
 it was found that patients after vd had higher mean physical hrqol scores than after cs while mean mental hrqol were similar among vd and cs groups . in another study , 
 however , careful assessment of the predictor variables of poorer physical and mental health after childbirth may improve the quality of postpartum care . 
 the aim of this study was to explore the impact of delivery mode on women 's postpartum physical and mental health related qol . 
 a prospective study was performed in the period august 2007 to october 2008 of pregnant women who admitted for prenatal care in the health center . 
 a total of 10 health center were selected randomly in kashan city in iran . after applying inclusion and exclusion criteria , 
 there were 365 consecutive women that agreed to take part in the study and all women gave their informed consent before entering the study . of whom 342 women entering the study after delivery consist of 175 ( % 51.2 ) mothers after vd and 167 ( % 48.8 ) mothers after cs ( elective or emergency cs ) , in follow - up 2 months after delivery ( time 1 assessment ) 162 women after vd and 159 after cs completed qol questionnaire . 
 of whom 150 women in each type of delivery completed qol questionnaire 4 months after delivery again ( time 2 assessment ) in health centers . in these analyses , we have included all women with singleton , live born infant , term pregnancy , prenatal care started before 20 weeks gestation , having uncomplicated pregnancies , parity 1 - 3 and the study population was ethnically iranian , not having : others pre - existing diseases , history of infertility , and divorce . 
 excluding criteria were instrumental delivery , birth weight less than 2,500 g , child or fetal dead , child abnormality , not breast feeding and medical problems in child and mother . 
 all patients were interviewed by the trained midwives and a structured questionnaire was used to evaluate women 's socio - demographic characteristics and obstetric variables . 
 also we used a generic hrqol instrument to measure qol ( categorized into physical and mental health ) . 
 qol was assessed using the iranian version of short form-36 health survey social functioning ( sf-36 ) . 
 sf-36 is a standard and well - known generic health related qol instrument and proved to be highly feasible , reliable and is a good choice to measure health related qol after delivery . 
 it consists of 36 items , organized into eight scales : physical functioning ( pf ) , role limitation due to physical problems or role physical ( rp ) , bodily pain ( bp ) , general health ( gh ) , vitality ( vt ) , social functioning ( sf ) , role limitation due to emotional problems or role emotional ( re ) , and emotional well - being ( ew ) . 
 the scores on each subscale range from 0 to 100 with higher scores indicating a better condition , the sf-36 furthermore provides physical health ( based on the pf , rp , bp , and gh scales ) and a mental health ( relating to vt , sf , re , and ew ) . 
 we compared women 's socio - demographic characteristics and physical and mental health scores between women with vd and cs that completed qol questionnaire at 2 and 4 months after delivery ( 150 vd and 150 cs ) . 
 furthermore score differences within each mode of delivery between time 2 and time 1assessment were evaluated . 
 differences in means were analyzed using the student 's t - test and the mann 
 the study protocol was approved by the local research committee in kashan university of medical sciences . 
 there were no significant differences between vd and cesarean groups with regard to these variables . 
 maternal age was between 20 and 40 and vd was performed up to % 80 with episiotomy or laceration . 
 the physical and mental health parameters related qol at 2 months follow - up has shown in table 2 ; total physical health score in vd women was higher than cs group . 
 mean scores was 60.52  13.28 and 57.37  12.20 ( p = 0.034 ) respectively . on performing a detailed analysis , statistically significant differences in 2 parameters ; 
 pf ( p = 0.001 ) and rp ( p = 0.026 ) were found . 
 total mental health score in cs women was generally higher than vd , but differences were not significant . comparing physical and mental health parameters at 4 months postpartum 
 results showed that the vd group in compared cs group had a higher score in total physical health ( 61.37  13.05 and 58.36  14.09 p = 0.05 ) and total mental health was ( 64.99  12.44 and 61.83  13.58 p = 0.036 ) respectively . in comparing subscales ; 
 differences statistically significant were found for 1 parameter in physical health ( physical function p = 0.01 ) and for 2 parameter in mental health ( sf p = 0.036 and ew p = 0.042 ) . 
 to compare the findings within each group results demonstrated that the vd group showed more improvements on total mental hrqol from time 1 to time 2 assessment [ table 4 ] . 
 the mean score differences ( 4 months scores minus 2 months scores ) in vd and cs groups were 4.70  16.29 and 0.3  13.81 ( p = 0.01 ) respectively . in comparing subscales differences 
 statistically significant were found for 2 parameter ( sf p = 0.03 and ew p = 0.01 ) . 
 general characteristics of patients according to mode of delivery distribution of postpartum physical and mental health according to mode of delivery at 2 months after delivery distribution of postpartum physical and mental health according to mode of delivery at 4 months after delivery the mean score differences within each mode of delivery ( 4 months scores  2 months scores ) * 
 our study showed that physical hrqol at 2 and 4 months after delivery is better in women with vd . 
 there were significant differences in the pf and rp parameters in time 1 and pf in time 2 assessments . 
 also mental hrqol at 4 months after delivery and improvement on mental hrqol from time 1 to time 2 assessments was better in vd group ; differences were significant in sf and ew . 
 mental hrqol at 2 months after delivery was better in cs group but differences were not significant in each of parameters ; a reason for this occurred is mothers after cs supported better due to surgical intervention . also , 
 in our study , nearly % 80 of vd was performed with episiotomy or laceration . in a study , the median time to restart intercourse in the normal vd with episiotomy was 40 days and in the cs group was 10 days postpartum and there was significant decreases in the sexual functioning scores after vd when compared to cs group . concerning its effects on postpartum sexual functioning . 
 sexual health is perceived as an integral part of gh as it can affect postpartum depression . 
 there are many studies that assess morbidity resulting from vd and cs , but a few studies have focused on women 's postpartum hrqol . 
 however , our results are consistent with several studies but differ from some of the previous studies ; in a prospective study 100 women were interviewed ( 50 with normal delivery and 50 with cs ) . 
 qol was measured using the sf-36 at two points ( time 1 : 6 - 8 weeks after delivery and time 2 : 12 - 14 weeks after delivery ; result showed vd group had a better qol for almost all subscales in both assessment times , the differences were significant for vt , mental health and pf . 
 another study was performed to investigated fatigue and hrqol in women after vd , elective cs and emergency cs . 
 a total of 141 women ( 71 after vd , 36 after elective cs and 34 after emergency cs ) completed the hrqolsf-36 questionnaires ; result showed patients after vd had higher mean physical hrqol scores than after cs , but the mean mental hrqol scores of the study groups were similar . 
 in contrast these results a cross - sectional study evaluated the effect of delivery mode on women 's postpartum qol in rural china . in this study , 
 none of six dimensions and total score of qol displayed significant difference between women with normal delivery and cs . in this study , cs rate was 70.0% , and most of them ( 59.7% ) were selected by maternal request . 
 the most important in this data being a high frequency of cs due to maternal request also other factors such as lower education level and male gender of infant were associated with poor qol and whereas , in our study , these factors were similar in two groups . however , socio - cultural determinants may contribute in influencing postnatal qol . 
 safarinejad et al . in a cohort study showed the qol parameters by using sf-36 questionnaire in planned cs women were generally higher than vd . 
 there were differences significant for two physical health domains ( gh and rp ) and all of the mental health domains . in this study planned cs compared to vd and all of the women were parity 1 and younger age . 
 one might argue the findings were influenced by the fact that women with elective or emergency cesarean may experience rather different qol during the postnatal period . 
 furthermore in primipara women other reasons are associated with poor qol . in our study , cs consist of elective or emergency and parity was 1 - 3 . in another study dunn and oherlihy compared satisfaction levels by early postnatal questionnaire among 140 women who had a vaginal birth after delivery ( vbac ) with women who had cesarean section after vaginal delivery . 
 the vbac group experienced minimal pain after delivery and had felt better prepared for delivery . in our study , 
 subscale pain at 2 month after delivery was similar in two groups ; one reason was high frequency of episiotomy in vd group , it is important to pay attention to this risk factor to be able to offer good care and treatment to women during postpartum where necessary . in a prospective study in mothers who requested cesarean section in the absence of medical indication , their reason for the request , self - estimated health and experience of delivery were investigated . 
 results showed women requesting cesarean section experienced their health as less good and were more often planning for one child only . 
 they more often reported anxiety for lack of support during the labor , for loss of control and concern for fetal injury or death . 
 after planned cesarean section women in this group reported a better birth experience compared to women planning a vaginal birth . 
 there were no differences in signs of postpartum depression between the groups 3 months after birth . 
 however intended caesarean delivery was found to be an independent risk factor for complications and is associated with an increased risk of maternal rehospitalization . 
 furthermore mean duration of sickness and mean days required for returning to normal activities were also higher in case of caesarian section also it is expensive than normal vaginal delivery . 
 our research was a prospective study , data were obtained from the participants by trained questionnaires and study population was ethnically uniform . 
 unfortunately , we only used a general instrument and this might be considered as a limitation . 
 another limitation of the study was that the type of cesarean section was not considered , women with elective or emergency cesarean may experience rather different qol during postpartum . 
 the findings suggest that vd lead to a better physical health at 2 months after delivery and physical and mental health at 4 months after delivery . 
 furthermore , we recommend the future studies include both general and specific questionnaire in assessing postpartum qol among women with vd , elective cs and emergency cs , in addition other studies needed for recognizing social and cultural factors that are related to physical and mental hrqol after delivery .","background : the postpartum period is a critical life event for women leading physical , emotional , and social changes . 
 postpartum quality - of - life may be affected by delivery mode . 
 the purpose of this study was to determine the association between postpartum health related quality - of - life ( hrqol ) and mode of delivery.materials and methods : in a prospective study , 300 women consisting of 150 vaginal deliveries ( vd ) and 150 cesarean sections ( cs ) were recruited between august 2007 and october 2008 from health centers . 
 stratified random sampling was performed to select 10 health centers in kashan city , iran . 
 physical and mental hrqol was measured using the sf-36 questionnaire and compared between vd and cs groups 2 and 4 months after delivery . 
 data were analyzed by using the student 's t - test , mann  whitney u - test , and chi - square test.results:results showed physical hrqol at 2 months after vd was better than cs significantly ; there were significant differences in the physical functioning and role physical subscales . 
 furthermore mental hrqol at 4 months after vd was better than cs significantly ; there were significant difference in the social function and emotional health subscales.conclusion:the findings demonstrated that vd leads to a better physical health at 2 months after delivery and mental health at 4 months after delivery . 
 efforts should be made to reduce cs .",pubmed
"maslow 's hierarchy of needs has been a well - known theory since abraham maslow proposed it in 1943   and in his book motivation and personality in 1954 , in which he proposed that human needs can be portrayed in the shape of a pyramid , with the most fundamental levels of needs at the bottom . from bottom to the top are the needs : physiological needs , safety , belongingness and love , esteem , and self - actualization . 
 physiological needs are the physical requirements for the survival of individual and the animal kind , such as food and sex , and safety needs are personal security , financial security , and health and well - being . 
 maslow proposed that if the physiological needs are relatively satisfied , there then emerges a new set of needs , which may be categorized roughly as the safety needs . 
 the organism may be wholly dominated by them , which may serve as the almost exclusive organizers of behavior , recruiting all the capacities of the organism in their service , and we may then fairly describe the whole organism as a safety - seeking mechanism . 
 but maslow also found that  practically everything looks less important than safety , even sometimes the physiological needs which being satisfied , are now underestimated . 
 safety needs are personal security , financial security , and health and well - being , which are more fundamental than physiological needs ( figure 1 ) . for example , the deer can not eat ( physiological needs ) on the wild prairie when the wolves chase them ( safety ) . 
 let me take one example from cosmides and tooby ( 2000 ) , who propose that the emotions serve to regulate behavior . 
 he wrote about fear like this :  imagine walking alone at night and hearing some rustling in the brush . 
 your energies are aroused to be ready for action , you become acutely aware of sounds that could indicate that you are being stalked , the threshold for detecting movements is lowered , you no longer feel pangs of hunger , attracting a mate is the farthest thing from your mind .  . 
 whether physiological needs or safety needs are more important will affect our opinions about mental disorders , for example , freud proposed libido ( physiological needs ) more important . 
 safety needs can block physiological needs ; the classical example is miller 's avoidance and approach experiments ( figure 2 ) . in 1961 , 
 neal miller used behavioral measures to assess motivational disposition in rodents ( figure 2 ) . 
 as the animal moves closer to the potential reward ( e.g. , food ) , the force exerted to obtain the reward increases . 
 similarly illustrated is the avoidance : the force the animal exerted to avoid the aversive stimulus ( a shock ) also increases as the animal comes closer , and furthermore , the slope of the avoidance gradient tended to be steeper than that of approach . 
 later experiments by ito found that the organisms tend to be more sensitive to the threatening information and generally process such information faster than the rewarding information . 
 he called this phenomenon negativity bias and attributed it as a protective strategy through evolution , since even a single failure to respond adaptively to a survival threat may preclude passing on genetic information . 
 he found that  as a potential treat looms , the adaptive response of the brain is to amplify these threats and initiate appropriate behavioral responses , such as fleeing , freezing , or attacking .  and he found that the negativity bias can be seen across all levels of the neuraxial organization . 
 these data support the notion that safety needs are faster and more fundamental than hedonic needs . 
 maslow thought adults usually inhibit the reaction for safety needs , so he used infants as an example and found that the child 's need for safety is his preference for some kind of undisrupted routine or rhythm . 
 maslow mentioned ,  he seems to want a predictable , orderly world . for instance , 
 injustice , or inconsistency in the parents seems to make a child feel anxious and unsafe . 
 this attitude may be not so much because of the injustices per se or any particular pains involved , but rather because this treatment threatens to make the world look unreliable , or unsafe , or unpredictable .  . 
 maslow also mentioned ,  confronting the average child with new , unfamiliar , strange , unmanageable stimuli or situations will too frequently elicit the danger or terror reactions . 
 this is consistent with the behaviorists , who propose that behavior is a process - form  stimulus - reaction  , while cognition scientists extend it to  stimulus - opinion - reaction . 
 is stimulus that has the hedonic value , which fits into our personal physiological needs . 
 and it also has another feature : happening in an expected way , or unexpected way , which is related to threat and can be called safety value . 
 we draw these two features of stressful events in two dimensions : hedonic value and safety value ( figure 1 ) . 
 the safety value has nothing to do with the hedonic value , for both the liked things and disliked things can induce unsafety . 
 for example , we worry about losing the liked things and also worry about getting the disliked things ; and we also will not be angry if we lose the good thing as expected and also get the disliked thing as expected . 
 even though it is something you liked , if it is unexpected , you still feel afraid and angry . therefore the safety is related to unexpectancy 
 . one feature of the safety need induced emotion is the rapid detection of potential threats and can initiate appropriate approach / avoidance behaviors . 
 prediction error . 
 the studies were done from electrophysiological recordings of dopamine neurons in awake , behaving monkey in schultz 's lab . 
 these phased activation does not discriminate different types of rewarding stimuli . and it is quite unexpected that the reward delivery will not elicit dopamine neuron firing , once the animal has learned the stimulus and reward association . 
 therefore , dopamine neurons are related to expectation about external stimulus rewarding , especially when it is uncertain or prediction error [ 6 , 7 ] . 
 the studies of emotions have been expanded exponentially by two prominent researchers : magda arnold and richard lazarus , who proposed appraisal theory . 
 appraisal theory states that emotions result from people 's interpretations and explanation of their circumstances . in the structural model of appraisal theory , 
 lazarus borrowed the concept of appraisal from arnold and elaborated the concept as a key factor for emotions : emotional processes depend on the predictability of the stressful events . 
 he distinguishes two basic forms of appraisal , primary and secondary appraisal , and he proposed that the primary appraisal and its induced emotions are a faster activating , automatic process , which is similar to the safety need . 
 indeed , lazarus distinguishes three types of stressful events : harm , threat , and challenge , which are related to primary appraisal . 
 the secondary appraisal is concerned with coping options , which include blame or credit , coping potential , and future expectations . 
 it seems that the primary appraisal is related to fear and the secondary is related to anger ( figure 3 ) . 
 lazarus mentioned that if a person appraises a situation as motivationally relevant , motivationally incongruent and also holds a person other than himself accountable , the individual would most likely experience anger in response to the situation . process model of the appraisal theory is more accurate to explain the safety needs , for personal safety , which are related to the  unexpected ways of stimulus occurring . 
 the process model proposed two main appraisal processes : perceptual stimuli and associative processing and reasoning . 
 perceptual stimuli are what the individual picks up from his surroundings , such as sensation of pain or pleasure . 
 then , the individual performs two main appraisal processes : associative processing , which is memory based , and reasoning , which is a slower and more deliberate process that involves logical thinking about the stimulus . 
 for example , when you meet a car that quickly passes and stops before you , you will be firstly scared and then blame the car . 
 let us take another example from izard 's paper ,  when rafe was hit from the back by a wheel chair , the first reaction of him was scared and angry , and showed angry expression and clenched fist . 
 but after he turned back to see rebecca , a person with hemiplegia whose wheelchair had gone out of control and cause her to crash into rafe . 
 so when something unexpected occurs , you will first evaluate its threat ( fear / anger ) and next evaluate its hedonic value ( happy / sad ) ( figure 3 ) . 
 similar emotional flow happens in our lives all the time : everything in our lives is normally calm as expected ; but you will first feel scared ( fear ) when something unexpected occurs , and then you will blame ( anger ) the un - expectancy after fear is gone . and 
 afterwards you might feel happy after successfully coping with the stressful events or feel sad if you failed to cope with them . finally , the stressful events go away , and people calm down . 
 this kind of emotional flow , big or small , long or short , constitutes our everyday emotions . 
 so fear - anger - happiness - sadness - calm might constitute the rainbow of emotions or emotional flow in our everyday life . 
 the amygdala has been proved to be the neural basis for fear , and it is also recognized as the neural basis for stress elicited fear and anxiety [ 12 , 13 ] . 
 in addition , electrical stimulation of the amygdala promotes autonomous reactions and stress - like behavioral , whereas amygdala ablation induced a marked tameness increase , motivation loss , and fear decrease to aversive stimuli [ 14 , 15 ] . 
 amygdala is one of the most important limbic structures that link to fear , which was first suggested by klver & bucy in 1937 , who demonstrated that the lesion of the medial temporal lobe resulted in a wide range of odd behaviors , such as approaching normally to fearful objects . 
 and about 20 years later , weiskrantz ( 1956 ) found that it is the amygdala whose impairment resulted in the odd behaviors , which are called klver and bucy syndrome . 
 ledoux puts amygdala as the emotional computer to work out the emotional significance of stimuli . 
 he demonstrated two neural pathways of sensory information from the thalamus to the cortex : ( 1 ) a slow - acting  thalamus - to - cortex circuit ,  whose function is to analyze sensory information in detail , and ( 2 ) a fast - acting  thalamus - amygdala circuit ,  whose major function is to analyze simple stimulus features , which bypass the cortex . 
 these two pathways possibly underlie the two evaluation systems : the fast one for the fear / anger and the slow one for hedonic . 
 other reports , such as findings from ohman and soares ( 1994 ) also support a fast - acting system for threat detection that involves only minimal cortical processing [ 19 , 20 ] . 
 ( 2001 ) also reported a patient whose primary visual cortex was impaired and therefore showed no conscious visual perception but showed significant fearful reports . 
 in all , the fast - acting thalamus - amygdala circuit is important for our ancient ancestors to rapidly recognize dangers to help with survival . 
 it is an evolutionary adaptation for our ancestors to better cope with the unexpected environment with the fast activing thalamus - amygdala circuit . 
 in addition to amygdala , the ne / lc system is important to direct behaviors of the animal to cope with the dangerous environment , and the well - known function of ne / lc system is to induce  fight or flight  behaviors . take a deer in the wild as an example . 
 when a deer meets a lion , the reaction of the deer is flight ( fear ) , while the reaction of a lion is fight ( anger ) . 
 so the emotion fear and anger might also be derived from the same neurotransmitter ne and the same stressful event ( figure 4 ) . 
 ne is released from the locus coeruleus ( lc ) in the brain to keep the brain alert , which has been described as increasing the  signal to noise  of the sensory inputs . 
 lc has been reported to be activated at stressful events : increased lc neuron firings were observed at visual threat . and 
 lc sends projections to amygdala , which is the most important limbic structure that links to fear . 
 therefore , amygdala and ne / lc system might constitute the neural structure for safety needs . 
 our ancestors navigating rich environments had to face very complicated environments , with many forms of uncertainties . 
 so they evolved an adaptive mechanism to first do a safety check for everything around them . 
 if it would happen in an anticipated way , they will feel calm ; instead if something happened surprisingly , they will be scared and angry 
 . therefore , fear and anger are due to things happening in an unexpected way ( figure 4 ) . of note , the unexpectancy will also increase the tension of hedonic emotions . with the same kind of hedonic stimulus , 
 if it comes in an unexpected way , people will feel excited ; instead , people will feel happy . 
 one reason for the clearer appearance of the threat or danger reaction in infants , is that they do not inhibit this reaction at all , whereas adults in our society have been taught to inhibit it at all costs . 
 thus even when adults do feel their safety to be threatened we may not be able to see this on the surface . 
 infants will react in a total fashion  in infants we can also see a much more direct reaction to bodily illnesses of various kinds  for instance , vomiting , colic or other sharp pains .  . 
 even the adults can inhibit our reactions , they still can react in some mental disorders . 
 maslow wrote ,  some neurotic adults in our society are , in many ways , like the unsafe child in their desire for safety , although in the former it takes on a somewhat special appearance . 
 their reaction is often to unknown , psychological dangers in a world that is perceived to be hostile , overwhelming and threatening . 
 the neurotic individual may be described in a slightly different way with some usefulness as a grown - up person who retains his childish attitude toward the world . 
 this is to say , a neurotic adult may be said to behave as if he were actually afraid of a spanking , or of his mother 's disapproval , or of being abandoned by his parents , or having his food taken away from him . 
 it is as if his childish attitude of fear and threat reaction to a dangerous world had gone underground , and untouched by the growing up and learning processes , were now ready to be called out by any stimulus that would make a child feel endangered and threatened . 
 fear - phobia . 
 if something happens surprisingly , people will be scared and angry ; and if it would happen in an anticipated way , they will feel calm . 
 so for the phobia patients , their problems might be that they can not successfully accomplish the emotional flow ( fear - anger - happiness - sadness - calm ) . 
 the best way to remove fear is anger , these patients are too timid to show anger , so their emotions are checked at emotional flowing from fear to anger . 
 therefore , anger might be the best treatment for these patients and ne is the neural substrate for them . 
 depression is characterized by unrelenting sadness accompanied by an inability to derive pleasure from positively hedonic situations . 
 therefore , depression might be related to the primary appraisal , the worrying about safety instead of physiological satisfaction . 
 indeed , excessive self - blame and feeling worthless are symptoms of major depression episodes across cultures , which is similar to lazarus 's secondary appraisal . 
 so the depressed patients have problems with anger or with coping appraisal or their problem is due to inability to cope with the unsafe stressful situation and showed inward anger . 
 the difference between fear and anger is the direction of the behavior : fear is to throw oneself away from the stimulus and anger is to throw the stimulus away . 
 for these patients , they do not have the ability to throw the outside stimulus due to repeated helplessness , they want to kill themselves . 
 safety needs are the most fundamental needs for the human kind , which include personal security , financial security , and health and well - being . 
 safety is the major reason for mental disorders , such as anxiety , phobia , depression , and ptsd . 
 the neural basis for safety is amygdala and lc / ne system , which can be regarded as a  safety circuitry ,  whose major behavior function is  fight or flight  and  fear and anger  emotions , or conditioned learning for these emotions . fear and anger are due to the safety needs , while joy and sadness are due to the physiological needs , which should come after safety needs in maslow 's hierarchy of needs . 
 fear and anger are two sides of one sword , for they will act in different directions : fear is to flight away from the danger and anger is to fight the danger away . 
 they are all due to the stressful events : normally everything is as expected , and life is calm . 
 when something unexpected happens , the individuals first feel scared and then blame the unexpectancy ; this is the first safety check . 
 afterwards the individual will have a hedonic need to see if it fits their personal needs and get the happy or sad emotions . 
 finally , everything comes to an end , and people return to calmness or miss the lost things and worry for the uncertain bad things . 
 so the emotional rainbow ( or emotion flow ) fear - anger - happiness - sadness - missing constitutes our emotions in everyday life .","safety first ,  we say these words almost every day , but we all take this for granted for what maslow proposed in his famous theory of hierarchy of needs : safety needs come second to physiological needs . here 
 we propose that safety needs come before physiological needs . 
 safety needs are personal security , financial security , and health and well - being , which are more fundamental than physiological needs . 
 safety worrying is the major reason for mental disorders , such as anxiety , phobia , depression , and ptsd . 
 the neural basis for safety is amygdala , lc / ne system , and corticotrophin - releasing hormone system , which can be regarded as a  safety circuitry ,  whose major behavior function is  fight or flight  and  fear and anger 
 emotions . 
 this is similar to the appraisal theory for emotions : fear is due to the primary appraisal , which is related to safety of individual , while anger is due to secondary appraisal , which is related to coping with the unsafe situations . if coping is good , the individual will be happy ; if coping failed , the individual will be sad or depressed .",pubmed
"an imposing body of 
 studies , spanning 
 more than three decades , has cogently elucidated the involvement of 
 epoxyeicosatrienoic acids ( eets ) in a wide array of critical physiological 
 functions , inter alia , blood pressure regulation , nociception , adipogenesis , anti - inflammatory activity , organ regeneration , insulin 
 potentiation , podocyte integrity , and cellular responses to bacterial infection . 
 the most prominent regioisomer , 14,15-epoxyeicosa-5(z),8(z),11(z)-trienoic acid ( 14,15-eet ) , along with other 
 members of this autacoid family , arise via metabolism of arachidonic 
 acid by cytochromes p450 , especially members of the 2c and 2j families . 
 the ratio of eet isomers and their stereocomposition are cyp p450 
 isoform - dependent and , thus , species- 
 and tissue - specific . 
 numerous chemical 
 and metabolic factors conspire to complicate 
 investigations into the roles of 14,15-eet and restrain its potential 
 therapeutic utility ( figure 1 ) . 
 its susceptibility toward aerial oxidation or 
 auto - oxidation , i.e. , a nonenzymatic , free radical process involving 
 the 1,4-dienyl substructures characteristic of polyunsaturated fatty 
 acids , necessitates handling and/or storage under strict conditions 
 that minimize exposure to oxygen and trace transition metals . 
 a precis of inactivating enzymatic processes 
 includes esterification , metabolism by 
 other pathways of the arachidonate cascade , conjugation , -oxidation , chain elongation , and hydrolysis of the epoxide . in biological 
 milieu , 
 hydration of the epoxide by soluble epoxide hydrolase ( seh ) 
 is a major determinant in maintaining the steady state levels of 14,15-eet , whose half - life has been estimated at no more 
 than seconds to minutes . 
 the pioneering 
 studies of hammock and others confirmed inhibition of seh can elevate eet 
 levels both in vitro and in vivo , thus offering an indirect means 
 for pharmacologic intervention in eet - mediated processes . 
 however , 
 this strategy might prove less efficacious than an agonist replacement 
 therapy whenever endogenous eet biosynthesis is compromised , for instance , 
 as a consequence of disease , inflammation , radiotherapy , aging , and/or 
 exposure to xenobiotics and drugs that inhibit cytochromes p450 . 
 to address 
 some of the stability limitations of natural eets , our 
 laboratories previously prepared several iterations of eet surrogates 
 with improved stability . 
 advanced versions were 
 evaluated for relaxation of precontracted bovine coronary artery rings 
 and for in vitro inhibition of recombinant human seh . 
 depending upon the bioisostere and its position along the 
 carbon chain , varying levels of vascular relaxation and/or seh inhibition 
 were observed . 
 generally , oxamides and n - ipr - amides displayed useful 14,15-eet agonist activities 
 but were modest to poor seh inhibitors . 
 the in vitro 
 success of this generation of analogues prompted us to consider further 
 structural iterations . 
 the surrogates 
 described herein modify the free carboxylic acid 
 of the previous generation of 14,15-eet mimics while , for the most 
 part , retaining some key structural features of the pharmacophore 
 identified earlier ( i.e. , cis--olefin and an epoxide bioisostere ) . 
 they were evaluated as described 
 before in precontracted bovine coronary artery rings for ( i ) % vasorelaxation 
 at 10 m relative to a 14,15-eet control , ( ii ) ec50 for vasorelaxation , and ( iii ) ic50 for seh inhibition 
 ( table 1 ) . 
 interestingly , 
 the simple expedient of conjugating the carboxylate with a short poly(ethylene 
 oxide ) ( peg ) unit improved both % vasorelaxation and the ec50 somewhat compared with the parent free acids regardless the type of epoxide bioisostere , viz . , n - ipr - amide 1 , urea 2 , and oxamide 3 ; on the other hand , the soluble 
 epoxide hydrolase ( seh ) inhibitory activity of urea 2 was significantly compromised versus the parent free acid ( ec50 7.5 m , ic50 46 nm ) . 
 linkage of the carboxylate 
 to the nitrogen of glycine ( 4 and 5 ) or 
 aspartic acid ( 6 ) resulted in a modest boost to the vasoactivities 
 while the seh ic50 of 6 dropped by an order 
 of magnitude compared to the parent free acid . 
 conversion to the n - phenyl and n - methylsulfonimides 7 and 8 , respectively , left the biological activities 
 virtually unchanged in all categories while the simplified phenylsulfonamide 9 led to an improvement in the ec50 by a factor 
 of 2 and smaller improvements in the other parameters . at 10 m 
 , 14,15-eet induces 
 85% of maximum vasorelaxation and its ed50 is 2.2 m . 
 for recombinant human seh , 
 given the generally lackluster behavior 
 of the esters and amides , 
 our focus changed to replacement of the carboxylate with a variety 
 of heterocyclic bioisosteres identified from literature sources . 
 for phosphonate 10 and sulfonate 11 , the increase in polarity did not improve potency as seen 
 with the peg esters . 
 interestingly , sulfonate 11 retained 
 its ability to suppress seh , in contrast to phosphonate 10 and peg ester 2 . 
 vasorelaxation by s - alkylthiocatechol 12 was poor , but its ic50 for seh was pushed down into the single digits . 
 the sulfone variant 13 regained some vasopotency , 
 probably due to an increase in the acidity of the phenol but not sufficiently 
 to be viable . 
 replacement of the phenol moiety in 12 with 
 a tetrazole ring produced 14 and was encouraging , but this trend was not continued in triazole 15 . 
 sequential oxidation of the sulfur to sulfoxamide 16 and sulfonamide 17 , as seen for 12  13 , incrementally improved the ec50 . 
 the ed50 jumped in the tetrazole bioisostere series 1821 , achieving a submicromolar value 
 for urea 19 ( 12-fold better than 14,15-eet ) , while the 
 % vasorelaxation versus 14,15-eet peaked . 
 factoring in a very respectable 
 low nanomolar ic50 , 19 is the best dual activity 
 analogue in the study and a leading candidate for further development . 
 notably , all three parameters began to erode , albeit minimally , in 
 the one - carbon homologue 20 and further in oxamide 21 . 
 the performance of another five - membered heterocycle , oxadiazol-5-one 22 , while acceptable , 
 was not comparable to urea 19 . 
 the closely related oxathiadiazole-2-oxides23 and 24 , on the 
 other hand , demonstrated good vasorelaxant activities ; seh inhibition 
 potency was consistent with our previous observations that ureas  n - ipr - amides . 
 the isomeric thioxo-1,2,4-oxadiazol25 registered a 6-fold improvement 
 in ed50 over 14,15-eet yet was a comparatively poor seh 
 inhibitor and , thus , is a good option whenever an eet agonist with 
 minimal impact on seh function is required . 
 the corresponding urea 
 thioxo - oxadiazol,26 , could 
 not sustain the vasopotency ; its ed50 sagged by almost 
 an order of magnitude versus 25 while at the same time 
 its seh ic50 improved by more than an order of magnitude . 
 a final variant on this heterocyclic theme , 5-thioxothiadiazol27 , fell short of expectations 
 on all levels and further study of this particular heterocycle was 
 terminated . despite its success in the glitazone series of antidiabetic 
 drugs , the 2,4-thiazolidinedione modification 
 proved disappointing when incorporated into 28 . 
 given 
 the larger size of the n-(4-hydroxy-2-benzothiazolyl)acetamide 
 bioisostere , the carbon chain utilized 
 in most of the analogues ( figure 1 ) was trimmed 
 by four carbons before attachment via the phenolic oxygen to generate 29 . 
 following inspection of molecular models , the carbon 
 chain was shortened even further and the olefin was deleted , leading 
 to n - ipr - amide 30 , 
 urea 31 , and oxamide 32 , whose total chain 
 length more closely resembled that of 14,15-eet . as hoped , the ec50 values increased relative to 29 , although the 
 % vasorelaxation was variable . 
 contrary to expectations , 
 there was no evident correlation between 
 the pka of the eet analogues and the ed50 for vasorelaxation ( figure 2 ) , suggesting 
 factors other than ionic interactions are involved in binding the 
 carboxylate at the putative eet binding site . plot of calculated pka vs ed50 ( m ) of analogues 
 in table 1 . 
 the protonated 
 form of analogues 6 , 11 , and 12 was used for the calculation . 
 the syntheses of tetrazole 19 and 
 thioxo-1,2,4-oxadiazol 25 are summarized in scheme 1 and illustrate the approach used to prepare the 
 other analogues . 
 semihydrogenation of the known acetylene 34 using p-2 nickel and h2 led to cis - olefin 35 that was subjected 
 to azidation using diphenylphosphoryl azide ( dppa ) under mitsunobu 
 conditions . 
 staudinger reduction of the product , azide 36 , led to primary amine 37 that was reacted without purification 
 with n - pentyl isocyanate to furnish urea 38 . 
 an uneventful series of functional group interchanges proceeding 
 through alcohol 39 , bromide 40 , and ending 
 with nitrile 41 set the stage for the zinc bromide mediated 
 annulation with sodium azide that delivered 
 tetrazole 19 . 
 reagents and conditions : ( a ) 
 p2-ni/(h2nch2)2 , h2 ( 1 
 atm ) , etoh , rt , 1 h ( 96% ) ; ( b ) diad / ph3p / ph2p(o)n3 , thf , 20 to 23 c , 4 h ( 72% ) ; ( c ) 
 ph3p , h2o / thf , rt , 12 h ( 76% ) ; ( d ) c5h11nco , thf , rt , 3 h ( 76% ) ; ( e ) nbu4nf , thf , rt , 12 h ( 8289% ) ; ( f ) cbr4/ph3p , ch2cl2 , rt , 2 h ( 8384% ) ; 
 ( g ) kcn , dmso , rt , 12 h ( 7881% ) ; ( h ) nan3/znbr2 , iproh / h2o ( 1:3 ) , 110 c , 
 18 h ( 76% ) ; ( i ) ho(co)2nh(ch2)3ch3 , edci , dmf , rt , 12 h ( 73% ) ; ( j ) h2noh 
 / na2co3 , meoh / h2o ( 4:1 ) , 60 c , 18 h ( 62% ) ; 
 ( k ) im2c(s ) , thf , rt , 45 min ( 63% ) . 
 following the same sequence of transformations as described above , 42 was converted into nitrile 45 . 
 condensation 
 of hydroxyimine 46 , obtained by addition of hydroxylamine to 45 , with 1,1-(thiocarbonyl)diimidazole 
 yielded thioxo-1,2,4-oxadiazol 25 as a crystalline solid . 
 final compounds were 
 judged 95% pure by hplc using a zorbax eclipse c18 column 
 ( 250 mm  4.6 mm ; agilent ) connected to an agilent 1200 api / lc - ms 
 using acetonitrile / water combinations as eluent unless otherwise noted . 
 nuclear magnetic resonance ( nmr ) spectra were recorded on varian 300 , 
 400 , or 500 spectrometers at operating frequencies of 300/400/500 
 mhz ( h ) or 75/100/125 mhz ( c ) in cdcl3 with tms as internal standard , unless otherwise stated . 
 h nmr data are reported as follows : chemical shift ( ppm ) , 
 multiplicity ( s = singlet , br s = broad singlet , d = doublet , t = 
 triplet , q = quartet , app q = apparent quartet , qn = quintet , app 
 qn = apparent quintet , m = multiplet ) , and coupling constant ( hz ) . 
 high resolution mass spectra ( hrms ) were obtained at ut - arlington 
 using a shimadzu it - tof mass spectrometer or at the medical college 
 of wisconsin by prof . 
 infrared ( ir ) spectra were 
 obtained using a perkinelmer spectrum 1000 fourier transform spectrometer . 
 melting points were measured using an optimelt from stanford research 
 systems and are uncorrected . 
 analytical thin layer chromatography 
 ( tlc ) used emd chemicals tlc silica gel 60 f254 plates ( 0.0400.063 
 mm ) with visualization by uv light and/or kmno4 or phosphomolybdic 
 acid ( pma ) solution followed by heating . 
 all oxygen and/or moisture 
 sensitive reactions were performed under an argon atmosphere using 
 oven - dried glassware and anhydrous solvents . 
 extracts were dried over 
 anhydrous na2so4 and filtered prior to removal 
 of all volatiles under reduced pressure . 
 chromatographic purifications 
 utilized preparative tlc or flash chromatography using prepacked sio2 columns on a combiflash rf200 chromatograph ( teledyne 
 isco ) . unless otherwise noted , yields refer to isolated , purified 
 material with spectral data consistent with assigned structures or , 
 if known , were in agreement with published data . 
 reagents were purchased 
 at the highest commercial quality available and used without further 
 purification , unless otherwise noted . 
 anhydrous solvents were dried 
 using a glass contours solvent system by passage through columns of 
 activated packing material under argon immediately prior to use . a mixture of 1-(12-cyanododec-5(z)-enyl)-3-n - pentylurea ( 41 ) . 
 ( 500 mg , 1.55 mmol ) , sodium azide ( 100 mg , 1.55 mmol ) , and zinc 
 bromide ( 335 mg , 1.48 mmol ) was heated at 110 c in 2-propanol / h2o ( 1:3 , 8 ml ) while stirring vigorously in a sealed tube . after 18 h , 
 the mixture was cooled to room temperature 
 and the ph was adjusted to 1 using aq hcl ( 3 n , 4 ml ) . 
 ethyl acetate 
 ( 10 ml ) was added , and the stirring was continued until no solid was 
 present . 
 the organic layer was isolated and the aqueous layer extracted 
 with etoac ( 2  25 ml ) . 
 the combined organic fractions were washed 
 with water ( 3  25 ml ) , dried , and concentrated in vacuo . 
 the 
 residue was purified by silica gel column chromatography to give the 
 tetrazole 19 ( 431 mg , 76% ) as a colorless solid , mp 205.6205.8 
 c . 
 h nmr ( cd3od , 
 300 mhz )  5.405.30 ( m , 2h ) , 3.063.11 ( m , 4h ) , 
 2.93 ( t , j = 8.0 hz , 2h ) , 1.982.10 ( m , 4h ) , 
 1.701.82 ( m , 2h ) , 1.241.50 ( m , 16h ) , 0.90 ( t , j = 7.6 hz , 3h ) . 
 c nmr ( cd3od , 75 
 mhz )  160.16 , 156.81 , 129.77 , 129.47 , 39.81 , 39.68 , 29.88 , 
 29.80 , 29.35 , 28.99 , 28.69 , 28.55 , 27.48 , 26.85 , 26.81 , 26.68 , 22.96 , 
 22.31 , 13.22 . 
 hrms calcd for c19h37n6o [ m + 1 ] 365.3029 , found 365.3030 . a mixture of n-(13-amino-13-(hydroxyimino)tridec-5(z)-enyl)-n - n - butyloxalamide ( 46 ) ( 100 mg , 0.27 mmol ) and 1,1-(thiocarbonyl)diimidazole ( 57 
 mg , 0.32 mmol ) in dry thf ( 5 ml ) was stirred at room temperature for 
 45 min . 
 the mixture was diluted with water ( 20 ml ) and extracted with 
 ethyl acetate ( 3  10 ml ) . 
 the combined organic extracts were 
 washed with water and dried , and the solvent was evaporated in vacuo . 
 the residue was dissolved in acetonitrile ( 5 ml ) to which was then 
 added dbu ( 61 mg , 0.40 mmol ) . after stirring at room temperature for 
 1 h , the mixture was diluted with water ( 10 ml ) , adjusted ph  
 4 with 1 n hcl , and extracted with ethyl acetate ( 3  10 ml ) 
 to give thioxo-1,2,4-oxadiazol 25 ( 71 mg , 63% ) as a white 
 solid , mp 110.6110.8 c . 
 h nmr ( 400 mhz )  8.90 ( br s , nh , 1h ) , 7.52 ( br s , nh , 
 2h ) , 5.285.40 ( m , 2h ) , 3.203.40 ( m , 4h ) , 2.59 ( t , j = 7.5 hz , 2h ) , 1.982.10 ( m , 4h ) , 1.211.70 
 ( m , 16h ) , 0.92 ( t , j = 7.3 hz , 3h ) . 
 c nmr ( 100 mhz )  160.12 , 160.08 , 153.31 , 130.62 , 129.46 , 39.93 , 
 39.85 , 31.35 , 29.33 , 28.94 , 28.89 , 28.68 , 27.02 , 26.84 , 26.69 , 23.96 , 
 20.23 , 13.90 . 
 nabh4 ( 82 
 mg , 2.28 mmol ) was added in portions with vigorous stirring to a room 
 temperature solution of ni(oac)24h2o ( 567 
 mg , 2.28 mmol ) in absolute ethanol ( 20 ml ) under a hydrogen atmosphere 
 ( 1 atm ) . after 15 min , 
 freshly distilled ethylenediamine ( 0.30 ml , 
 4.56 mmol ) was added to the black suspension , followed after a further 
 15 min by a solution of 12-(tert - butyldiphenylsilyloxy)dodec-5-yn-1-ol ( 34 ) ( 4.0 g , 9.16 mmol ) in absolute 
 etoh ( 10 ml ) . 
 after 1 h , the reaction mixture was diluted with et2o ( 20 ml ) and passed through a small bed of silica gel . 
 the 
 combined ethereal filtrates were concentrated under reduced pressure 
 to afford alcohol 35 ( 3.85 g , 96% ) as a colorless oil 
 sufficiently pure to be used directly in the next step . 
 h 
 nmr ( 300 mhz )  7.647.68 ( m , 4h ) , 7.347.42 ( m , 
 6h ) , 5.425.28 ( m , 2h ) , 3.63 ( t , j = 6.4 hz , 
 4h ) , 2.081.96 ( m , 4h ) , 1.501.60 ( m , 4h ) , 1.401.24 
 ( m , 10h ) , 1.04 ( s , 9h ) . 
 c nmr ( 100 mhz )  135.81 , 
 134.40 , 130.61 , 129.71 , 129.60 , 127.80 , 64.21 , 63.14 , 32.78 , 32.60 , 
 29.98 , 29.27 , 27.42 , 27.14 , 27.10 , 26.08 , 25.92 , 19.48 . 
 diisopropyl azodicarboxylate 
 ( diad ; 1.46 ml , 7.35 mmol ) was added dropwise to a 20 c 
 solution of pph3 ( 2.10 g , 8.0 mmol ) in dry thf ( 45 ml ) 
 under an argon atmosphere . after 10 min , a solution of 12-(tert - butyldiphenylsilyloxy)dodec-5(z)-en-1-ol 
 ( 35 ) ( 3.20 g , 7.35 mmol ) in dry thf ( 10 ml ) 
 after 30 min , the mixture was warmed to 0 c and diphenylphosphoryl 
 azide ( 1.58 ml , 7.35 mmol ) was added dropwise . after stirring 4 h 
 at rt , 
 the reaction mixture was quenched with water ( 150 ml ) and extracted 
 with etoac ( 2  100 ml ) . 
 the combined organic extracts were washed 
 with brine ( 100 ml ) , dried ( na2so4 ) , and concentrated 
 under reduced pressure . 
 the residue was purified by sio2 column chromatography eluting with 4% etoac / hexane to afford azide 36 ( 2.45 g , 72% ) . 
 h nmr ( 400 mhz )  7.647.68 
 ( m , 4h ) , 7.347.42 ( m , 6h ) , 5.285.42 ( m , 2h ) , 3.70 
 ( t , j = 5.8 hz , 2h ) , 3.27 ( t , j = 
 6.3 hz , 2h ) , 1.962.10 ( m , 4h ) , 1.241.64 ( m , 12h ) , 
 1.04 ( s , 9h ) . 
 c nmr ( 100 mhz )  135.84 , 134.41 , 
 130.93 , 129.75 , 129.12 , 127.83 , 64.22 , 51.62 , 32.81 , 29.93 , 29.30 , 
 28.68 , 27.46 , 27.14 , 27.02 , 26.90 , 25.96 , 19.49 . 
 ir ( neat ) 2930 , 2783 , 
 2331 , 2097 , 1106 cm . 
 triphenylphosphine ( 1.18 
 g , 4.50 mmol ) was added to a stirring solution of 1-tert - butyldiphenylsilyloxy-12-azidododec-7(z)-ene ( 36 ) ( 1.90 g , 4.10 mmol ) in thf ( 12 ml ) containing 10 drops 
 of deionized water . 
 after 12 h , the reaction mixture was diluted with 
 ch2cl2 ( 10 ml ) , dried , and concentrated in vacuo 
 to give amine 37 ( 1.36 g , 76% ) as a viscous , colorless 
 oil that was used directly in the next reaction without further purification . 
 h nmr ( 400 mhz )  7.627.68 
 ( m , 4h ) , 7.327.40 ( m , 6h ) , 5.305.40 ( m , 2h ) , 3.63 
 ( t , j = 5.2 hz , 2h ) , 2.62 ( t , j = 
 4.8 hz , 2h ) , 1.922.06 ( m , 4h ) , 1.401.58 ( m , 4h ) , 1.201.40 
 ( m , 8h ) , 1.03 ( s , 9h ) . 
 c nmr ( 100 mhz )  135.79 , 
 134.37 , 130.42 , 129.70 , 127.78 , 64.19 , 42.28 , 33.44 , 32.77 , 29.93 , 
 29.28 , 27.40 , 27.21 , 27.10 , 25.92 , 19.44 . 
 a solution of 1-tert - butyldiphenylsilyloxy-12-aminododec-7(z)-ene ( 37 ) ( 1.32 g , 3.0 mmol ) in thf ( 5 ml ) 
 was added dropwise to a stirring solution of n - pentyl 
 isocyanate ( 0.386 ml , 3.0 mmol ) in thf ( 10 ml ) . 
 after 3 h stirring 
 at room temperature , all volatiles were removed under reduced pressure 
 and the residue was purified by sio2 column chromatography 
 eluting with 20% etoac / hexane to afford urea 38 ( 1.26 
 g , 76% ) as a viscous oil . 
 h nmr ( 300 mhz )  7.607.70 
 ( m , 4h ) , 7.357.42 ( m , 6h ) , 5.285.42 ( m , 2h ) , 5.16 
 ( br s , nh , 2h ) , 3.65 ( t , j = 6.5 hz , 2h ) , 
 3.083.20 ( m , 4h ) , 1.962.08 ( m , 4h ) , 1.221.60 
 ( m , 18h ) , 1.02 ( s , 9h ) , 0.89 ( t , j = 7.3 hz , 3h ) . 
 c nmr ( 100 mhz )  159.23 , 135.80 , 134.24 , 130.52 , 129.74 , 
 129.49 , 127.82 , 64.22 , 40.62 , 40.54 , 32.80 , 30.33 , 29.95 , 29.37 , 29.32 , 
 27.46 , 27.34 , 27.18 , 27.11 , 25.97 , 22.71 , 19.46 , 14.29 . 
 hrms calcd 
 for c34h55n2o2si [ m + 
 1 ] 551.4033 , found 551.4032 . a mixture of 1-(12-(tert - butyldiphenylsilyloxy)dodec-5(z)-enyl)-3-n - pentylurea ( 38 ) ( 1.12 g , 2.0 mmol ) and tetra - n - butylammonium fluoride 
 ( 2.20 ml of 1 m soln in thf , 2.2 
 mmol ) in dry thf ( 10 ml ) was stirred at room temperature under an 
 argon atmosphere for 12 h and then evaporated to dryness in vacuo . 
 the residue was dissolved in etoac ( 50 ml ) and washed with water ( 30 
 ml ) , brine ( 30 ml ) , dried , and evaporated in vacuo . 
 purification of 
 the residue via sio2 column chromatography gave alcohol 39 ( 0.56 g , 89% ) as a colorless solid , mp 63.763.8 
 c . 
 h nmr ( 300 mhz )  5.255.42 ( m , 2h ) , 
 4.48 ( br s , nh , 2h ) , 3.64 ( d , j = 6.5 hz , 
 2h ) , 3.083.20 ( m , 4h ) , 1.962.14 ( m , 4h ) , 1.221.60 
 ( m , 18h ) , 0.88 ( t , j = 7.0 hz , 3h ) . 
 c nmr ( 125 mhz )  159.26 , 130.23 , 129.62 , 63.72 , 40.33 , 40.29 , 
 32.92 , 30.30 , 30.26 , 29.74 , 29.35 , 29.13 , 27.26 , 27.20 , 27.13 , 25.82 , 
 22.69 , 14.27 . 
 cbr4 ( 0.55 g , 1.66 
 mmol ) and pph3 ( 0.43 g , 1.66 mmol ) were added to a 0 c 
 solution of 1-(12-hydroxydodec-5(z)-enyl)-3-n - pentylurea ( 39 ) ( 0.43 g , 1.38 mmol ) in ch2cl2 ( 20 ml ) . 
 after 2 h at room temperature , the 
 reaction mixture was concentrated in vacuo and the residue was purified 
 via sio2 column chromatography to give 1-(12-bromododec-5(z)-enyl)-3-n - pentylurea ( 40 ) ( 0.43 g , 83% ) as a solid , mp 46.746.8 c . 
 h 
 nmr ( 300 mhz )  5.225.42 ( m , 2h ) , 4.40 ( br s , 2h ) , 3.42 
 ( t , j = 9.3 hz , 2h ) , 3.103.20 ( m , 4h ) , 1.982.10 
 ( m , 4h ) , 1.801.90 ( m , 2h ) , 1.251.55 ( m , 16h ) , 0.92 
 ( t , j = 7.2 hz , 3h ) . c nmr ( 100 mhz ) 
  159.51 , 130.14 , 129.69 , 40.48 , 40.39 , 34.20 , 32.96 , 30.34 , 
 29.67 , 29.36 , 28.58 , 28.25 , 27.31 , 27.27 , 27.17 , 22.68 , 14.26 . 
 hrms 
 calcd for c18h36brn2o [ m + 1 ] 375.2011 , found 375.2014 . a mixture of potassium cyanide 
 ( 0.23 g , 3.54 mmol ) and 1-(12-bromododec-5(z)-enyl)-3-n - pentylurea ( 40 ) ( 0.90 g , 2.40 mmol ) was stirred 
 in dmso ( 5 ml ) at room temperature . 
 after 12 h , the reaction mixture 
 was diluted with water ( 20 ml ) and extracted with ethyl acetate ( 2 
  50 ml ) . 
 the combined organic extracts were washed with water 
 ( 2  25 ml ) , brine ( 25 ml ) , dried ( na2so4 ) , and passed through a silica gel column to give nitrile 41 ( 0.62 g , 81% ) as a colorless solid , mp 5657 c . 
 h nmr ( 300 mhz )  5.295.40 ( m , 2h ) , 4.27 ( br 
 s , nh , 2h ) , 3.103.20 ( m , 4h ) , 2.34 ( t , j = 7.0 hz , 2h ) , 1.982.08 ( m , 4h ) 1.241.70 ( m , 18h ) , 
 0.89 ( t , j = 7.0 hz , 3h ) . 
 c nmr ( 125 
 mhz )  159.41 , 129.94 , 129.86 , 120.14 , 40.45 , 40.35 , 30.30 , 
 29.50 , 29.33 , 28.70 , 28.51 , 27.26 , 27.16 , 25.47 , 22.66 , 17.28 , 14.24 . 
 ir ( neat ) 2930 , 2281 , 2184 , 2042 , 1936 , 1613 , 1197 , 1042 cm . 
 hrms calcd for c19h36n3o [ m + 
 1 ] 322.2858 , found 322.2867 . a mixture of 
 2-(n - butylamino)-2-oxoacetic acid ( 0.40 g , 2.70 mmol ) , 1-tert - butyldiphenylsilyloxy-12-aminododec-7(z)-ene ( 37 ) ( 1.20 g , 2.70 mmol ) , 1-hydroxybenzotriazole 
 ( hobt ; 0.44 g , 3.30 mmol ) , and [ 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide 
 hydrochloride ] ( edci : 0.63 g , 3.30 mmol ) in dry dmf ( 5 ml ) was stirred 
 at room temperature overnight . 
 the reaction mixture was quenched with 
 water ( 30 ml ) and extracted into ethyl acetate ( 3  20 ml ) . 
 the 
 combined organic extracts were washed with water ( 2  10 ml ) 
 and brine ( 10 ml ) , dried , and concentrated in vacuo . 
 the residue was 
 purified by sio2 column chromatography to give n - n - butyl - n-(12-(tert - butyldiphenylsilyloxy)dodec-5(z)-enyl)oxalamide ( 42 ) ( 1.10 g , 73% ) . 
 h nmr ( 400 mhz )  8.05 ( br s , nh , 2h ) , 7.667.74 
 ( m , 4h ) , 7.327.42 ( m , 6h ) , 5.305.42 ( m , 2h ) , 3.67 
 ( t , j = 3.9 hz , 2h ) , 3.31 ( q , j = 
 5.2 hz , 4h ) , 1.962.10 ( m , 4h ) , 1.501.64 ( m , 6h ) , 1.221.44 
 ( m , 10h ) , 1.06 ( s , 9h ) , 0.92 ( t , j = 7.8 hz , 3h ) . 
 c nmr ( 100 mhz )  160.33 , 135.80 , 134.35 , 130.73 , 129.74 , 
 129.20 , 127.83 , 64.17 , 39.89 , 39.69 , 32.79 , 31.48 , 29.94 , 29.29 , 29.07 , 
 27.46 , 27.23 , 27.14 , 27.0 , 25.96 , 20.29 , 19.46 , 13.96 . 
 hrms calcd 
 for c34h53n2o3si [ m + 
 1 ] 565.3826 , found 565.3824 . 
 n - n - butyl - n-(12-(tert - butyldiphenylsilyloxy)dodec-5(z)-enyl)oxalamide ( 42 ) ( 1.20 g , 2.12 mmol ) 
 was desilylated as described above for 39 to give n - n - butyl - n-(12-hydroxydodec-5(z)-enyl)oxalamide 
 ( 43 ) ( 0.568 g , 82% ) as a colorless solid , mp 102.8102.9 
 c . 
 h nmr ( 400 mhz )  7.69 ( br s , 2h ) , 5.205.35 
 ( m , 2h ) , 3.56 ( t , j = 4.2 hz , 2h ) , 3.26 ( q , j = 5.6 hz , 4h ) , 2.17 ( br s , 1h ) , 1.952.02 ( m , 4h ) , 
 1.441.56 ( m , 6h ) , 1.201.40 ( m , 10h ) , 0.87 ( t , j = 7.2 hz , 3h ) . 
 c nmr ( 100 mhz )  160.15 , 
 130.66 , 129.21 , 62.98 , 39.80 , 39.63 , 32.93 , 31.39 , 29.77 , 29.18 , 28.95 , 
 27.26 , 27.0 , 26.88 , 25.80 , 20.18 , 13.85 . 
 n - n - butyl - n-(12-hydroxydodec-5(z)-enyl)oxalamide ( 43 ) ( 330 mg , 1.0 mmol ) was 
 brominated as described above for 40 to give n-(12-bromododec-5(z)-enyl)-n - n - butyloxalamide ( 44 ) ( 330 mg , 84% ) as a white solid , mp 46.046.3 c . 
 h nmr ( 400 mhz )  7.79 ( br s , nh , 1h ) , 7.77 
 ( br s , nh , 1h ) , 5.205.32 ( m , 2h ) , 3.32 ( t , j = 6.4 hz , 2h ) , 3.22 ( q , j = 7.2 hz , 4h ) , 
 1.902.00 ( m , 4h ) , 1.721.82 ( m , 2h ) , 1.421.56 
 ( m , 4h ) , 1.201.40 ( m , 10h ) , 0.85 ( t , j = 
 7.3 hz , 3h ) . 
 c nmr ( 100 mhz )  160.17 , 160.15 , 
 130.40 , 129.34 , 39.77 , 39.59 , 34.12 , 32.93 , 31.40 , 29.62 , 29.0 , 28.54 , 
 27.25 , 27.24 , 27.0 , 26.91 , 20.18 , 13.85 . 
 n-(12-bromododec-5(z)-enyl)-n - n - butyloxalamide ( 44 ) ( 250 mg , 0.642 mmol ) was treated 
 with potassium cyanide as described above for 41 to give n - n - butyl - n-(12-cyanododec-5(z)-enyl)oxalamide 
 ( 45 ) ( 168 mg , 78% ) as a colorless solid , mp 83.083.3 
 c . 
 h nmr ( 400 mhz )  7.45 ( br s , nh , 
 2h ) , 5.305.40 ( m , 2h ) , 3.34 ( q , j = 8.6 hz , 
 4h ) , 2.32 ( t , j = 7.6 hz , 2h ) , 1.982.08 ( m , 
 4h ) , 1.301.68 ( m , 16h ) , 0.92 ( t , j = 7.2 
 hz , 3h ) . 
 c nmr ( 100 mhz )  160.03 ( 2c ) , 130.03 , 
 129.08 , 120.10 , 39.88 , 39.42 , 31.22 , 29.40 , 28.82 , 28.60 , 28.42 , 27.07 , 
 27.06 , 26.82 , 25.54 , 20.06 , 17.01 , 13.80 . 
 hrms calcd for c19h34n3o2 [ m + 1 ] 336.2651 , 
 found 336.2650 . to a suspension of n - n - butyl - n-(12-cyanododec-5(z)-enyl)oxalamide 
 ( 45 ) 
 ( 420 mg , 1.29 mmol ) in meoh / h2o ( 4:1 ; 
 12 ml ) was added h2nohhcl ( 228 mg , 3.28 mmol ) and 
 na2co3 ( 344 mg , 3.25 mmol ) . 
 the reaction mixture was heated at 60 c for 18 h 
 then cooled to room temperature , and all volatiles were removed in 
 vacuo . 
 the residue was diluted with water ( 30 ml ) and extracted into 
 ethyl acetate ( 2  25 ml ) . 
 the combined organic extracts were 
 washed with water ( 2  10 ml ) and brine ( 10 ml ) , dried , and purified 
 via silica gel column chromatography to give n-(13-amino-13-(hydroxyimino)tridec-5(z)-enyl)-n - n - butyloxalamide ( 46 ) ( 287 mg , 62% ) as a colorless solid , 116.3116.4 c . 
 h nmr ( cd3od , 400 mhz ) 
  5.285.40 ( m , 2h ) , 3.24 ( t , j = 6.4 
 hz , 4h ) , 1.982.00 ( m , 6h ) , 1.501.60 ( m , 6h ) , 1.261.40 
 ( m , 10h ) , 0.92 ( t , j = 7.3 hz , 3h ) . 
 c nmr ( cd3od , 100 mhz )  160.55 ( 2c ) , 156.31 , 130.05 , 
 129.18 , 39.23 , 39.09 , 31.18 , 30.63 , 29.51 , 28.83 , 28.69 , 27.10 , 26.87 , 
 26.59 , 19.88 , 12.88 . 
 the influence of eicosanoids and analogues 
 on coronary vascular tone was measured by the induced changes in isometric 
 tension of bovine coronary artery rings precontracted with the thromboxane - mimetic , 
 u46619 , as previously described . 
 all assays were conducted in triplicate 
 or greater and are means 10% sd of the reported value . 
 recombinant human seh was produced in a baculovirus expression system and was purified by affinity chromatography . 
 human seh ( 1 nm ) was incubated with inhibitors ( 0.4 < 
 [ i]final < 100000 nm ) for 5 min in 25 mm bis - tris / hcl 
 buffer ( 200 ml , ph 7.0 ) at 30 c before the substrate , cyano(2-methoxynaphthalen-6-yl)methyl trans-(3-phenyl - oxyyran-2-yl]methyl carbonate ( cmnpc ; [ s]final = 5 mm ) , was added . 
 activity was assessed by measuring 
 the appearance of the fluorescent 6-methoxynaphthaldehyde product 
 ( em = 330 nm , ex = 465 nm ) at 
 30 c during a 10 min incubation ( spectramax m2 , molecular device , 
 inc . , 
 ic50s refer to the concentrations of inhibitor that reduced activity 
 by 50% and are the averages of three replicates .","the cytochrome p450 eicosanoid 14,15-epoxyeicosa-5,8,11-trienoic 
 acid ( 14,15-eet ) is a powerful endogenous autacoid that has been ascribed 
 an impressive array of physiologic functions including regulation 
 of blood pressure . because 14,15-eet is chemically and metabolically 
 labile , structurally related surrogates containing epoxide bioisosteres 
 were introduced and 
 have become useful in vitro pharmacologic tools 
 but are not suitable for in vivo applications . a new generation of 
 eet mimics incorporating modifications to the carboxylate were prepared 
 and evaluated for vasorelaxation and inhibition of soluble epoxide 
 hydrolase ( seh ) . 
 tetrazole 19 ( ed50 0.18 m ) 
 and oxadiazole-5-thione 25 ( ed50 0.36 m ) 
 were 12- and 6-fold more potent , respectively , than 14,15-eet as vasorelaxants ; 
 on the other hand , their ability to block seh differed substantially , 
 i.e. , 11 vs > 500 nm . these data will expedite the development of 
 potent 
 and specific in vivo drug candidates .",pubmed
"in response to the increasing demand for an improved healthcare system in the united states , the american academy of family physicians , the american academy of pediatrics , the american college of physicians , and the american osteopathic association developed the joint principles of the patient - centered medical home ( table 1 ) . the patient - centered medical home ( pcmh ) is an extension of internationally employed edward wagner 's chronic care model ( ccm ) . 
 the ccm was developed to address the increasing rate of patients with chronic conditions in the united states using team - based care . 
 the rate of chronic conditions is currently estimated to be 2.2 conditions for individuals having 60 years old and up , on average . in its implementation , the ccm has proven to reduce patients ' healthcare costs and improve patient care quality , two elements directly aligned with the goals of the pcmh . 
 the pcmh model strives to provide quality , coordinated , and cost - effective care to patients and to increase access to services . 
 in addition , it aims to increase practice efficiency and subsequently provider and patient satisfaction . within this paper , we follow the process of implementing the pcmh model within primary care practices and discuss the difficulties these practices have encountered in the transition as well as potential solutions . 
 our goal is to provide future pcmhs with an insight into the transition process and to remove the likelihood of these problems reoccurring .   in spite of difficulty with the transition process , over 1,500 sites and 7,700 clinicians across the united states 
 have successfully completed the national committee for quality assurance ( ncqa ) recognition process and are functioning as patient - centered medical homes . 
 they see pcmh as a way to better serve their patients , to address the crisis in primary care , and to seize evolving payment opportunities . 
 encouraged by the expansion of the pcmh across the united states , in june 2010 , medical practitioners and healthcare administrators ( military providers , civilian doctors and nurses , researchers , hospital staff , and administration ) met in alexandria , virginia , to discuss their experiences with the transition . from the conference , we were able to collect detailed experiences that will provide a unique insight and data from the transition process . from the panel of attendees , two specific healthcare providers  carillion clinic and the air force  contributed the vast majority of the examples and experiences discussed here . 
 carillion clinic is a large healthcare organization located in southwest virginia , providing healthcare to individuals in both urban and rural settings . 
 their organization comprises over 600 physicians in multicare - specialty group practices and eight not - for - profit hospitals . 
 the examples in this paper are primarily taken from their experiences transitioning their urban primary care practices into pcmhs . 
 more specifically , their experiences were directly pulled from practices with multiple physicians ( carilion clinic does group certifications of pcmhs by region ) . 
 the air force has employed their version of the pcmh , termed the air force patient - centered medical home ( previously the family health initiative ) , at several of their bases within the united states . 
 these air force pcmh practices are within the air force bases themselves , and each of them has a patient panel of military beneficiaries ( active duty members , retirees , and families ) creating a unique healthcare environment because the air force operates under a complex healthcare and insurance system with both military care and purchased care ( our focus in this paper is on the onbase care provided by the air force and does not pertain to care purchased offbase through the air force 's tricare program ) .   from these healthcare organizations ' experiences , 
 combined with a literature review of empirical work , this paper addresses the challenges and successes encountered in the transition to pcmh . 
 the purpose of this paper is to realize the difficulties that arise in the transition into a pcmh across various settings and to provide solutions for practices to follow in their transition given their particular needs . 
 this way these practices can provide better quality of care to patients , decrease patient 's health care costs , and improve the system as a whole . 
 lessons learned  discusses the most common concerns among primary care practices who have initiated the transition to a pcmh , which are promoting physician buy - in , changing office culture , care coordination , staffing and space allocations , and leveraging electronic medical records ( emrs ) . under each of these topics , difficulties and successes are examined using specific examples provided by the pcmh workshop and previous studies . 
 the first step in the transition process is to understand what procedures and standards a primary care practice must follow in order to obtain recognition . 
 recognition as a pcmh increases the likelihood of reimbursement for the pioneering pcmh procedures that are currently undercompensated . 
 as more studies discuss the positive results of pcmh , new reimbursement methods become more of an obtainable goal . for a practice 
 to become recognized as a pcmh by the ncqa , the practice must provide documentation of the practice 's guidelines for implementation ( table 2 , ) . 
 however , ncqa standards do not offer instructions for practices to follow in making the transition . 
 this flexibility in the ncqa standards is necessary due to the uniqueness of each practice as it would be difficult to create one that addresses the implementation of all the standards of the pcmh for each practice type . 
 consequently , each practice must create its own written policies that adhere to pcmh principles and fit the specific practice structure . 
 practices must account for their patient panels , location , and financial resources when creating policies . 
 the practices must then decide whether it would better suit their needs to attempt the transformation incrementally or all at once . 
 once these policies have been written , implemented , and then the outcomes documented , the practice can apply for ncqa recognition through an online survey that collects information regarding its guidelines about administration ( appointments , access , telephone calls ) , clinical services ( patient satisfaction , tracking critically important conditions ) , and performance tracking [ 8 , 9 ] . 
 once these steps are completed successfully as determined by the ncqa review process , the practice is awarded one of three tiers of recognition by the ncqa ( the ncqa is currently debating reframing the recognition system to a two - tiered recognition system . some supporters of the pcmh feel that the implementation of the model should be to the fullest extent possible and only acknowledged in by the ncqa in these cases . however , as discussed within this paper , there are difficulties that arise when attempting to administer all of the changes necessary and not all practices can achieve the standards required for these recognition levels ) . 
 each tier reflects how many performance elements under each ncqa standard the practice has satisfied . as with the introduction of any innovative healthcare model 
 , the transition and recognition of a pcmh can be time consuming and expensive , but rewards range from better patient care to a more coordinated practice structure and also potentially reducing the overall cost of care for the patient in their lifetime through preventative and care management services , the latter of which is becoming more and more of a concern for primary care practices due to the current debate over reimbursement schedules . 
 primary care physicians typically leave the workforce earlier in their careers than specialists with complaints of being overworked and poorly compensated . therefore , 
 selling the model to physicians , with their already hectic schedules , so that their participation and contribution to pcmh is pivotal in a successful transformation 
 . however , many physicians involved in the transformation resist the change due to a lack of the appropriate pcmh training , misaligned financial incentives , underreimbursement , and time - consuming procedures . 
 the pcmh model recommends that practice staff and providers ( nurses and physicians ) take time to analyze patients ' needs as a whole . 
 the model encourages practice leaders to empower ancillary staff to enlist protocols when meeting chronic , acute , and health maintenance needs . 
 this team - based care is delivered prior to or after the provider encounter during a visit . 
 the air force gave the following example of team - based collaboration when addressing a patient with a sprained ankle . 
 first , the technician evaluates the patient and then performs the ottawa ankle rules , which clearly delineate whether the patient requires an x - ray . 
 next , nurses will order x - rays based upon the results of the ottawa ankle rules the technician provides . 
 subsequently , the skilled nurses return to educate the patient on ankle exercises and necessary bracing . training a nurse to do this initial triage and intervention 
 enables the provider to meet with the patient after x - rays are complete , which allows for a more efficient appointment . 
 the team - based care creates a routine in which it is no longer necessary for a provider to address the patient each time they come to the practice . 
 many physicians are reluctant to release these face - to - face encounters with patients to their staff . 
 this can be explained by the lack of training specific to pcmh practices , as well as the current reimbursement schedule , which encourage physicians to treat patients based on volume of care , rather than quality . 
 practices must emphasize that the reallocation of responsibilities across the practice 's medical team can create more time for them to complete other requirements and make the practice more efficient as seen in the above air force example . 
 second , the current form of financial reimbursement creates misaligned incentives for the physicians with their new responsibilities . 
 for instance , according to the medicare resource - based relative value scale ( rbrvs ) , physicians are only reimbursed for a patient visit if they can report a 15-minute office evaluation , discouraging the physicians from relinquishing any of their patients ' visits to their nurses . restructuring the reimbursement scheme so that such face - to - face visits between physicians and patients are not required would allow physicians to provide care within the best interest of the patient and their practice . however 
 , this modification may involve many hours of lobbying and petitioning the federal government and insurance companies . with many successful pcmhs across the united states , in order to sustain the model payment schemes will eventually need to adjust to reimburse physicians for improving patients ' quality of care and overall health , rather than for just the volume of services they provide . aside from physicians ' reimbursement , 
 other components of the pcmh policies , such as care coordination and team - based care , require the use of resources not currently reimbursed in most health insurance packages . 
 in addition to the above - mentioned responsibilities of the physicians and their staff , the pcmh model introduces team - based care to primary care practices . team - based care requires staff to address patient treatment decisions collectively . in advance of each appointment 
 , a previsit meeting is conducted to allocate resources , to ensure that adequate health maintenance is performed , and to see that chronic care management is addressed by the care team ( nurses , care coordinators , physicians , other providers in the practice , and front office staff ) . 
 however , it is important to emphasize that previous studies show the use of previsit huddles has resulted in improved decisionmaking and care for patients , as well as patient satisfaction [ 15 , 16 ] . the studies also show that patients provided with team - based care had increases in their satisfaction levels from 5 to 10 points in five out of six satisfaction care scales . 
 in addition , their research shows that once providers accepted the team - based care structure , their satisfaction levels increased as well . 
 in spite of the current difficulty with physician buy - in to pcmh , we found that many physicians are inspired by their ability to provide patients with the quality care they deserve through pcmh . 
 a lead primary care physician from a carillion clinic has seen an improvement in the health of patients due to the coordination and team - based care pcmh offersthe pcmh care model has helped us provide more comprehensive care to our patient population . 
 the use of registries and care coordinators have enabled us to focus on patients that are not getting needed care or are not returning for follow - up appointments as they should . 
 the pcmh care model has helped us provide more comprehensive care to our patient population . 
 the use of registries and care coordinators have enabled us to focus on patients that are not getting needed care or are not returning for follow - up appointments as they should . 
 the transformation of a primary care practice into a pcmh requires significant changes in office culture . in this section 
 , we will discuss several areas that need to be addressed by the primary care practice , including nurse and patient experience . 
 implementing the pcmh model is not as simple as assigning new staff roles and restructuring old staff with new titles . 
 the healthcare organization must understand how changing positions within the practice brings new experiences to all of their current members and how these experiences can potentially affect their capability of delivering care . 
 for instance , in the air force , many nurses in the pcmh transition shift from positions where they have not been exposed to patients to being required to evaluate patients face to face regularly ; this new interaction created anxiety among these nurses . the air force has addressed this issue through additional training programs so nurses can better understand their new relationship with patients . 
 in addition to providing new training for nurses , it is important also to train front office staff in their new roles as they play a part in carrying out several pcmh principles , such as ensuring greater access and increased communication . 
 both clerical and clinical staff members are encouraged to develop a more sophisticated level of decision - making skills , which may require additional training on the pcmh principles , new workflow processes in the office , and increasingly integrated responsibilities to patients and each other . 
 every healthcare model must take into account patients ' response to the new healthcare structure . 
 patients value the respect , quality , and concern of their healthcare provider in addition to accessibility ; their reaction to the change in structure significantly affects the ability of a team to provide care . 
 it is equally important that patients understand that the ultimate goal of pcmh is to improve their quality of life and participate in achieving this objective . 
 pcmh focuses not only on the manner in which care is administered , but also educating patients regarding their health and ensuring they have the knowledge necessary to improve it . 
 national pcmh demonstration projects have shown patient education has increased their participation in their own care and subsequently their quality of life . 
 patients are encouraged to interact with practice staff and their providers regularly during appointments and through new means of communication , such as online patient portals , and to take advantage of health education opportunities within the community to which they are referred by staff . 
 in pcmh , practices are tasked with improving care coordination and documenting patient engagement in self - management . 
 this requirement has prompted many pcmhs to create a care manager or care coordinator position . 
 this staff member ( e.g. , a nurse or nonclinical staff member ) performs tasks that were previously delivered inconsistently or not at all , or that were formerly the responsibility of the physician , such as patient followups and patient education ( when introducing the care coordinator to the patients , we encourage caution to avoid dramatization of past inadequacies in care delivery during patient and physician interactions , which could undermine their relationship with the physician . ) throughout the pcmh conference , discussants emphasized their success with care coordinators in their practices , many of whom have received positive feedback from their patients . 
 for instance , a diabetic patient from one of carilion clinic 's roanoke , virginia practices reportedi have recently been diagnosed with diabetes . while this was initially very upsetting for me , i was able to receive immediate counseling about my diabetes from the care coordinator . 
 we reviewed some initial diet and exercise information , and then she set me up to see a dietitian in the diabetes management program.this patient 's improvement is unquestionable and shows the benefits this new position can provide . 
 however , problems arise with the adoption of multidisciplinary team members in the education and support of patients , their time , salary , responsibilities , and workload are not defined in the cost or structure of a typical practice . 
 additionally , many practices that hire care coordinators face the challenge of reimbursing their work ; this is because their work is typically not covered by health insurance at all or only to a limited extent . 
 while this was initially very upsetting for me , i was able to receive immediate counseling about my diabetes from the care coordinator . 
 we reviewed some initial diet and exercise information , and then she set me up to see a dietitian in the diabetes management program . 
 in addition to self - management coaching of those patients with chronic conditions , the care coordinators at carilion clinic have been assigned the task of population management . 
 the care coordinators use registries ( registries are a list of patients with a chronic disease displayed with many clinical features to stratify level of disease control ) , to focus on specific chronic diseases , such as high blood pressure and diabetes . 
 care coordinators use predetermined protocols to contact those patients in poor disease control or those who are due for a follow - up visit . 
 once these patients come back into the office , a number of things happen as they reengage with their care team . 
 the nurse updates the health maintenance care in the chart , the provider focuses on the acute or chronic disease issues , and the care coordinator offers to meet with the patient , further identifying barriers they face that may interfere with disease self - management . a sophisticated emr or free - standing disease registry facilitates this task . 
 these technologies are used to organize and target the entire patient panel to track health indicators and in turn , isolate the patients of interest . 
 this disciplined approach to population management is not typical in most primary care offices ; therefore , this activity tends not to be included in the budget , and time is not allocated for it . 
 if the transitioning pcmh practice is committed to carrying out population management work , there needs to be added space for this individual to work along with a private consultation area to perform the self - management coaching .   along with refocusing the office culture to adhere to the new comprehensive level of care , pcmh connects patients with resources in the healthcare system and the community . 
 primary care practices need to take time to build collaborative relationships with other professionals , such as social workers , nutritionists , and health educators , to provide needed services . 
 it is not feasible for most pcmh practice sites to have space on hand for these professionals to work side by side at all times . 
 the pcmh model does not require that a specific staff member document the services offered , ordered , or rendered , and neither does ncqa . 
 rather , what is important is that there is a workflow process in place for such documentation in patients ' medical records . 
 for instance , at carilion , ambulatory nurses have taken on roles in both chronic disease management and health maintenance . in some practices , 
 nurses have taken on the documentation of all health maintenance needs of patients , while in others , this responsibility is shared among team members . 
 no matter who plays what role , they need to be clearly within individual practice workflows and protocols .   there may be a period when nurses will need to familiarize themselves with the new structure . during this time 
 for example , the air force experienced a backlog problem with the transition of nurses from floor nurses to disease managers . 
 the disease manager started as one of the nurses in the clinic but was taken out of patient treatment and put into this new position . 
 the problem is that this created backfill , so the disease management nurses were not able to concentrate on the task at hand but instead needed to address the problems that arose in the practice during their training . 
 once the air force 's disease management nurses were able to focus on disease management , there was significant improvement in the quality of care for patients . 
 it was also found in an outside study that patients who participated in chronic disease management had less frequent hospitalizations and on average spent 0.8 fewer nights in the hospital once admitted . 
 carilion clinic has also implemented disease management and has found that scanning the patient registry on a regular basis uncovers patients who would benefit from intensive disease management . in particular , 
 one patient under carilion 's care , a 70-year - old woman with diabetes , coronary artery d status after coronary artery bypass grafting , and congestive heart failure , was oxygen dependent and wheelchair bound . 
 she was contacted after the care coordinator saw in her emr that she had visited the emergency room three times within the past eight months and one visit resulted in a prolonged hospital and intensive care unit stay . following the practice 's protocol , her care team referred her to cardiac rehab . 
 subsequently , she has not visited the emergency room in over a year , has lost 30 pounds , no longer needs oxygen , and walks without assistance . 
 the size of the primary care practice can be an obstacle in implementing the pcmh model . here , the term small is used to refer to primary care practices with only one or two physicians and an average - sized patient panel ( it is not uncommon for a primary care practice with one physician to have a couple thousand patients ; however , practices discussed within this paper do not have over 2,000 patients in their panel ) . 
 approximately 32% of healthcare physicians practice in either an individual or a partnership practice , and 60% of practices function with 50 physicians or fewer . 
 smaller practices typically can not employ the same resources as larger facilities due to budget constraints , and therefore , they may have a harder time making the changes necessary to become a pcmh and have to find ways around their limited resources . 
 for instance , rather than full - time coordinators in small practices , carilion employs shared coordinators in sites where there are only one or two full - time providers ; as such these smaller practices can not rationalize adding a full - time staff member . 
 national pcmh demonstrations have shown that these smaller practices with such resource constraints have a more difficult time transforming to a high - functioning pcmh ; they may qualify as level 1 , rather than level 3 , which means they only meet the minimum requirements of the ncqa [ 1719 ] . 
 an emr is not required by the ncqa for a practice to apply for pcmh recognition , but having one may facilitate the practice 's goals for care coordination . implementing an emr is not particularly difficult ; instead , the concern is who will service , update , and sustain it . 
 regardless of the size of practices , pcmh principles encourage the use of electronic medical records to increase the efficiency of care coordination , although it is not required . this change in record keeping 
 removes the need for medical record storage space within the practice and in turn allows for clinical space to be optimized . 
 prior to fully transitioning to the pcmh model , healthcare organizations must address the issue of space . 
 for instance , if a practice employs a care coordinator , they must be provided with space to communicate with patients in private , to perform their health assessments and individual disease coaching on the phone or in person , and to prepare plans for care , such as materials for the pre - visit huddle . 
 it is uncommon for the traditional primary care practices to have a location for these types of private conversations and activities . 
 empty exam rooms or doctors ' offices may just be sufficient , especially since these conversations taking place in waiting rooms or more public areas can easily violate confidentiality standards and the health insurance portability and accountability act of 1996 . 
 a fully integrated emr helps pcmhs conduct team - based care for chronic conditions by allowing members to visualize the patient instantly , through the use of problem lists , immunization history , and medication lists . 
 however , creating an emr that administers the principles of pcmh has been a difficult task for primary care practices in transition . in a 2006 study , 
 it was reported that only 42% of primary care practices found it easy to generate a disease registry , while only 18% felt it was easy to create special follow - up appointments based on high - risk medications patients were taking . 
 many of the pcmh conference attendees had complaints about their difficulty in creating an emr that suited their specific practice 's needs . for practices transitioning from a paper - based system to an emr 
 , it takes staff members a great deal of time to input all of the information into a computerized system 
 . practices sometimes find the sheer volume of information needing to be recorded overwhelming and difficult to decipher , because there may not have been a pcmh template in paper charts . 
 if the practice is unable to employ the emr to its advantage , many of the goals of pcmh are very difficult to satisfy . for instance , care coordination and disease management are very difficult for large practices to handle using a paper - based system . 
 once employed , emrs can be used to improve patient care and therefore patient health by monitoring their health status to anticipate necessary treatments . using the emr , 
 care coordinators and nurses are able to more efficiently search for specific patient types by viewing the patient 's medical record on the computerized database and to acknowledge those who require immediate attention and contact them in a timely fashion . 
 staff then uses population management tools in the emr to contact these individuals and encourage them to make an appointment . in a review of the pcmh model , 
 the authors were encouraged by pcmh physicians who are using proactive methods to assist unhealthy patients , rather than waiting for patients to seek help . 
 carilion clinic has created a structured method of administering disease management resulting in the optimization of their emr . 
 their approach is to first search and then contact their patients who have a priority one rating . 
 their searches are focused on patients with a chronic disease , which can be sorted in the emr based on their conditions . thus far , in their transition to a fully integrated emr , carillion clinic has formed registries for asthma , congestive heart failure ( chf ) , diabetes ( dm ) , and high blood pressure ( htn ) . within each of the registries , the patient 's clinical information , such as blood pressure , 
 their registries can be sorted by the necessity of care or the priority as determined by carillion clinic . 
 priority one patients are those who have at least one of three predefined health conditions determined by carilion clinic ( carillion developed these predefined health conditions from nationally recognized medical guidelines ) . 
 an example of a priority one patient , as defined by carilion clinic , would be a diabetic ( dm ) with an a1c over 10 . 
 another example of a priority one patient would be a patient who is hypertensive , or a patient with a systolic bp over 160 . the carillion staff contacts patients with these conditions as soon as possible . between the months of march and 
 may 2010 , of the diabetic patients and hypertensive patients contacted , 43% committed to an appointment with carillion clinic and their care team to address their current health status ( as of june 11 , 2010 , 63% of the appointments made have been kept ) . without a fully functioning emr with capabilities that allow carilion clinic and other health care providers to search for these patients efficiently 
 , they would remain invisible to the practice and may not receive the care they need . 
 this paper has examined lessons learned from the implementation of pcmh at several sites , both military and civilian . 
 pcmh demonstrations show there is still much work to be done , particularly through educating the medical community about the benefits of the model . 
 current pcmh demonstrations have shown that this method of care can improve the quality of life for patients , such as reducing the patient 's frequency of er visits and hospital admissions , as well as reducing health care costs [ 25 , 26 ] . 
 overall , the results illustrate that pcmh decreases the gap between what providers say they want to do in order to improve patient care and what they can actually do with their current resources .   as with any new healthcare structure 
 , current pcmh practices are still developing methods to improve the quality of care provided to their patients , such as care coordination , team - based care , and the use of emr to promote care coordination . 
 there continues to be a struggle for transitioning pcmhs in determining how to implement the guidelines that the ncqa provides , which is a concern because ncqa is the most widely recognized organization in the united states for certifying practices as pcmhs . 
 ncqa recognition of pcmh does not require primary care practices to excel at every standard , but rather it requires a system of documentation about the quality of care being provided , and a plan for continuous improvement that follows each of the principles . 
 however , this does not guarantee a successful practice ; a successful practice should be defined as one that realizes pcmh recognition is not an end at all , but there is always room for improvement and maturation . with a great deal of enthusiasm from adopters thus far , pcmh can certainly have a permanent place in the future us healthcare system . 
 healthcare professionals and organizations will be under pressure to prove the  value  of their services . in implementing pcmh , 
 one will track hba1c , bmi , and/or other parameters as indicators of quality , but at what point would a pcmh be qualified to receive additional payment for their improvement in the quality of patient care ? without funding for additional resources , practices will continue with the discrepancy between the current volume - based healthcare system and a system that focuses on the quality of care . 
 the question remains of whether pcmh is the direction healthcare should go in , and many studies show that preventative care improves the quality of life . 
 pcmh holds a great deal of potential in addressing the broken healthcare system in the united states , but at the same time , it also faces many obstacles in terms of payment reform , professional support , and patient participation .","the patient - centered medical home ( pcmh ) is a primary care model that provides coordinated and comprehensive care to patients to improve health outcomes . 
 this paper addresses practical issues that arise when transitioning a traditional primary care practice into a pcmh recognized by the national committee for quality assurance ( ncqa ) . 
 individual organizations ' experiences with this transition were gathered at a pcmh workshop in alexandria , virginia in june 2010 . 
 an analysis of their experiences has been used along with a literature review to reveal common challenges that must be addressed in ways that are responsive to the practice and patients ' needs . 
 these are : ncqa guidance , promoting provider buy - in , leveraging electronic medical records , changing office culture , and realigning workspace in the practice to accommodate services needed to carry out the intent of pcmh . 
 the ncqa provides a set of standards for implementing the pcmh model , but these standards lack many specifics that will be relied on in location situations . 
 while many researchers and providers have made critiques , we see this vagueness as allowing for greater flexibility in how a practice implements pcmh .",pubmed
"incremental improvements in the survivorship from cpr occurred as more and more people were trained in cpr and as defibrillators became portable and were deployed in more locations . 
 finally , most people who had suffered from cardiac arrest did not survive to leave the hospital or did so in a neurological devastated state . in the early of 21 century , the survival rate of cardiac arrest outside of the hospital remained 78% . 
 about one - quarter of patients regained pulses after cpr , and about one - third of the patients with those initial successes survived hospitalization . the severe brain defect after cpr stimulated many investigations into the pathophysiology of , and treatments for , global brain ischemia . 
 for the protection of patient 's brain , mild hypothermia therapy has been proposed . in many animal experiments 
 for example , it can increase the survival rate and improve neurological function of animal . 
 besides , it reduces metabolic rate and the expression of some pro - apoptosis proteins . in clinical trials , 
 the effectiveness of hypothermia also has been proved . hence , 2010 american heart association guidelines for cardiopulmonary resuscitation and emergency cardiovascular care recommended , comatose patients with out - of - hospital ventricular fibrillation cardiac arrest were cooled to 3234c for 12 or 24 h. however , recent years some other clinical trials showed that mild hypothermia can not improve the prognosis of patients after cpr . 
 therefore , we conducted a meta - analysis of all relevant published studies to evaluate the effectiveness and safety of mild hypothermia on patients with cardiac arrest . 
 studies were considered for inclusion if they met the following criteria : ( 1 ) all published randomized controlled trials ( rcts ) . ( 2 ) we included studies in adult patients who suffered from cardiac arrest and were successfully resuscitated . 
 ( 3 ) the intervention was therapeutic mild hypothermia , no matter how to lower the body temperature . 
 the temperature should remain between 32c and 34c . and the treatment for the control group was according to the standard treatment after cardiac arrest without hypothermia . 
 the secondary outcomes were adverse events , such as rearrest , renal failure , pulmonary edema and so on . 
 ( 5 ) the relative risks ( rrs ) with their corresponding 95% confidence intervals ( cis ) were reported . 
 studies were considered for exclusion if they met the following criteria : ( 1 ) we excluded studies on children and pregnant women . 
 ( 2 ) cardiac arrest patients under the treatment of mild hypothermia received other drugs in combination therapy . 
 ( 3 ) there was a history of central nervous system depressant drug medication prior to cardiac arrest . 
 an extensive literature search was conducted using electronic databases , manual searching , and correspondence with authors of included studies . 
 the cochrane library , pubmed , web of science , embase , cnki , and wan fang data were searched from the date of their establishment to october 2014 . 
 search terms used were  cooling ,   hypothermia ,   cpr ,   cardiac arrest  and  rct . 
 two reviewers independently reviewed the citations , abstracts , and full - text articles and determined the eligibility of all the studies identified in the initial search . 
 when the entire process was completed , the two cross - checked with each other . in cases of disagreements , a third reviewer was consulted . 
 the following details were extracted : authors , year of publication , sample size , interventions , and outcomes . 
 assessment of the quality of the included studies was performed using the methodology recommended by cochrane collaboration . 
 this method comprised assessments of the risk of potential bias in six domains : random sequence generation ( correct , incorrect or unclear ) , allocation concealment ( correct , incorrect or unclear ) , blinding of outcome assessment ( correct , incorrect or unclear ) , incomplete outcome data ( complete , incomplete or unclear ) , selective reporting ( yes , no or unclear ) , other bias ( yes , no or unclear ) , such as the baseline , source of funding , and academic biases . if all quality criteria were met , the trial was considered to have a low risk of bias ( score : a ) . 
 if one or more of the quality criteria were only partially met , the trial was considered to have moderate risk of bias ( score : b ) , and if one or more criteria not met , the trial was considered to have high risk of bias ( score : c ) . 
 statistical analysis was performed using review manager version 5.2 ( cochrane collaboration , oxford , uk ) software . 
 the heterogeneity of the qualitative analysis was assessed by chi - square test , and the significant level was set to p = 0.1 . 
 if p > 0.1 , i < 50% , the different rcts can be regarded as homogeneous . 
 if p < 0.1 , i  50% , the different rcts can be regarded as heterogeneity . 
 we used weighted mean deviation ( wmd ) and 95% ci to represent the continuous data . and 
 studies were considered for inclusion if they met the following criteria : ( 1 ) all published randomized controlled trials ( rcts ) . ( 2 ) we included studies in adult patients who suffered from cardiac arrest and were successfully resuscitated . 
 ( 3 ) the intervention was therapeutic mild hypothermia , no matter how to lower the body temperature . 
 the temperature should remain between 32c and 34c . and the treatment for the control group was according to the standard treatment after cardiac arrest without hypothermia . 
 the secondary outcomes were adverse events , such as rearrest , renal failure , pulmonary edema and so on . 
 ( 5 ) the relative risks ( rrs ) with their corresponding 95% confidence intervals ( cis ) were reported . 
 studies were considered for exclusion if they met the following criteria : ( 1 ) we excluded studies on children and pregnant women . 
 ( 2 ) cardiac arrest patients under the treatment of mild hypothermia received other drugs in combination therapy . 
 ( 3 ) there was a history of central nervous system depressant drug medication prior to cardiac arrest . 
 an extensive literature search was conducted using electronic databases , manual searching , and correspondence with authors of included studies . 
 the cochrane library , pubmed , web of science , embase , cnki , and wan fang data were searched from the date of their establishment to october 2014 . 
 search terms used were  cooling ,   hypothermia ,   cpr ,   cardiac arrest  and  rct . 
 two reviewers independently reviewed the citations , abstracts , and full - text articles and determined the eligibility of all the studies identified in the initial search . 
 when the entire process was completed , the two cross - checked with each other . in cases of disagreements , a third reviewer was consulted . 
 the following details were extracted : authors , year of publication , sample size , interventions , and outcomes . 
 assessment of the quality of the included studies was performed using the methodology recommended by cochrane collaboration . 
 this method comprised assessments of the risk of potential bias in six domains : random sequence generation ( correct , incorrect or unclear ) , allocation concealment ( correct , incorrect or unclear ) , blinding of outcome assessment ( correct , incorrect or unclear ) , incomplete outcome data ( complete , incomplete or unclear ) , selective reporting ( yes , no or unclear ) , other bias ( yes , no or unclear ) , such as the baseline , source of funding , and academic biases . if all quality criteria were met , the trial was considered to have a low risk of bias ( score : a ) . if one or more of the quality criteria were only partially met , the trial was considered to have moderate risk of bias ( score : b ) , and if one or more criteria not met , the trial was considered to have high risk of bias ( score : c ) . 
 statistical analysis was performed using review manager version 5.2 ( cochrane collaboration , oxford , uk ) software . 
 the heterogeneity of the qualitative analysis was assessed by chi - square test , and the significant level was set to p = 0.1 . 
 if p > 0.1 , i < 50% , the different rcts can be regarded as homogeneous . 
 if p < 0.1 , i  50% , the different rcts can be regarded as heterogeneity . 
 we used weighted mean deviation ( wmd ) and 95% ci to represent the continuous data . and 
 a total of 1613 potentially relevant studies were identified from the following databases in our initial articles search : two hundred and twenty - six from pubmed , 412 from embase , 161 from cochrane library , 292 from web of science , 245 from cnki , 237 from wan fang data . after screening the titles or abstracts , 
 the full text of the remaining 60 studies was retrieved and assessed for eligibility . of the retrieved studies , 
 54 studies were excluded because of other type of publications ( review , case report , retrospective studies , prospective studies ) . finally , a total of six studies met the inclusion criteria [ figure 1 ] . 
 the six rcts included 531 adult patients who suffered from cardiac arrest and were successfully resuscitated . 
 the original articles compared the therapeutic effect under different conditions ( hypothermia or normothermia ) . 
 characteristics of the rcts included in the meta - analysis rcts : randomized controlled trials , vf : ventricular fibrillation , ca : cardiac arrest . 
 ( 1 ) all of the six studies mentioned  random ,  but only three studies described the method of generating a random sequence correctly . 
 ( 2 ) only two studies showed us they use a sealed envelope to conduct allocation concealment . 
 ( 3 ) six studies mentioned blinding in which five studies were double - blind . 
 ( 4 ) all of the six studies described the case of which quit or lost to follow - up . 
 the number of quit or lost to follow - up of each study were < 20% of the total number . 
 the methodological quality of the rcts included in the meta - analysis rcts : randomized controlled trials 
 . the survival rate of patients who suffered from cardiac arrest and were successfully resuscitated is one of the primary outcomes which we concerned . 
 all of the six studies reported the survival rate in which three studies reported survival rate at hospital discharge ( subgroup 1 ) , the other three studies reported survival rate after 6 months ( subgroup 2 ) . 
 there was no heterogeneity from the outcome in subgroup 1 ( p = 0.50 , i = 0 ) and subgroup 2 ( p = 0.32 , i = 12% ) . 
 the pooled results showed [ figure 2 ] no significant difference in the survival rate at hospital discharge between treatment group and control group ( rr = 1.35 , 95% ci : 0.872.10 , p = 0.18 ) . 
 however , there was significant difference in the survival rate after 6 months ( rr = 1.23 , 95% ci : 1.021.48 , p = 0.03 ) 
 . summarized the above six studies , no heterogeneity among them ( p = 0.60 , 
 i = 0 ) , and the pooled results showed hypothermia does improve the survival rate ( rr = 1.25 , 95% ci : 1.051.49 , p = 0.01 ) . 
 the neurological function of patients who suffered from cardiac arrest and were successfully resuscitated is another primary outcome which we concerned . in clinical , cerebral performance category ( cpc ) 
 the cpc scale ranges from 1 to 5 , with 1 representing good cerebral performance ( conscious , alert , capable of normal life ) or minor disability which do not significantly compromise cerebral or physical function , 2 moderate cerebral disability ( function is sufficient for independent activities of daily life ) , 3 severe cerebral disability , dependent on others for daily life support , 4 coma or vegetative state ( not conscious , unaware of surroundings , no cognition ) , and 5 brain death . included studies considered cpc 12 as good neurological recovery and cpc 35 as bad neurological recovery . hence , these data can be converted to dichotomous data . all of the six studies reported the neurological function in which three studies reported neurological function at hospital discharge ( subgroup 3 ) , the other three studies reported neurological function after 6 months ( subgroup 4 ) . 
 there was no heterogeneity from the outcome in subgroup 3 ( p = 0.30 , i = 18% ) and subgroup 4 ( p = 0.23 , 
 the pooled results showed [ figure 3 ] no significant difference in the neurological function at hospital discharge between treatment group and control group ( rr = 1.53 , 95% ci : 0.952.45 , p = 0.08 ) . however , there was significant difference in the neurological function after 6 months ( rr = 1.33 , 95% ci : 1.081.65 , p = 0.007 ) 
 . summarized the above six studies , no heterogeneity among them ( p = 0.38 , i = 6% ) , the pooled results showed hypothermia does improve the neurological function ( rr = 1.37 , 95% ci : 1.131.66 , p = 0.001 ) 
 . summary of data on neurological function for mild hypothermia versus normothermia . in the included studies , only four studies mentioned rearrest . 
 meta - analysis [ figure 4 ] demonstrated mild hypothermia does not influence the incidence of rearrest ( rr = 1.19 , 95% ci : 0.871.61 , p = 0.27 ) . 
 the result [ figure 5 ] showed no significant difference between treatment group and control group ( rr = 0.88 , 95% ci : 0.481.61 , p = 0.68 ) . 
 other adverse events such as pulmonary edema , pneumonia , bleeding , showed the same results ( data not shown ) . because only a single article mentioned the above adverse events , we did not put them into meta - analysis . 
 most studies at the top of the funnel plot , it meant the studies ci is narrow and their precision is high . to evaluate the influence of any single study on the pooled rr and ci 
 we omitted two high - risk studies from the overall analysis , the pooled rr ( 95% ci ) ranged from 1.25 ( 1.051.49 ) to 1.21 ( 1.011.45 ) for studies reporting the rrs by survival rate and from 1.37 ( 1.131.66 ) to 1.30 ( 1.061.59 ) for studies reporting the rrs by neurological function . 
 funnel plot compares log relative risk ( rr ) versus the standard error of log rr for survival rate and neurological function (  : at hospital discharge ;  : at 6 months ) . 
 a total of 1613 potentially relevant studies were identified from the following databases in our initial articles search : two hundred and twenty - six from pubmed , 412 from embase , 161 from cochrane library , 292 from web of science , 245 from cnki , 237 from wan fang data . after screening the titles or abstracts , 
 the full text of the remaining 60 studies was retrieved and assessed for eligibility . of the retrieved studies , 
 54 studies were excluded because of other type of publications ( review , case report , retrospective studies , prospective studies ) . finally , a total of six studies met the inclusion criteria [ figure 1 ] . 
 the six rcts included 531 adult patients who suffered from cardiac arrest and were successfully resuscitated . 
 the original articles compared the therapeutic effect under different conditions ( hypothermia or normothermia ) . 
 characteristics of the rcts included in the meta - analysis rcts : randomized controlled trials , vf : ventricular fibrillation , ca : cardiac arrest . 
 ( 1 ) all of the six studies mentioned  random ,  but only three studies described the method of generating a random sequence correctly . 
 ( 2 ) only two studies showed us they use a sealed envelope to conduct allocation concealment . 
 ( 3 ) six studies mentioned blinding in which five studies were double - blind . 
 ( 4 ) all of the six studies described the case of which quit or lost to follow - up . the number of quit or lost to follow - up of each study were < 20% of the total number . 
 the methodological quality of the rcts included in the meta - analysis rcts : randomized controlled trials . 
 the survival rate of patients who suffered from cardiac arrest and were successfully resuscitated is one of the primary outcomes which we concerned . 
 all of the six studies reported the survival rate in which three studies reported survival rate at hospital discharge ( subgroup 1 ) , the other three studies reported survival rate after 6 months ( subgroup 2 ) . 
 there was no heterogeneity from the outcome in subgroup 1 ( p = 0.50 , i = 0 ) and subgroup 2 ( p = 0.32 , i = 12% ) . 
 the pooled results showed [ figure 2 ] no significant difference in the survival rate at hospital discharge between treatment group and control group ( rr = 1.35 , 95% ci : 0.872.10 , p = 0.18 ) . 
 however , there was significant difference in the survival rate after 6 months ( rr = 1.23 , 95% ci : 1.021.48 , p = 0.03 ) 
 . summarized the above six studies , no heterogeneity among them ( p = 0.60 , i = 0 ) , and the pooled results showed hypothermia does improve the survival rate ( rr = 1.25 , 95% ci : 1.051.49 , p = 0.01 ) . 
 the neurological function of patients who suffered from cardiac arrest and were successfully resuscitated is another primary outcome which we concerned . in clinical , cerebral performance category ( cpc ) 
 the cpc scale ranges from 1 to 5 , with 1 representing good cerebral performance ( conscious , alert , capable of normal life ) or minor disability which do not significantly compromise cerebral or physical function , 2 moderate cerebral disability ( function is sufficient for independent activities of daily life ) , 3 severe cerebral disability , dependent on others for daily life support , 4 coma or vegetative state ( not conscious , unaware of surroundings , no cognition ) , and 5 brain death . included studies considered cpc 12 as good neurological recovery and cpc 35 as bad neurological recovery . 
 all of the six studies reported the neurological function in which three studies reported neurological function at hospital discharge ( subgroup 3 ) , the other three studies reported neurological function after 6 months ( subgroup 4 ) . 
 there was no heterogeneity from the outcome in subgroup 3 ( p = 0.30 , i = 18% ) and subgroup 4 ( p = 0.23 , i = 31% ) . 
 the pooled results showed [ figure 3 ] no significant difference in the neurological function at hospital discharge between treatment group and control group ( rr = 1.53 , 95% ci : 0.952.45 , p = 0.08 ) . 
 however , there was significant difference in the neurological function after 6 months ( rr = 1.33 , 95% ci : 1.081.65 , p = 0.007 ) 
 . summarized the above six studies , no heterogeneity among them ( p = 0.38 , i = 6% ) , the pooled results showed hypothermia does improve the neurological function ( rr = 1.37 , 95% ci : 1.131.66 , p = 0.001 ) . 
 meta - analysis [ figure 4 ] demonstrated mild hypothermia does not influence the incidence of rearrest ( rr = 1.19 , 95% ci : 0.871.61 , p = 0.27 ) . 
 the result [ figure 5 ] showed no significant difference between treatment group and control group ( rr = 0.88 , 95% ci : 0.481.61 , p = 0.68 ) . 
 other adverse events such as pulmonary edema , pneumonia , bleeding , showed the same results ( data not shown ) . because only a single article mentioned the above adverse events , we did not put them into meta - analysis . 
 most studies at the top of the funnel plot , it meant the studies ci is narrow and their precision is high . to evaluate the influence of any single study on the pooled rr and ci , we performed a sensitivity analysis . 
 we omitted two high - risk studies from the overall analysis , the pooled rr ( 95% ci ) ranged from 1.25 ( 1.051.49 ) to 1.21 ( 1.011.45 ) for studies reporting the rrs by survival rate and from 1.37 ( 1.131.66 ) to 1.30 ( 1.061.59 ) for studies reporting the rrs by neurological function . 
 funnel plot compares log relative risk ( rr ) versus the standard error of log rr for survival rate and neurological function (  : at hospital discharge ;  : at 6 months ) . 
 as the cpr technology became more and more mature , a growing number of patients with cardiac arrest were saved . 
 in the half past century especially the last 10 years , hypothermia is the only method that approved by a large amount of clinical trials which can improve the prognosis of patients . 
 meta - analysis can evaluate the effectiveness and safety of hypothermia which results are quite credible . in this study , we used meta - analysis to integrate these different independent researches , in order to get more reliable analysis about the efficacy of mild hypothermia for the treatment of patients with cardiac arrest . for patients rescued from cardiac arrest , we pay most attention to their prognosis . 
 the data of three studies were measured at hospital discharge , the other three were measured after 6 months . in order to avoid the heterogeneity first we analyzed the data by subgroups . 
 the mild hypothermia therapy can not improve survival rate and neurological function at hospital discharge . 
 we speculated the possible reasons are as follows : ( 1 ) whole - body hypothermia influences all organ systems , and any potential benefit should be balanced against possible side effects ; ( 2 ) in clinical treatment , combination hypothermia with many other drugs may bring about some unknown influences , for example it will reduce the rate of drug metabolism and so on ; ( 3 ) hypothermia revives neurons is a long and slow process , for some other reasons patients die before neurons recovery . 
 the pooled results showed us that mild hypothermia does improve survival rate and neurological function of patients with cardiac arrest . 
 for example : hypothermic blanket , ice packs , cold saline intravenous infusion and so on . 
 however , some other studies or meta - analysis hold the view that early initiation of rapid cooling can not improve the prognosis . 
 the possible mechanisms include : ( 1 ) hypothermia can reduce cerebral oxygen consumption and energy metabolism ; ( 2 ) it can reduce reactive oxygen species generation and release of excitatory amino acids ; ( 3 ) it also can preserve the integrity of the blood - brain barrier , regulate the gene expression of inflammatory protein and apoptotic proteins and reduce cell death . 
 although there are many advantages of mild hypothermia , we should not ignore the side effects . 
 in the included six studies , the main adverse events were re - arrest , renal failure , pulmonary edema and so on . 
 hence , we drew a conclusion mild hypothermia therapy for cardiac arrest patients is safe . 
 however , the study also suffers from several limitations : ( 1 ) the number of trial and the study size may be inadequate . 
 therefore , the precision of the outcome parameters obtained is generally low ; ( 2 ) another potential limitation of this meta - analysis is clinical and methodological heterogeneity . according to the six studies , we knew cardiac arrest patients with different first recorded cardiac rhythm are included . besides , the time from start of cooling to target temperature was different , the duration of keeping hypothermia was different and the methods of cooling body temperature were also different in these studies . 
 all of the above factors would lead to inaccurate results . in this meta - analysis 
 , we conducted a subgroup analysis according to different situations , although there was almost no heterogeneity . 
 but in order to get more accurate results , further research should include high - quality studies to analyze the impact of different cooling measures or different hypothermia duration on the survival rate and neurological function . in conclusions , 
 this meta - analysis demonstrated mild hypothermia can not improve survival rate and neurological function of patients at hospital discharge . 
 the pooled results showed mild hypothermia does improve prognosis of patients . and it does not influence the incidence of adverse events compared with the control group .","background : therapeutic hypothermia has been recommended for the treatment of cardiac arrest patients who remain comatose after the return of spontaneous circulation . 
 the aim of this study was to evaluate the effectiveness and safety of mild hypothermia on patients with cardiac arrest by conducting a meta-analysis.methods:the relevant trials were searched in cochrane library , pubmed , web of science , embase , cnki and wan fang data from the date of their establishment to october 2014 . 
 thereafter , the studies retrieved were screened based on predefined inclusion and exclusion criteria . 
 data were extracted , and the quality of the included studies was evaluated . 
 a meta - analysis was conducted using the cochrane collaboration review manager 5.2 software.results:six randomized controlled trials involving 531 cases were included , among which 273 cases were assigned to the treatment group and the other 258 cases to the control group . 
 the meta - analysis indicated that mild hypothermia therapy after cardiac arrest produced significant differences in survival rate ( relative risk [ rr ] = 1.23 , 95% confidence interval [ ci ] : 1.021.48 , p = 0.03 ) and neurological function ( rr = 1.33 , 95% ci : 1.081.65 , p = 0.007 ) after 6 months compared with normothermia therapy . 
 however , no significant differences were observed in the survival to the hospital discharge ( rr = 1.35 , 95% ci : 0.872.10 , p = 0.18 ) , favorable neurological outcome at hospital discharge ( rr = 1.53 , 95% ci : 0.952.45 , p = 0.08 ) and adverse events.conclusions:the meta - analysis demonstrated that mild hypothermia can improve the survival rate and neurological function of patients with cardiac arrest after 6 months . on the other hand , regarding the survival to hospital discharge , favorable neurological outcome at hospital discharge , and adverse events , our meta - analysis produced nonsignificant results .",pubmed
"for several years , a male factor has been recognized in the diagnosis and treatment of all infertility ; approximately 40% of marital infertility is due to a male factor . 
 it is a condition characterized by persistent enlargement and elongation of the pampiniform plexus , venous convolution , and sinus formation with transient or permanent backflow . 
 the etiopathogenesis of varicocele remains unclear and , at present , there is still no unequivocal explanation of their formation and effect on the excretory and secretory functions of the testis . 
 the left testicular vein runs vertically and joins the left renal vein opposite the origin of the lower adrenal vein . 
 thus , blood turbulence occurs in the vicinity of the opening of the testicular vein and , in consequence , retrograde flow of renal metabolites and adrenal hormones to the testis is observed . 
 the elevated hydrostatic pressure within the testicular vein leads to venous stasis within the testis resulting in testicular hyperemia and compromised testicular oxygen concentration [ 1 , 2 ] . 
 one of the hypotheses of spermatogenesis impairment is the concept of atrophic changes within convoluted tubules due to increased scrotal temperature or tissue hypoxia ; in consequence , testicular growth is arrested in children and adolescents on the affected side [ 3 , 4 ] . 
 a high  grade varicocele may be detected during a meticulous andrologic examination involving inspection and palpation of the genital organs while standing up . 
 grading the varicocele is usually based on the dubin  amelar 1 to 3 scale . 
 we believe that a diagnosis based on inspection and palpation should subsequently be confirmed by accessory ultrasound . 
 doppler examination allows the assessment of pampiniform plexus diameters , presence and character of backflow , as well as objective and comparative analysis of the size of both testes [ 1 , 5 , 6 ] . 
 a palpable varicocele with testicular hypotrophy on the affected side , confirmed by color doppler , is an indication for surgical repair . 
 from 1996 to 2011 , eighty  two adolescents were operated on for idiopathic varicocele with testicular hypotrophy . 
 sixty  eight patients were subjected to the current analysis ( 14 men were lost in follow  up ) . 
 the age of the patients was 13 to 17 years ( mean 15.3 years ) . 
 this is a retrospective study , where data from patients charts were analyzed . as the study is not an experimental one it did not need approval from the ethics committee . 
 the clinical diagnosis was established on the basis of physical and andrologic examinations with an assessment of testicular size and varicocele severity on the dubin  amelar 1 to 3 scale . 
 color doppler ultrasound was performed in all patients ; testicular size was measured according to costabile with an orchidometer . in subjects with varicocele disease , 
 a difference in testicular volume of 2 ml was used as the criterion of testis hypotrophy . 
 laparoscopic repair was performed via a transperitoneal approach with division of the testicular vein only . 
 the evaluation of surgical outcome was carried out at 1 to 9 years following laparoscopic varicocele repair , and was based on ultrasound evidence of varicocele subsidence and increase of testicular volume on the affected side . according to who directions , all subjects had their semen parameters checked twice after they had turned eighteen . 
 the abnormal values of semen parameters were consistent with who 2010 guidelines also adopted in poland [ 12 , 13 ] . 
 blood tests to determine levels of testosterone ( t ) , follicle stimulating hormone ( fsh ) , and lutenizing hormone ( lh ) were also performed and presented as qualitative variables ( normal / abnormal ) . 
 abnormal adopted levels of the abovementioned hormones were : less than 300 ng / dl for testosterone and less than 1.5 iu / l for both lh and fsh . 
 statistical analysis was carried out using the t  student test ( statistica version 7.0 ) . 
 for dependent variables , the paired test was applied and for independent variables , the unpaired t 
 a grade 2 varicocele was found in 32 ( 47.1% ) subjects and a grade 3 was found in 36 ( 52.9% ) ( table 1 ) . before laparoscopic repair , mean left and right testicular volumes were 16.2 ( sd = 4.3 ) cm and 19.7 ( sd = 6.4 ) cm , respectively . 
 thus , left testicular volume was significantly lower compared to that of the right testis ( p = 0.03 ) . 
 an increase in left testicular volume when compared to the contralateral testis was found in 25 ( 78.1% ) subjects with clinical grade 2 varicocele , and in 32 ( 88.8% ) subjects with grade 3 abnormality . 
 patients divided according to preoperative varicocele severity and postoperative improvement in testicular diameter and semen parameters number in brackets reflects % of the whole group in patients with unilateral grade 2 varicocele mean baseline left and right testicular volumes were 16.7 ( sd = 4.2 ) cm and 19.8 ( sd = 2.4 ) cm , respectively . 
 after surgery the respective volumes were 20.9 ( sd = 3.7 ) cm and 21.4 ( sd = 1.6 ) cm ; the increase was statistically significant ( p = 0.01 ) . in patients with unilateral grade 3 varicocele , mean initial left and right testicular volumes were 16.2 ( sd = 2.3 ) cm and 18.7 ( sd = 3.9 ) cm , respectively . 
 after surgery the respective volumes were 19.9 ( sd = 2.5 ) cm and 20.2 ( sd = 3.3 ) cm ; the increase was statistically significant ( p = 0.03 ) . 
 an increase in left testicular volume was found in 46 ( 85.1% ) of 54 patients with unilateral varicocele and in 12 ( 85.7% ) of 14 subjects operated on for bilateral disease . in patients with bilateral varicocele 
 mean baseline left and right testicular volumes were 15.5 ( sd = 6.6 ) cm and 17.9 ( sd = 3.2 ) cm , respectively . 
 after surgery the respective volumes were 19 ( sd = 6.9 ) cm and 20.2 ( sd = 5.6 ) cm . 
 the left testicular volume increases were comparable irrespective of whether unilateral or bilateral repair was performed . 
 bilateral hypotrophy was seen in two ( 14.2% ) of fourteen subjects with bilateral disease ; they had no testicular volume increase at follow  up . 
 semen analysis was performed in all subjects who had undergone laparoscopic varicocele repair in adolescence . 
 the check revealed normozoospermia in 58 ( 85.2% ) of our 68 patients while the remaining 10 ( 14.7% ) had oligozoospermia ( < 15 million sperm / ml ) or low percentage of motile spermatozoa , ie . 
 , asthenozoospermia ( < 40% ) or low percentage of normally shaped sperm , ie . , teratozoospermia ( < 4% ) with persistent testicular hypotrophy . 
 normozoospermic men also had normal fsh , lh , and t levels as opposed to 10 subjects with different variants of spermatogenesis abnormalities , who showed fsh and lh elevation with low t levels . 
 varicocele recurrence was only observed in two ( 2.9% ) patients ; they underwent repeat repair . 
 complications developed in six ( 8.8% ) of patients , and included a hydrocele testis  five patients ( 7.3% ) , and epididymitis 
 hospital stay was 1 to 3 days ( mean 2.1 days ) and overall convalescence was 7 to 12 days ( mean 8.2 days ) . 
 the varicocele was described in antiquity ; since the end of the xix century it has been considered one of the causes of male factor infertility . 
 although several convincing hypotheses exist to explain the etiology and pathomechanisms of varicoceles , there is no consensus about the advisability of early surgical repair and harmful effect of the vascular lesion on male fertility potential . 
 we agree with greenfield , cayan , and other authors that in the case of suspicion or preliminary diagnosis of genital abnormality , the patient should be referred to a urologist or andrologist [ 2 , 6 , 14 , 15 , 16 ] . 
 an increase in testicular volume on the affected side indicates the efficiency of surgical repair . 
 we also agree that varicocele with testis hypotrophy on the affected side , confirmed by color doppler ultrasound , is an indication for surgical repair [ 2 , 9 , 15 , 19 , 20 , 21 ] . 
 laparoscopic varicocele repair resulted in significant increase in hypotrophic testicular volume in 58 ( 83.8% ) of our subjects . 
 numerous surgical techniques have been developed for treatment of varicocele , which are aimed at closing incompetent veins of the spermatic cord , thus preventing retrograde blood flow towards the testis and venous stasis in dilated vessels of the pampiniform plexus . 
 surgical repair of varicocele can be achieved by conventional open varicocelectomy , laparoscopic , microsurgical intervention , or transdermal sclerotherapy and embolization of the testicular vein . 
 technological progress of the last twenty years has allowed the introduction of a new minimally invasive surgical modality , ie . , 
 we believe that laparoscopic varicocele repair as described by palomo ( ligation and division of both testicular vein and artery ) or bernardi ( testicular artery sparing ) should be recommended as the treatment of choice for the vascular lesion . 
 however , opinions of experts on the efficacy of the above  mentioned modalities are divided [ 2 , 9 , 17 , 18 ] . 
 while recognizing the value of both procedures for varicocele correction , it is assumed that the laparoscopic bernardi technique should be given priority in boys and adolescents . 
 axis occur earlier than their physical manifestation , ie . , decreased testicular volume and consistence especially on the varicocele  affected side 
 . concomitant damage to the right testis results when a immunological factor is present as it is in the case of unilateral cryptorchidism or unilateral testicular torsion . 
 hormonal regulation of the reproductive system in adolescent boys and men of procreative age depends on sertoli and leydig cells stimulation in the testis by pituitary 
 lh causes leydig cells to synthesize and , subsequently , secrete testosterone , which controls meiosis and spermatid transformation into a spermatozoon . 
 fsh acts with testosterone to induce the secretory activity of sertoli cells ; they synthesize proteins indispensable for normal spermatogenesis . 
 clinical observations and results of experimental research also suggest important roles of estradiol ( e2 ) and prolactin ( prl ) in the process [ 1 , 2 , 15 , 20 , 23 , 24 ] . 
 postoperative follow  up of young men after they turn 18 facilitates objective evaluation of the efficacy of surgical varicocele repair . 
 our results , based on sperm examination at 1 to 9 years after laparoscopic intervention , mostly revealed normal sperm count , motility , and morphology . 
 other authors also report a significant improvement of sperm parameters following surgical varicocele repair , thus confirming the advisability of surgical correction for the vascular lesion . 
 normal fsh , lh , and t levels found in young men operated on in adolescence are also significant ; late diagnosis and treatment frequently result in spermatogenesis disturbances and abnormal concentrations of sex hormones [ 2 , 23 , 25 , 26 ] . 
 laparoscopic varicocele repair resulted in significant increase of hypotrophic testicular volume in 83.8% of our subjects . 
 assessment of the efficacy of surgical varicocele repair should include hormonal tests and semen analysis in procreative age group .","introductionfailure to perform surgical repair of varicocele before puberty is among the common causes of male infertility . 
 the purpose of this study was to evaluate the testicular volume and fertility potential in men after laparoscopic varicocelectomy conducted in adolescence due to varicocele and concomitant testicular hypotrophy.material and methodsfrom 1996 through 2011 , eighty  two adolescents were operated on for unilateral primary varicocele with testicular hypotrophy . 
 sixty  eight patients were subject to the current analysis . 
 the age of the patients was 13 to 17 years ( mean 15.3 years ) . 
 clinical diagnosis was established on the basis of andrologic examination and ultrasonography with an assessment of testicular size and varicocele severity . 
 laparoscopic surgical repair was performed by a transperitoneal approach with division of testicular vein only.resultsan increase in left testicular volume when compared with the contralateral testis was found in 25 ( 78.1% ) young men with clinical grade 2 varicocele ( p = 0.02 ) and in 32 ( 88.8% ) subjects with grade 3 abnormality ( p = 0.04 ) . 
 an increase in left testicular volume was found in 46 ( 85.1% ) of 54 patients with unilateral varicocele and in 12 ( 85.7% ) of 14 subjects operated on for bilateral disease . 
 a left testicular volume increase was comparable independent of the use of uni or bilateral repair . 
 fifty  eight ( 85.2% ) of our 68 patients had normozoospermia.conclusionslaparoscopic varicocele repair resulted in a significant increase of hypotrophic testicular volume in 83.8% of our subjects .",pubmed
"radical retropubic prostatectomy is the surgical treatment of choice for patients with localized prostate cancer . 
 numerous studies have been published demonstrating excellent tumor control , good functional results , and low morbidity associated with the procedure ( 1 - 4 ) . 
 however , the major long - term morbidity from radical retropubic prostatectomy that remains is erectile dysfunction , despite advances in surgical techniques . 
 prior to studies done by walsh and donker ( 5 ) using male fetuses , the cause of erectile dysfunction after radical prostatectomy was not well understood . 
 they proposed and eventually demonstrated that erectile dysfunction occurred secondary to the injury of cavernosal nerves . 
 these nerves were identified branching from the pelvic plexus and running as a plexus of small nerves within a prominent neurovascular bundle on the posterolateral border of the prostate , terminating in the region of the membranous urethra . 
 these nerves are mainly concentrated laterally to the prostate and these neural structures are referred to as neurovascular bundles . 
 nerve sparing radical retropubic prostatectomy has led to improvements in potency rates , with studies reporting a 16 to 76% recovery of erectile function in men whose bilateral neurovascular bundles were preserved and 0 to 56% in those with unilateral neurovascular bundle preservation ( 6 , 7 ) . 
 this variability can be attributed to the fact that there was no definite and exact anatomy of periprostatic nerve fibers , especially the cavernosal nerves . 
 although some papers about the anatomy of periprostatic nerves were published recently , there are still controversies about detailed descriptions of the exact periprostatic nerve distribution in the literature . 
 thus , in the present study , we described periprostatic nerve distribution and constructed a 3-dimensional model of nerve distribution . 
 the prostate , together with seminal vesicle , bladder , and rectum , was removed en bloc from five adult male human cadavers ( age at death 43 - 72 yr ) after filling urinary bladder with 40% formaldehyde using urethral catheter . 
 each specimen was sliced by 5 mm step sections from the base of the prostate to the apex , and a total of eight slices of whole - mount histologic sections were embedded in paraffin , and prepared , and then stained with anti - s-100 antibody neural stain to delineate the precise location of this neural tissue . 
 immunostaining with anti - s-100 protein antibody was performed by dewaxing the slides in xylene three times for three minutes and then rehydrating in ethanol ( two changes of three minutes in 100% ethanol and two changes of three minutes in 90% ethanol ) and distilled water . 
 the rabbit polyclonal primary antibody to s-100 protein ( immunon , pittsburgh , pa , usa ) was applied in a 1:500 dilution and incubated for one hour at room temperature . 
 the samples were then washed in pbs , subjected to envision - hrp kit ( dako , carpinteria , ca , usa ) , and the reaction product was visualized with chromogen 3 , 3'-diaminobenzidine ( dab ) for three minutes . 
 the stained whole - mount section slide was divided into four arbitrary sectors : two laterals , one ventral , and one dorsal ( rectal side ) according to one fourth the circumference of whole - mount specimens . 
 whole - mount specimens were then divided by two axes , such as from 1.5 to 7.5 o'clock and 10.5 to 4.5 o'clock direction ( fig . 
 each slide was taken from the base , mid - part , and apex of the prostate . 
 nerve fibers 100 micrometer or greater in diameter were counted in 200 magnification microscopically ( olympus bx51 ; tokyo , japan ) , in addition to the number of nerve fibers , the distance from nerve fiber to the true prostate capsule , and nerve fiber diameter on each sector from representative slides . 
 the three - dimensional periprostatic nerve distribution model was made by connecting the stained nerve fibers from the same location of the same sectors of the eight whole - mount slides from each prostate and reconstructing its image after taking a picture of the three - dimensional model ( fig . 
 the difference in variables was assessed using anova and kruskal - wallis nonparametric anova . statistical significance in this study was considered at p<0.05 . 
 the average number of nerve fibers located in the ventral part of the prostate was 5.751.20 , and comparatively , the average number in the dorsal ( rectal ) part was 28.502.17 . 
 there were even fewer nerve fibers in the ventral part of the prostatic base area , numbering 3.751.38 . 
 in the right lateral part the average number of nerve fibers was 25.259.92 , and in the left lateral part 25.7512.76 ; thus the number of nerve fibers in the dorsal ( rectal ) , right lateral , and left lateral parts of the prostate capsule showed no significant difference , and nerve fibers were distributed evenly around the prostatic capsule microscopically in the posterior , right lateral , and those in left lateral areas . 
 however , nerve fibers in the ventral part were small in number compared to the dorsal , right lateral , and left lateral parts at all levels ( p=0.001 ) ( table 1 ) . moving from base to apex , the average number of nerve fibers increased in the ventral area , but this increase was not statistically significant ( p=0.386 ) . 
 the average distance from prostatic capsule to nerve fibers was 1.730.43 mm in the ventral part , 1.800.16 mm in the dorsal ( rectal ) part , 1.580.26 mm in the right lateral part , and 1.230.29 mm in the left lateral part . 
 there was no difference in the average distances when comparing the four different parts statistically ( p=0.266 ) . 
 also there was no significant difference among the base , mid - part , and apex levels ( p=0.673 ) ( table 2 ) . 
 the average thicknesses of nerve fibers in the ventral and dorsal parts were 0.190.02 mm and 0.300.03 mm , and those in the right and left lateral parts were 0.300.03 mm and 0.280.03 mm , respectively . 
 there was no difference in nerve fiber thickness according to level such as base , midpart , and apex ( p=0.180 ) . 
 however , the average thickness of the nerve fibers in the ventral part was significantly decreased as compared to the other parts ( p=0.005 ) ( table 3 ) . the three - dimensional model of the periprostatic nerve fibers revealed an almost even distribution and nearly similar thickness of periprostatic nerve fibers on dorsal and both lateral parts . 
 furthermore , there was absence of a cord - like structure , such as neurovascular bundle , noted on the study . however , the nerve fibers in the ventral part were substantially fewer in number and decreased in thickness . 
 traditionally , it was thought that cavernosal nerves branched from the pelvic plexus and ran as a plexus of small nerves within a prominent cord - like neurovascular bundle on the posterolateral border of the prostate ( 5 , 8) . 
 new advancements in surgery , including the use of laparoscopic and robotic modalities and magnifying visual devices in open surgery , have enabled precise nerve dissection . however , despite all these advancements in nerve preservation or restoration , potency rates have remained unsatisfactory ( 9 - 11 ) . 
 recently , several descriptions of the neurovascular bundle have been proposed : the neurovascular bundle , in contrast to the usual concept , appears to contain few pelvic splanchnic nerve components at the bladder - prostate junction . 
 the pelvic splanchnic nerve branches demonstrate a spray - like distribution , and caudal branches reach the dorsolateral portion of the prostate at levels more than 20 mm inferior to the bladder - prostate junction . 
 therefore , the cavernous nerves appear to be spread and located beyond the neurovascular bundle ( 12 ) . 
 ( 13 ) found , in addition to the main neurovascular bundles , other nerve branches running between the prostatic capsule and endopelvic fascia , and called them distal accessory nerve pathway . 
 according to these authors , this pathway could explain the unpredictable response to erection recovery and the lack of correlation between surgeon perception of the quality of neurovascular sparing and functional results after radical prostatectomy . 
 recently , several descriptions of the neurovascular bundle have shown variations in the distributions of autonomic fibers between different individuals . 
 about half of the patients have been reported as having a ' fan - like ' nerve fiber distribution on the lateral aspect of the prostate ( 14 ) . as mentioned above , the traditional knowledge of the neurovascular bundle has been challenged . 
 therefore , we performed this study to distinguish microscopic features and anatomic navigation of periprostatic nerves using male adult cadavers . 
 we found that the number of periprostatic nerve fibers was distributed evenly to dorsal and both lateral area of the prostate in our study . unlike the study of kiyoshima et al . 
 ( 14 ) , no cord - like neurovascular bundle was observed in any cadaver case in our study . 
 in addition , the distances from the prostatic capsule were almost the same in both the lateral and dorsal ( rectal ) parts of the base , middle , and apex levels of the prostate . 
 these data , including numerical values about periprostatic nerve fibers , have not been reported and will serve as a guide for improved results for nerve sparing radical prostatectomy . 
 several urologists reported surgical techniques according to the anatomy of the neurovascular bundle related to postoperative sexual dysfunction ( 14 , 15 ) . 
 ( 16 ) suggested a modified technique of nerve sparing called "" curtain dissection "" , which involves incising the periprostatic fascia and dissecting the neurovascular bundle far more anteriorly than previously described . 
 ( 2 ) reported the technique of open retropubic nerve - sparing radical prostatectomy . in their study , the parapelvic fascia of the prostate is incised at the lateroventral aspect of the prostate at 10 o'clock and 2 o'clock . 
 they noted the importance of starting the incision high up on the ventral aspect of the prostate to preserve a maximum number of nerve fibers because a substantial number of nerve fibers are located at the ventral area . 
 it has been shown that potency rates strongly correlate with the number of preserved nerve fibers . 
 had introduced their modified technique of robotic radical prostatectomy , named vattikuti institute prostatectomy ( 17 ) . 
 the features of their technique are early transaction of the bladder neck , preservation of the prostatic fascia ( veil of aphrodite ) , and control of the dorsal vein complex after dissection of the prostatic apex . 
 they reported that the veil nerve sparing procedure offers superior erectile function compared with conventional nerve - sparing surgery without compromising cancer control ( 18 ) . 
 ( 19 ) performed a histologic study of comparison between the veil of aphrodite technique and standard nerve - sparing technique during a robotic prostatectomy . 
 they concluded that the veil of aphrodite technique is a safe procedure that effectively preserved the lateral prostatic fascia , which contains nerve bundles that run along the surface of the anterolateral zones . in their study , the nerve bundle counts of the anterolateral zones was different between the two techniques with statistical significance . 
 our result means modified nerve - sparing techniques by incising the periprostatic fascia more anteriorly are feasible for saving periprostatic nerves . 
 but an excessive high up incision ( over 2 , 10 o'clock ) would not be effective because the number of nerve fibers of both lateral parts is much more than that of the ventral part of the periprostatic area . 
 these findings are differernt from eichelberg 's reports that the sum of the median percentage of nerves detected in anterior half of the prostate was more than 20% ( 20 ) . 
 our result revealed that the mean percentage of nerves in ventral part was 6.7% ( range : 4.5 - 9.4% ) ( table 1 ) . according to costello et al . 
 ( 21 ) , the majority of nerves which exist on the anterior aspect of the prostate laterally are not functionally significant parasympathetic nerves . 
 so , there is little evidence to support higher incisions in the lateral prostatic fascia anatomically . based on this study , 
 the number of periprostatic nerve fibers is evenly distributed on both lateral and dorsal ( rectal ) parts from base to apex of the prostate without forming any cord - like structures . because the number of nerve fibers on the ventral part is few in comparison , an excessive high up incision over 2 , 10 o'clock on the ventral part of the prostate is insignificant during nerve - sparing radical prostatectomy .","we investigated the distribution and navigation of periprostatic nerve fibers and constructed a 3-dimensional model of nerve distribution . 
 a total of 5 cadaver specimens were serially sectioned in a transverse direction with 0.5 cm intervals . 
 hematoxylineosin staining and immunohistochemical staining were then performed on whole - mount sections . 
 three representative slides from the base , mid - part , and apex of each prostate were subsequently divided into 4 sectors : two lateral , one ventral , and one dorsal ( rectal ) part . 
 the number of nerve fibers , the distance from nerve fiber to prostate capsule , and the nerve fiber diameters were analyzed on each sector from the representative slides by microscopy . 
 periprostatic nerve fibers revealed a relatively even distribution in both lateral and dorsal parts of the prostate . 
 there was no difference in the distances from the prostate capsule to nerve fibers . 
 nerve fibers in the ventral area were also thinner as compared to other areas . in conclusion , 
 periprostatic nerve fibers were observed to be distributed evenly in the periprostatic area , with the exception of the ventral area . as the number of nerve fibers on the ventral part is fewer in comparison , 
 an excessive high up incision is insignificant during the nerve - sparing radical prostatectomy .",pubmed
"rheumatoid arthritis ( ra ) is a progressive , autoimmune disease , characterized by chronic inflammation of synovial membrane and consistently degradation of articular cartilage and subchondral bone . 
 serum amyloid a ( saa ) is a highly conserved acute - phase protein , released in response to inflammation or infection . 
 production of acute - phase saa ( a - saa ) is stimulated by proinflammatory cytokines , such as interleukin-6 ( il-6 ) , il-1 , tumor necrosis factor ( tnf ) , interferon- , and transforming growth factor- ( tgf- ) . 
 the concentration of a - saa increases dramatically during acute inflammation and injury , reaching within 5 - 6 hours levels that are 1000-fold greater than normal [ 13 ] . as with other acute - phase reactants , 
 however , extrahepatic production of saa by several tissues and cell types has been described in patients with chronic diseases , for example , alzheimer 's disease , cancer , diabetes , obesity , insulin resistance , metabolic syndrome , and atherosclerosis [ 46 ] . 
 it has been even suggested that saa may play an important , pathogenic role in the proinflammatory cascade in the course of ra . 
 saa is not only an acute - phase protein but also a kind of apolipoprotein , relevant in cholesterol metabolism . 
 normally , saa circulates in low levels bound to high - density lipoprotein ( hdl ) , but during inflammation saa can contribute up to 80% of hdl apolipoprotein ( apo ) composition , exceeding apo - a1 in quantity and impairing the protective function of hdl . 
 saa may also increase the oxidation of low - density lipoprotein ( ldl ) and thus may be associated with cardiovascular ( cv ) disease ( cvd ) and atherogenesis [ 5 , 7 ] . 
 saa is also the precursor of amyloid a protein , a fibrillar , insoluble product deposited in major organs , which carries a risk of organ failure and premature death in the course of secondary amyloidosis . 
 aa amyloidosis is a late complication of ra , connected with active , long - standing , and disabling disease . 
 the prevalence of aa amyloidosis in ra reported in different studies is highly variable because the complication frequently remains undetected due to lack of clinical manifestations in many patients . 
 persistently high concentration of saa is a prerequisite for aa amyloidogenesis [ 1 , 3 ] . 
 high levels of saa correlate with the progression of amyloidosis and low levels are associated with its regression [ 2 , 9 ] . 
 it was reported that , in aa amyloidosis patients , serum saa concentration was the most important predictive parameter of the risk of death , associated with renal function . 
 the aim of the study was to assess the concentration of saa in ra patients with different disease activity , with reference to other inflammatory , metabolic parameters , as well as cv and renal disease risk factors . 
 the study group consisted of 140 ra patients admitted consecutively and treated in the department of rheumatology and connective tissue diseases , medical university of lublin . 
 the patients were qualified for the treatment in order to make a decision whether to maintain or modify the ongoing therapy , depending on an assessment of the disease activity . 
 the ethical committee of the medical university of lublin approved the design of the study . 
 demographic and clinical information was obtained through structured interview , review of medical records , self - report questionnaires , physical examination , laboratory tests , and high - resolution b - mode ultrasonography . 
 disease activity was measured using the disease activity score ( das ) based on evaluation of 28 joints ( das28 ) , calculated with the number of tender joint counts ( tjc ) and swollen joint counts ( sjc ) , erythrocyte sedimentation rate ( esr ) value , and patient 's global assessment ( pga ) of disease activity in visual analogue scale ( vas ) . 
 ability to perform daily activities was measured using the modified health assessment questionnaire ( m - haq ) with the range 03 ( a score 0 representing no impairment of function ) . 
 erosive form of ra was diagnosed in those patients , who presented erosions on joint surfaces of bones in radiograms of hands and/or feet , according to steinbrocker et al . 
 extra - articular symptoms were noted , which occurred during the whole course of the disease . 
 blood was collected after an overnight fasting to determine the complete blood cell count , erythrocyte sedimentation rate ( esr ) , serum concentration of crp , creatinine ( cr ) , total protein , albumin , fibrinogen , total cholesterol ( tc ) , hdl - cholesterol [ hdl - c ] , ldl - cholesterol [ ldl - c ] , and triglycerides ( tg ) at the university hospital central laboratory . 
 serum level of crp was measured by immunoturbidimetric assay , with the upper limit of the normal range at 5  mg / l . 
 concentrations of tc , hdl - c , and tg were measured using the standard enzymatic technique ( biomaxima ) ; ldl - c was calculated according to the friedewald formula . 
 modification of diet in renal diseases ( mdrd ) was calculated for every patient to estimate the glomerular filtration rate ( gfr ) using serum cr concentration and demographic factors . 
 blood samples were also stored at 80c for further assessment of cystatin - c ( cys - c ) and saa . 
 cys - c is used as an endogenous marker for gfr , more precise than creatinine . 
 serum cys - c was measured using quantitative enhanced immunonephelometry method ( with commercially available assay developed by dade behring ) . 
 the producer recommended the normal range of cys - c between 0.53 and 0.95  mg / l independently of sex , age , and body mass . 
 serum concentration of saa was determined by a commercial enzyme - linked immunosorbent assay ( elisa ) , with detection limit 0.005  mg / l ( human saa ; biosource europe s.a . 
 igm - rheumatoid factor ( rf - igm ) was determined using the antirheumatoid factor elisa ( igm ) test ( euroimmun ) with the recommended upper limit of the normal range 20 units ( ru)/ml . 
 anticitrullinated protein antibodies ( acpa ) were determined using the quanta lite ccp3.1 igg / iga elisa assay ( inova diagnostics ) with negative results < 20  u / ml . height and weight were measured barefoot wearing light clothes . 
 body mass index ( bmi ) was calculated as the ratio of weight and squared height . during physical examination blood pressure ( bp ) 
 was performed in every patient , with assessment of corrected qt interval ( qtc ) . 
 the 10-year risk of fatal cvd using the systemic coronary risk evaluation ( score ) model according to the eular recommendations was estimated in every patient . 
 carotid intima - media thickness ( cimt ) was measured using high - resolution b - mode ultrasound ( logiq 7 ge ) . in every subject imt 
 was assessed bilaterally in the three regions : common carotid artery ( cca ) , carotid bulb ( bulb ) , and internal carotid artery ( ica ) . 
 the average of the maximal imt from all 6 carotid segments ( defined as mean cimt ) was used in the analyses . 
 it has been reported that cimt  0.6  mm is a marker of subclinical atherosclerosis . 
 plaques were defined as a distinct protrusion , greater than 1.5  mm into the vessel lumen . 
 results were expressed as mean ( standard deviation , sd ) or number ( % ) . 
 group differences were tested using student 's t - test and mann - whitney u test for normally and nonnormally distributed parameters , respectively . 
 spearman 's or pearson 's correlation test was used to determine the association between saa and clinical and laboratory variables . 
 multivariable analysis ( multiple linear regression ) was performed according to a forward selection procedure , introducing those variables that showed a statistically significant association with saa . 
 characteristics of patients with ra are presented in table 1 ; clinical and laboratory variables are presented in table 2 . 
 most patients were positive for rf - igm and acpa and had erosive form of ra , of low or moderate activity ( l / mda ) ( das28  5.1 ) at the time of assessment . 
 remission of ra according to das28 ( < 2.6 ) was observed in 8 patients ( 7 women and 1 men ) . 
 extra - articular symptoms during the course of ra were noted in 58 patients ( 41.4% ) and included rheumatoid nodules ( 40 patients ) , sicca syndrome ( 16 patients ) , interstitial lung disease ( 7 patients ) , amyloidosis ( 1 patient ) , and vasculitis ( 1 patient ) . at the time of examination disease modifying antirheumatic drugs ( dmards ) 
 were not used in 5 patients ( 3.6% ) . in the remaining 135 patients , treatment with at least 1 synthetic dmard 
 was administered : methotrexate ( mtx ) ( 58.6% of all patients ) , leflunomide , sulfasalazine , chloroquine , and cyclosporine . 
 biological dmards were used in 36 patients ( 25.7% ) ( adalimumab in 3 , etanercept in 15 , infliximab in 15 , and rituximab in 3 cases ) . 
 simultaneously , low - dose prednisone ( 10  mg / day ) was used in 108 patients ( 77.1% ) . 
 the mean saa concentration in the group of 140 patients was 327.0 ( 263.4 ) mg 
 positive , significant ( p < 0.05 ) correlations were found between saa and das28 , tjc , sjc , pga of the disease activity , morning stiffness , m - haq and esr value , concentration of crp , fibrinogen , and cys - c , as well as with white blood cell count ( wbc ) , platelet count ( plt ) , and score value . 
 negative , significant ( p < 0.05 ) correlations were found between saa and concentration of albumin , hemoglobin , and qtc value . 
 all the above - mentioned variables were included in the multiple linear regression analysis , which confirmed significant associations for crp , wbc , and qtc ( table 3 ) . 
 the mean saa concentration was significantly higher in men than in women ( p = 0.01 ) ( table 4 ) . 
 the group of men , when compared with women , was characterized by higher inflammatory parameters [ crp 27.4 ( 23.3 ) versus 17.4 ( 21.9 )  mg / l , p = 0.005 ; fibrinogen 5.4 ( 1.2 ) versus 4.6 ( 1.3 )  g / l , p = 0.006 ] , unfavorable lipid parameters [ hdl 50.7 ( 11.6 ) versus 62.3 ( 15.0 )  mg / dl , p = 0.0001 ; tc / hdl index 3.8 ( 1.0 ) versus 3.4 ( 0.8 ) , p = 0.01 ] , higher cv risk markers [ cimt 0.86 ( 0.17 ) versus 0.77 ( 0.14 )  mm , p = 0.01 ; score 4.3 ( 3.9 ) versus 1.1 ( 1.5 ) , p < 0.0001 ] , and higher cys - c concentration [ 0.83 ( 0.24 ) versus 0.75 ( 0.21 )  mg / l , p = 0.04 ] . 
 significantly higher concentrations of saa were found in patients with high disease activity ( hda ) versus l / mda ( p < 0.0001 ) and in patients currently treated versus not treated with glucocorticoids ( gcs ) ( p = 0.002 ) ( table 4 ) . 
 significantly higher concentration of saa was also observed in patients with advanced atherosclerosis when compared with those without atherosclerotic plaques ( p = 0.04 ) and in patients with increased ( 1.0  mg / l ) versus normal cys - c level ( p = 0.004 ) ( table 4 ) . 
 the mean saa concentration was significantly lower in women currently treated with biological dmards ( p = 0.01 ) ( table 4 ) . 
 there was no such a correlation in men . among female patients treated with biological dmards , 
 anti - tnf inhibitors were used in 32 ( 94% ) and rituximab was used in 2 cases . the normal saa level ( < 10 
 mg / l ) was observed in 11 patients ( 7.9% ) , 10 women , and 1 man . 
 the mean concentration of saa in this group was 6.4 ( 4.4 )  mg / l ( range 09.9 ) . 
 patients with normal versus increased saa concentration were characterized by lower concentration of crp [ 11.4 ( 18.2 ) 
 mg / l versus 20.2 ( 22.7 ) , p = 0.03 ] and fibrinogen [ 4.1 ( 1.6 ) versus 4.8 ( 1.3 ) 
 g / l , p = 0.05 ] , lower wbc [ 6.0 ( 1.2 ) versus 8.1 ( 2.5 )  10/l , p = 0.003 ] , and higher qtc duration [ 373.8 ( 52.8 ) versus 332.2 ( 57.2 )  ms , p = 0.04 ] . 
 there were no significant differences in methods of treatment between the two groups of patients . 
 the major finding of the study was that serum saa concentration was high above normal in the vast majority of ra patients . 
 the normal saa level was found only in < 10% of patients , characterized by lower inflammatory burden and normal qtc duration . elevated above normal saa levels 
 were found not only in patients with hda , but also in those with l / mda assessed with das28 . 
 this observation suggests that in spite of favorable clinical assessment , sustained inflammation persists in most of ra patients and may be connected with the disease complications . in this study 
 , we demonstrated a direct relationship between saa and ra activity ( with both clinical and laboratory parameters of activity ) . 
 significantly higher saa was associated with the current treatment with gcs , which are commonly used in patients with hda . 
 women treated with biological dmards had significantly lower saa concentration than women treated only with synthetic dmards . 
 these results confirm better therapeutic effects of biological dmards in comparison to synthetic dmards resulting in lower disease activity . 
 the mean saa level was significantly higher in patients with plaques as a manifestation of advanced atherosclerosis . 
 significantly higher mean saa concentration was noticed in patients with increased cys - c level as an early marker of chronic kidney disease ( ckd ) . 
 the results of our study indicate that , in ra patients , saa could be considered as a biomarker associated with both the current inflammatory disease activity and chronic complications , the risk of cv and renal involvement in the course of ra . 
 persistently elevated saa concentration increases the risk of amyloidosis development due to deposition of amyloid proteins in different tissues and organs . 
 our results are consistent with data in literature , which reported a strong correlation between saa and ra activity . the saa level correlated with clinical disease activity ( 28-joint sjc ) and 
 it has been demonstrated that saa may be also produced by rheumatoid synovial tissue ( but not in normal synovial membrane ) , inducing cell growth , angiogenesis , invasion and migration of cells , secretion of proinflammatory cytokines ( tnf , il-1 , il-6 , and il-8 ) , chemokines , reactive oxygen species , and matrix metalloproteinases ( mmp ) [ 2 , 6 , 20 , 21 ] . 
 therefore , saa may play a pathogenic role locally in joint destruction and cartilage damage [ 2022 ] . the correlation between saa level and wbc 
 may be associated with the reported function of saa as a priming agent , rendering neutrophils more responsive to opsonized particles , as well as inducing chemotaxis and adhesion to endothelial cells and cellular migration [ 23 , 24 ] . 
 significantly higher saa levels were found in patients currently treated with gcs , which seems to be a paradoxical effect . 
 however , gcs are commonly used in patients with hda , presenting increased inflammatory parameters . on the other hand , 
 it was reported that gcs induce extrahepatic saa expression , which has been shown in tissue cultures   and confirmed in patients with chronic obstructive pulmonary disease . 
 further prospective determinations of saa are necessary to define an accurate answer . according to our knowledge , 
 we also found significantly higher saa concentration in women not treated with biological dmards , which indicates more active disease in these cases . 
 however , patients in our group received therapy with different biological dmards ( one of the three tnf inhibitors or rituximab ) , which hinders a reliable assessment of relationship between saa and biological treatment . in literature , there is no data on the association between saa and rituximab . 
 there are few reports on the effect of anti - tnf [ 2731 ] and anti - il-6 [ 31 , 32 ] treatment on saa concentration , dealing mostly with patients with amyloidosis , secondary to ra . 
 the blockade of tnf prevents high systemic saa concentrations that are largely driven by tnf . 
 the effective and early dmards treatment works as the primary prevention of amyloidosis , as well as the secondary prevention in a subclinical phase of this complication . 
 however , even during anti - tnf therapy , excessive , extrahepatic expression of saa , mediated by il-6 and gcs , could probably be maintained in rheumatoid synovial tissue . in this case , saa may upregulate tnf itself and may be a potent upstream mediator of tnf , influencing response to existing anti - tnf therapy . 
 high level of saa during anti - tnf treatment might serve as a biomarker of persistent synovial inflammation , resistant to the therapy currently used . in our patients , 
 the mean saa concentration was significantly higher in men than in women , which may be related to significantly higher parameters of inflammation and unfavorable cv risk factors in men . according to literature , women have higher saa levels than men , for various reasons , due to hormonal status , interaction with leptin , or different body composition [ 33 , 34 ] . 
 men have larger intra - abdominal fat deposits , which produce significant amounts of cytokines ( tnf , il-6 ) , even in larger quantity than subcutaneous adipose tissue . 
 we observed significantly higher saa levels in patients with atherosclerotic plaques and increased cys - c serum concentration , which indicates the risk of cv and renal complications . 
 it has been demonstrated that cells within human atherosclerotic lesions ( foam and endothelial cells , smooth muscles ) could express saa and release to circulation at the site of plaque rapture [ 35 , 36 ] . 
 no independent effect of saa on cimt value was observed in one study . in literature , saa 
 is reported as a predictor of coronary artery disease and further coronary events , as well as biomarker of cerebrovascular disease [ 38 , 39 ] . 
 cys - c has been reported as a prognostic marker of risk for death , cv , and kidney outcomes in elderly patients , with and without ckd . in the whole group of our patients , 
 the mean qtc duration was below 350  ms and was inversely correlated with saa concentration . 
 intriguingly , in patients with normal saa level , the mean qtc duration was normal . according to our knowledge , 
 this is the first report on the association between saa and short qtc , which should be taken into account , considering a risk of life - threating arrhythmias , especially in patients with high saa concentration . 
 cardiovascular mortality is significantly increased in ra , with a risk of sudden death about twofold higher than in non - ra subjects [ 41 , 42 ] . 
 it has been suggested that this phenomenon may be associated with serious arrhythmias as a result of nonstructural cardiac alterations , such as reduced heart rate variability ( hrv ) or qt interval abnormalities . 
 shortening of qtc , recognized with the value < 350  ms , is associated with increased risk of atrial fibrillation with rapid ventricular tachycardia . 
 the study in patients with inflammatory arthritis demonstrated that high sensitivity crp ( hscrp ) was inversely correlated with hrv and directly correlated with qtc duration . 
 the study population consisted mostly of patients with spondyloarthropathies and only 25% of ra patients of unknown disease activity . 
 it is suggested that systemic inflammation may affect cardiac nervous system , irrespectively of other cv risk factors . 
 it seems that a short qt interval does not by itself predict risk of life - threating arrhythmias but rather should be taken in context of each individual patient . 
 the strengths are as follows : ( 1 ) relatively large sample of consecutive patients with ra , treated in our department , ( 2 ) the detailed characteristics of patients , who have been considered in all aspects of ra pathology including several inflammatory , cv , metabolic parameters , and assessment of renal function , ( 3 ) the design of the study which was based on clinical practice , and ( 4 ) according to our knowledge being the first study which evaluates the potential association of high saa level with qtc shortening , increasing the risk of serious arrhythmias and sudden death . 
 there are also some limitations including the following : ( 1 ) our patients were treated with different biological dmards ; that is why it is difficult to determine the exact association between saa and effect of therapy ; ( 2 ) ecg recording was performed routinely in every patient and qtc duration was assessed automatically , with no special procedure , and hrv was not assessed ; and ( 3 ) we did not collect detailed information about the ongoing treatment , which may influence heart rate . as a consequence , further investigation is needed to confirm the observed relationship , in relation to concurrent treatment . in our study , in patients with ra , 
 serum saa levels strongly correlated with the activity of the disease and were significantly higher in patients with high disease activity . 
 however , high saa concentrations were observed also in patients with low or moderate disease activity , in spite of clinical improvement . concurrent treatment with gcs was associated with higher saa and therapy with biological dmards was associated with lower saa concentration . 
 the mean saa level was inversely associated with qtc duration and markedly higher in patients with atherosclerotic plaques , emphasizing increased cv risk resulting from chronic inflammatory process . 
 we also found significantly higher saa levels in patients with increased cys - c as a marker of ckd , emphasizing increased risk of renal disease . 
 high saa concentration was strongly connected with the activity of the disease and the risk of cv and renal involvement in ra patients . 
 prospectively , repeated assessment of saa may facilitate searching ra patients with persistent inflammation and increased risk of extra - articular complications , as well as amyloidosis development .","objectives . 
 rheumatoid arthritis ( ra ) is a systemic , inflammatory disease . 
 serum amyloid a ( saa ) is an acute - phase protein , involved in pathogenesis of atherosclerosis . 
 the aim of the study was to assess serum concentration of saa in ra patients , with reference to other inflammatory parameters and markers of extra - articular involvement . 
 methods . 
 the study population consisted of 140 ra patients , low / moderate disease activity ( l / mda ) in 98 ( 70% ) patients and high disease activity ( hda ) in 42 ( 30% ) . 
 comprehensive clinical and laboratory assessment was performed with evaluation of electrocardiogram and carotid intima - media thickness . 
 results . the mean saa concentration [ 327.0 ( 263.4 ) 
 mg / l ] was increased highly above the normal value , even in patients with l / mda . 
 simultaneously , saa was significantly higher in patients with hda versus l / mda . 
 the mean saa concentration was significantly higher in patients treated with glucocorticoids , was inversely associated with qtc duration , and was markedly higher in patients with atherosclerotic plaques , emphasizing increased cv risk . 
 saa was significantly higher in patients with increased cystatin - c level . 
 conclusions . in ra patients , 
 high serum saa concentration was strongly associated with activity of the disease and risk of cv and renal involvement . 
 recurrent assessment of saa may facilitate searching patients with persistent inflammation and risk of extra - articular complications .",pubmed
"glucocorticoids have been extensively characterized as being anti - inflammatory   and/or immunosuppressive   in therapeutic settings and are generally discussed as opposed to the development of inflammation and limiting of production or maturation of some effector immune cell types , in a physiological context . 
 nevertheless , there is a well - established association between stress and allergic diseases , including asthma [ 4 , 5 ] . 
 glucocorticoids , which are an essential part of systemic stress responses , play coadjuvant roles in promoting inflammation and may promote th2-type immunity through differential effects on th1  th2 cytokine production [ 5 , 6 ] . chronically stimulated eosinophil production ( eosinopoiesis ) is an important feature of human asthma   and of murine allergic asthma models . in both cases , allergen challenge of sensitized subjects increases eosinopoiesis in the bone - marrow [ 7 , 8 ] . 
 this effect is antigen - specific and can be abolished by inducing oral tolerance to the allergen , which affects both eosinophils and neutrophils in bone - marrow . 
 the effects of oral tolerization in bone - marrow neutrophil and eosinophil granulocytes can be duplicated by transfer of splenic t lymphocyte subpopulations from tolerized / sensitized / challenged donors to histocompatible naive recipients . 
 these observations highlight the importance of acquired cellular immunity in regulating the hematological response to allergen sensitization and challenge . 
 they further suggest the possibility that granulopoiesis , encompassing both eosinophil and neutrophil production , might be regulated by lymphocyte populations in nonsensitized subjects as well . in an allergic asthma model , we demonstrated a critical role for endogenous glucocorticoids in the hematological response to allergen challenge : challenge induces a corticosterone surge that is paralleled by increased eosinophilia of bone - marrow in vivo and by increased responsiveness to il-5 , the major eosinopoietic cytokine , ex vivo ; the bone - marrow response to challenge is abolished by blockade of glucocorticoid signaling or glucocorticoid production . on the other hand , in the absence of allergen sensitization and challenge , we have also obtained evidence of a link between the corticosterone surge induced by mild surgical trauma and short - term bone - marrow eosinophilia ; again , blockade of endogenous glucocorticoid production or action abolished the bone - marrow eosinophilia induced by trauma . 
 these in vivo effects of corticosterone , an endogenous glucocorticoid released by the adrenal glands , are paralleled by those of exogenously provided corticosterone or dexamethasone on murine bone - marrow [ 12 , 13 ] and of other glucocorticoids on human hemopoietic cells . in balb / c mice , 
 dexamethasone increases eosinophil production in murine bone - marrow culture [ 12 , 13 ] and primes bone - marrow cells in vivo for increased ex vivo responses to il-5 . during further screening of inbred mouse strains for differences in the granulopoietic responses to dexamethasone , we observed bone - marrow eosinophilia in mice of the c57bl/6 ( b6 ) background injected with dexamethasone , which was undetectable in perforin - deficient b6 mutants   submitted to the same treatment . 
 perforin is a major mediator of cellular immunity [ 1519 ] , expressed in lymphocyte populations which fight viral   and bacterial pathogens [ 16 , 17 ] as well as malignant cells   in the context of both innate and acquired immune responses . 
 perforin is also expressed by murine bone - marrow neutrophils , which have a critical regulatory role in t cell - mediated contact hypersensitivity . 
 perforin deficiency is known to induce complex changes in leukocyte populations in humans and mice [ 16 , 17 , 20 ] , including a classical presentation of familial hemophagocytic lymphohistiocytosis , characterized by early life onset , high mortality , and multiple immunological defects , including uncontrolled activation and proliferation of cd4 + and cd8 + t cells , cytokine storm , macrophage activation and proliferation , pancytopenia , and anemia . here 
 we report that perforin deficiency also presents a selective defect in granulocyte production , which can be corrected by wild - type lymphocyte transfer . 
 sh30088.03 ) , rpmi1640 ( sh30011.01 ) , and imdm ( sh30228.01 ) were from hyclone ( logan , ut , eua ) ; l - glutamine ( g7513 ) , penicillin - streptomycin solution ( p4333 ) , essential amino acids solution ( 50x ) ( m5550 ) , methylcellulose ( m0387 ) , dexamethasone ( 21-phosphate , disodium salt , d1159 ) , mifepristone ( ru486 , m8046 ) , and histopaque-1083 ( 10831 ) were from sigma - aldrich corporation ( st . louis , mo , eua ) ; agar noble ( 0142 - 15/21422 ) was from difco ( detroit , mi , eua ) ; nonessential amino acids solution ( 100x ) , ( 11140 - 050 ) and mem vitamin solution ( 100x ) ( 11120 - 052 ) were from gibco life technologies ( carlsbad , ca , usa ) ; recombinant murine il-5 ( 405-ml-025 ) was from r&d systems ( minneapolis , mn , usa ) ; recombinant murine gm - csf ( 315 - 03 ) was from preprotech ( rocky hill , nj , usa ) ; rat antimouse cd8b ( clone : ebioh35 - 17.2 , 12 - 0083 - 82 , 0.2  mg / ml ) was from ebioscience ( san diego , ca , usa ) ; magnetic microspheres conjugated to antimouse cd4 ( l3t4 , 130 - 049 - 20 ) and to goat antirat igg ( 130 - 048 - 501 , as secondary antibody to primary rat antimouse cd8b ) were from miltenyi biotec ( ambriex , sp , brazil ) . 
 wild - type c57bl/6 , perforin - deficient ( pfp ) mutants of the b6 background , and wild - type b6.129 mice were bred by cecal - fiocruz , rio de janeiro , brazil . 
 the pfp stock was derived from the original b6.129s6-pfp stock   by backcrossing to c57bl/6 at fiocruz . 
 unless otherwise indicated , the wild - type controls for the experiments shown were c57bl/6 ; in selected experiments , b6.129 wild - type controls were used and yielded the same results as c57bl/6 ( not shown ) . 
 the animals were housed and handled following institutionally approved guidelines under license l-00209 from ceua - fiocruz . 
 routinely , female mice were used for the experiments , since male mice characteristically fight for dominance in the same cage , and the stress associated with fighting may confound the interpretation of the results . 
 we have no evidence , however , that the granulopoietic responses described here , including the strain differences , are restricted to females . dexamethasone in saline solution was injected i.p . 
 ( 200  l , i.p . , amounting to 5  mg / kg ; ) . 
 ru486 in 0.1% methylcellulose was given intragastrically 2  h before dexamethasone with a gavage needle ( 200  l , amounting to 100  mg / kg ) . 
 for lymphocyte transfers , 10 nylon - wool purified cells   from naive c57bl/6 donors in a 100  l volume of sterile saline were injected into the tail vein of pfp recipients , once . 
 controls received an equal number of cells from pfp donors . where lymphocytes depleted of cd4 + or cd8 + cells were used , the amount of cells injected was that recovered from 10 initial unseparated lymphocytes , in 100  l sterile saline , i.v . 
 dexamethasone was administered to the recipients , in both cases , 48  h after lymphocyte transfer . 
 where indicated , bone - marrow , peripheral blood from the abdominal vena cava , spleens , and whole thymuses were collected , for enumeration of cells or determination of relative weight of the thymus ( mg / g body weight ) [ 21 , 23 ] . 
 bone - marrow collected from both femurs of each mouse in 5  ml rpmi1640 medium/1% fbs with a 22-gauge needle and kept on ice was used for total and differential counts and cell culture ( see below ) . 
 spleen mononuclear cells were the source for isolation of lymphocytes ( see below ) [ 24 , 25 ] . 
 differential counts were carried out in cytocentrifugates after fixation in pbs-10% formaldehyde and staining for eosinophil peroxidase ( epo [ 26 , 27 ] ) followed by counterstaining with harris ' hematoxylin . 
 liquid bone - marrow cultures were established at 37c , with 10 bone - marrow cells in 1  ml rpmi1640/10% fbs , in 5% co2/95% air , plated in 24-well plates ( cat . 
 n : 142475 , nunc brand products ) with il-5 ( 1  ng / ml ; 7 days ) . where indicated , dexamethasone was added ( 10  l / well , to 10  m final concentration ) . 
 absolute numbers were calculated from total cell counts in turk 's solution multiplied by the % of epo+ cells ( eosinophil - lineage cells , both mature and immature ) in cytocentrifuge smears . 
 triplicate semisolid ( clonal ) bone - marrow cultures were established for 7 days with 2  10 cells adjusted to 1  ml of 1  :  1 imdm / agar mix , in 35  mm tissue culture plates ( nunc ) , in the presence of gm - csf ( 2  ng / ml final ) , with or without dexamethasone ( 10  m ) , at final 20% fbs and 0.3% agar concentrations . 
 total colonies ( defined as aggregates > 50 cells derived from a single progenitor cell ) [ 8 , 12 ] were scored at day 7 under an inverted microscope ( 400x , phase contrast ) . for lymphocyte isolation , spleens were collected and minced in rpmi1640/1% fbs on tissue culture plates . 
 spleen mononuclear cells ( 2  10 in 10  ml rpmi1640/1% sfb ) were isolated by centrifugation on a 1.083-density ficoll - hypaque cushion ( 3  ml , at 400  g , for 30 minutes , at room temperature , following manufacturer 's instructions ) [ 24 , 25 ] . 
 cells recovered from the medium / ficoll - hypaque interface were collected , washed in serum - free medium , resuspended , counted , and further separated on nylon - wool columns [ 24 , 25 ] at 4  10 cells/2  ml / g nylon wool . 
 cells eluted in a total 25  ml warm medium , dropwise , were washed and counted before cytocentrifugation / staining or incubation with antibodies . 
 depletion of cd4 + cells was done with 10  l l3t4/10 nylon - wool purified lymphocytes in 100  l serum - free rpmi1640 , on ice , following manufacturers ' instructions . 
 depletion of cd8 + cells was in two steps , with rat antimouse cd8b conjugated with pe , followed by goat antirat igg ( 20  l/10 lymphocytes in 80  l medium , on ice ) . 
 columns were eluted with 9  ml medium , dropwise , over a 5-minute period . 
 the numbers of experiments ( n ) are indicated in the caption of the figures , to avoid overcharging the figures and captions . for comparisons of two groups ( figures 1 and 2 ) , we used the two - tailed t - test with separate variances ( systat for windows , version 5 , systat inc . , 
 evanston , il ) . for multiple comparisons ( figures 3 , 4 , 5 , and 6 ) 
 , we used anova , with the tukey hsd correction for groups of equal size ( systat for windows ) or with bonferroni 's correction for groups of unequal size ( using prisma 5 for windows , graph pad , la jolla , ca ) , unless otherwise indicated in section 3 . 
 following preliminary experiments ( not shown ) that evidenced a positive response to dexamethasone in b6 wild - type mice , as well as the absence of any response , positive or negative , in pfp mutants of the b6 background , we reviewed data on freshly harvested bone - marrow from a large number of mice of both strains ( n = 44 and n = 48 , resp . ) , to look for evidence of strain differences in bone - marrow steady - state parameters , in the absence of dexamethasone exposure ( figure 1 ) . 
 a significant difference was observed in this large series , with pfp mice having lower bone - marrow cellularity than wild - type controls ( figure 1(a ) ; p < 0.001 ) 
 . the data available for epo+ cells ( figure 1(b ) ;  p = 0.033 ) and neutrophils ( figure 1(c ) ; p = 0.010 ) of the mice in the large series showed significant differences as well , with lower counts in pfp mutants . 
 as the two groups were defined only on the basis of genetic differences ( presence or absence of functional perforin genes ) , these differences could still be accounted for , in principle , by variance due to nongenetic factors within these groups , especially those which influence growth and development . because bone - marrow cellularity is roughly proportional to the size of the animal 
 , we next evaluated whether these differences would disappear in the comparison between groups of control and mutant mice matched by weight ( figures 1(d)1(f ) ) . 
 significant differences were still observed for total cells ( figure 1(d ) ; p = 0.001 ) , eosinophils ( figure 1(e ) ; p = 0.004 ) , and neutrophils ( figure 1(f ) ; p = 0.011 ) , all three parameters being lower in the mutant mice . because age might affect bone - marrow function through mechanisms unrelated to body weight gain ( as senescence may have an earlier onset in some strains ; furthermore , age is a major determinant of incidence of many pathological processes , such as malignancies ) , we next examined whether matching by age ( all animals at 12 weeks ) would eliminate the differences . 
 significant differences were still found for all three parameters ( for figures 1(g)1(i ) , resp . 
 , p = 0.001 , p = 0.006 , p = 0.001 ) , which were lower in the mutant mice . even matching by both weight and age ( for figures 1(j)1(l ) , resp . 
 , p = 0.022 , p = 0.032 , p = 0.001 ) failed to eliminate these significant differences between controls and mutants for any of the three parameters , which were all lower in the mutant mice . 
 overall , this suggests that bone - marrow cellularity , as well as bone - marrow eosinophil and neutrophil counts , is significantly lower in pfp mutants than in b6 mice and that this difference can not be dismissed as created by undue comparisons of two groups differing in body weight , age , or both . 
 we further examined ( figure 2 ) the counts of total cells , lymphocytes , neutrophils , and eosinophils in peripheral blood of weight - matched ( median 21  g , range 1923  g ) b6 and pfp mice . unlike bone - marrow 
 , peripheral blood total nucleated cell counts did not differ significantly between these strains ( figure 2(a ) , p = 0.795 ) , nor did lymphocyte counts ( figure 2(b ) , p = 0.417 ) . 
 by contrast , both neutrophil ( figure 2(c ) , p = 0.025 ) and eosinophil ( figure 2(d ) ; p = 0.030 ) counts were significantly different , and , like in bone - marrow , lower in pfp mice . 
 together , the data in figures 1 and 2 suggest that , even in the absence of dexamethasone , pfp mice have reduced granulocyte numbers both inside and outside of bone - marrow , relative to wild - type controls of comparable body weight . 
 our original observation of this strain difference was increased % eosinophils in cultured bone - marrow from b6 , but not pfp , mice when both il-5 and dexamethasone were present ( jones and cardoso de mendona , unpublished observations ) . 
 this is , however , insufficient to characterize a strain difference in response to dexamethasone , because the % of a given cell type in a bone - marrow sample , which has a highly heterogeneous composition , can be increased artifactually by a corresponding decrease in another cell type , rather than by a positive effect of dexamethasone on the cell type of interest . to confirm a positive effect of il-5 and dexamethasone in stimulation of wild - type and mutant eosinopoiesis , 
 liquid cultures were established from the same number of bone - marrow cells ( 10 ) from b6 or pfp donors , in the presence of il-5 , alone or associated with dexamethasone , and the absolute numbers of eosinophils in the culture were determined . 
 as shown in figure 3 , eosinophils were produced in cultures of il-5-stimulated wild - type and mutant bone - marrow ( figure 3(a ) ) . 
 control cultures lacking il-5 do not contain eosinophils at the end of the culture period ( not shown ; ) . 
 counts of epo+ cells recovered at the end of the culture were increased in the presence of dexamethasone ( 10  m ) , relative to il-5 controls , in cultures from b6 controls , but not from pfp mutants ( figure 3(a ) , p < 0.001 ) . nevertheless , the il-5 present was sufficient to sustain eosinopoiesis in the absence of dexamethasone by pfp bone - marrow to the same level observed in b6 control cultures . 
 on the other hand , data from semisolid cultures , which allowed us to examine the effects of dexamethasone on several classes of progenitors ( colony - forming cells ) , including the granulocyte ( g ) and granulocyte - macrophage ( gm ) progenitors , which produce neutrophils , are also shown in figure 3 . 
 the total counts of colonies formed by gm - csf - stimulated pfp bone - marrow progenitors , in the absence of dexamethasone , also differed significantly from those of b6 controls ( p  0.001 ) : even though identical numbers of bone - marrow cells were plated , less colonies were made by perforin - deficient bone - marrow ( figure 3(b ) ) . 
 furthermore , dexamethasone ( 10  m ) significantly stimulated colony formation by b6 bone - marrow ( p = 0.002 ) but failed to stimulate ( p = 0.558 ) pfp bone - marrow in the same conditions ( figure 3(b ) ) . 
 these observations show that bone - marrow progenitors from pfp mice differ significantly from those of b6 controls , because they form less colonies and do not respond to dexamethasone , which significantly enhances colony formation in the wild - type cultures . 
 we next examined the effect of dexamethasone ( 5  mg / kg injection ) on bone - marrow eosinophils and neutrophils , as well as on the relative weight of the thymus , which is significantly reduced by glucocorticoids . 
 as shown in figure 4 , dexamethasone injection increased the numbers of bone - marrow eosinophils ( figure 4(a ) , p = 0.003 ) and neutrophils ( figure 4(b ) , p = 0.019 ) in vivo , relative to saline - injected controls , in wild - type b6 mice , but not in pfp mice . 
 the effect of dexamethasone on eosinophil numbers in wild - type b6 mice was abolished by ru486 pretreatment , while ru486 had no effect of its own in the absence of dexamethasone , in either b6 or pfp mice ( not shown ) . 
 the relative weight of the thymus was significantly different ( p = 0.010 ) between body weight - matched pfp mice and wild - type controls , suggesting a decreased thymic cellularity in mutant thymus , even without exogenous glucocorticoid administration ( figure 4(c ) ) . 
 importantly , dexamethasone injection did reduce significantly the relative weight of the thymus in both wild - type ( p = 0.007 ) and mutant ( p = 0.001 ) mice ( figure 4(c ) ) . 
 again , the effect of dexamethasone on the thymus was abolished in both cases by ru486 pretreatment ( not shown ) . 
 together , these observations show that pfp bone - marrow is unresponsive to dexamethasone stimulation in vivo , although dexamethasone significantly increases eosinophil and neutrophil numbers in the bone - marrow of wild - type controls . 
 they also show that lack of responsiveness to dexamethasone in pfp bone - marrow is not due to a general lack of response to glucocorticoids , because the thymus of pfp mice responds to dexamethasone injection as expected . 
 because perforin is mainly expressed in various effector / regulatory lymphocyte subsets , it was important to test whether the perforin - related defect in bone - marrow response to dexamethasone could be corrected by introducing b6 lymphocytes in pfp mice . 
 we have done so with splenic lymphocytes from naive b6 donors , because both the baseline granulopoiesis defect and the defective granulopoietic response to dexamethasone were observed in the absence of allergic sensitization . the number ( 10 ) of lymphocytes for transfer into individual mice 
 was defined on the basis of similar reconstitution studies . to define whether elimination of a particular lymphocyte subpopulation defined by standard surface markers ( cd4/cd8 ) prevented reconstitution of the dexamethasone response , 
 10 total lymphocytes purified from spleen were submitted to alternative depletion protocols with marker - specific microbeads , and the depleted cells in the column effluent , corresponding to the number of cells negative for the selection marker present in the original lymphocyte sample , were injected . as shown in figure 5 , pfp recipients of wild - type lymphocytes ( unseparated ) show a significant in vivo response to dexamethasone injection , by increased total cell ( figure 5(a ) ; p = 0.032 , t - test ) , eosinophil ( figure 5(b ) ; p = 0.04 ) , and neutrophil ( figure 5(c ) ; p < 0.001 ) counts in the bone - marrow , sharply contrasting with observations in pfp mice in the absence of wild - type cell transfer ( compare figure 4 ) . on 
 the other hand , depletion of cd4 + or cd8 + cells in the wild - type lymphocyte preparation gave distinct results in the reconstitution assay , depending on the granulocyte population examined : while depletion of either subset abolished the ability of splenic lymphocytes to reconstitute the eosinopoietic response to dexamethasone ( figure 5(b ) ) , depletion of cd4 + cells did not prevent reconstitution of the neutropoietic response ( figure 5(c ) ; p = 0.013 ) , while depletion of cd8 + cells prevented reconstitution . as a further control , we performed transfer of pfp lymphocytes into pfp recipients and obtained no reconstitution of dexamethasone responses , judged by any of these three parameters ( figure 6 ) . in these controls , 
 dexamethasone reduced significantly the relative weight of the thymus , showing that dexamethasone was able to reach systemically active levels even if bone - marrow showed no evidence of responding to it . 
 overall , these observations suggest that lymphocytes from the spleen of naive wild - type mice , but not pfp mice , are able to reconstitute in the short term the granulopoietic responses to dexamethasone , but different cells may be involved in reconstitution of lineage - specific ( eosinophilic versus neutrophilic ) responses . 
 this is , to our knowledge [ 1520 ] , the first description of a selective defect in granulopoiesis associated with perforin deficiency in mice and of its correction by the intravenous transfer of wild - type lymphocytes . as such 
 , it extends the range of manifestations associated with perforin deficiency and raises the issue of how perforin contributes to regulation of granulocyte lineages in vivo . because the wild - type lymphocyte populations interact with a drug ( dexamethasone ) which initiates signaling through the ru486-inhibitable glucocorticoid receptor , to stimulate in vivo eosinophil and neutrophil production 
 , these observations may be relevant to processes in which bone - marrow is stimulated by immune responses   or trauma   with involvement of the same receptor and of endogenous glucocorticoids released by the adrenal glands . 
 perforin is a well - characterized effector protein , mainly ( but not exclusively ) expressed in lymphocytes which share the ability to induce cell - mediated cytotoxicity [ 1520 ] . 
 perforin deficiency was initially characterized by the loss of important cytotoxic lymphocyte functions [ 1520 ] . 
 however , it was soon realized that perforin deficiency entails other , more complex , functional consequences , leading to the development of type ii familial hemophagocytic lymphohistiocytosis [ 16 , 17 , 20 ] , which shares pathophysiological features with the macrophage activation syndrome , including interferon- ( ifn- )  overproduction . 
 hence , perforin deficiency has broader consequences in addition to impairment of cytotoxic lymphocyte function , and ifn- , alone or in association with other  cytokine storm  components , may transduce its effects on other leukocyte lineages . 
 one of the issues raised by our findings is whether the lower granulocyte numbers in blood and bone - marrow of pfp mice are due to the progression of the murine equivalent of the human familial hemophagocytic lymphohistiocytosis [ 16 , 17 , 20 , 28 ] . 
 it should be noted that the human familial hemophagocytic lymphohistiocytosis is a severe condition , presenting early in life in most cases and associated with important mortality [ 20 , 28 ] . 
 also , phenotypically distinct forms correspond to different mutations with perforin expressed at lower levels and/or with altered properties , a situation that is not applicable to the present study , which employed perforin null mice . the perforin - deficient mice in the original walsh et al . 
 study ( from which the mice used in this study descend ) were described as healthy for at least 5 months . in our study , mice were certified spf and maintained in microisolator units , with no evidence of mortality , stunting , or signs of infection during the entire observation period of up to 3 months ( 12 weeks ) . on the other hand , pfp defect in response to dexamethasone 
 could be corrected by transfer of as few as 10 lymphocytes 48  h before the stimulus . 
 the prompt reconstitution of this defect argues against the lack of dexamethasone response in pfp animals being caused by a chronic inflammatory disease of the bone - marrow , such as familial hemophagocytic lymphohistiocytosis , which in humans is challenging for therapy . 
 because correction was achieved by simple transfer of lymphocytes from naive donors , we think it is more likely that the transferred lymphocytes provided something that was lacking in the recipient . 
 neutrophils were reduced in bone - marrow and blood of pfp mice relative to b6 controls . 
 this not only shows that the neutrophil deficiency has peripheral expression but further argues against reduced numbers in bone - marrow being due to an increased export of neutrophils to the periphery . 
 the evidence from colony - forming assays suggests , instead , that production is greatly reduced in pfp relative to b6 mice , as there were less gm - csf - responsive progenitors in an identical number of bone - marrow cells . 
 reduced production would explain the decrease in granulocyte counts both outside and inside of bone - marrow . 
 on the other hand , normal numbers of lymphocytes and total leukocyte counts show that the defect is selective for granulocytes , which are not the major circulating leukocyte subpopulation in mice , so that reduction in their numbers does not have a major impact on total circulating leukocyte counts . 
 neutropenia is associated with increased susceptibility to bacterial and fungal infection , provided it is severe enough . 
 this was not observed in our study , so presumably the pfp mice were capable of coping with the microorganisms present in a rather clean environment ( spf conditions , microisolator housing ) . 
 it remains to be seen , however , whether following a more severe infectious exposure , such as that associated with sepsis induction , pfp mice would prove more vulnerable to bacterial dissemination . 
 this will be addressed in future studies , since emergency granulopoiesis , as opposed to baseline granulopoiesis , is driven by gm - csf and related hemopoietic cytokines . 
 the lack of appropriate response to gm - csf , both in the absence and in the presence of dexamethasone , would predict that emergency granulopoiesis would be defective in pfp mice and might therefore negatively influence the outcome of sepsis . 
 the hypothalamus - pituitary - adrenal axis is activated in sepsis , and therefore it is possible that emergency granulopoiesis is driven by endogenous glucocorticoids , in a way consistent with our observations . 
 the prediction , in this case , is that perforin would be required for normal host response to bacterial sepsis through an effect on glucocorticoid - mediated signaling in the bone - marrow . 
 the ability of lymphocyte preparations to reconstitute responses to dexamethasone was abolished by depletion of cd4 + and/or cd8 + cells before transfer , depending on the granulocyte population . for neutrophils , cd8 
 while this suggests that different mechanisms are involved for eosinophils and neutrophils , it certainly raises the issue of the lymphocyte subpopulation involved . since naive lymphocytes are sufficient , it is not likely that conventional t cells , cd4 + or cd8 + , are responsible , for they would not be activated in this isolation protocol [ 24 , 25 , 31 ] . 
 conventional nk cells would not be a strong candidate , since cd4 + or cd8 + lymphocyte depletion would probably not eliminate them [ 32 , 33 ] . 
 innate lymphocytes other than conventional nk cells , however , would be expected to be present in naive mouse spleens [ 32 , 33 ] . 
 natural killer t cells , which include subsets expressing cd4 and cd8 , are found in significant numbers in b6 spleen , present an activated memory phenotype without known activating exposure , and exert multiple regulatory functions through rapid secretion of large amounts of cytokines [ 32 , 33 ] , might perhaps account for some of our observations . 
 regardless of innate or adaptive lymphocyte population involved , two questions which require further study are as follows : ( a ) is perforin the missing component that is brought by the transferred lymphocytes ? and ( b ) do these lymphocytes act by homing to the recipients ' bone - marrow and interacting locally with granulocyte progenitors / precursors ? both questions , however , require a more complex experimental design and resources to allow us to detect and modify perforin expression inside living lymphocytes , as well as monitor their homing to the bone - marrow compartment . for this reason , 
 such experiments fall outside the scope of this paper , which is limited to the demonstration of the defect and of its correction . finally , it should be noted that lymphocyte transfer restored the short - term response to dexamethasone but did not bring the baseline counts of eosinophils and neutrophils to the same level of the b6 controls . 
 this might be interpreted as evidence that steady - state granulopoiesis and response to dexamethasone are unrelated processes ( i.e. , their perforin deficiency entails two distinct defects in granulopoiesis , rather than one defect with distinct manifestations ) . 
 we do not support this interpretation , however , because the duration of the experiment might be insufficient for an effect of lymphocyte transfer alone on steady - state granulocyte numbers to become noticeable . 
 it is also possible that demonstration of a durable effect on steady - state granulocyte numbers would require not only an extended observation period but also an increased or repeated input of wild - type lymphocytes and an interaction of exogenous lymphocytes with endogenous adrenal glucocorticoids as well .","exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone - marrow . 
 a hematological response dependent on endogenous glucocorticoids underlies bone - marrow eosinophilia induced by trauma or allergic sensitization / challenge . 
 we detected a defect in granulopoiesis in nonsensitized , perforin - deficient mice . 
 in steady - state conditions , perforin- ( pfp- ) deficient mice showed significantly decreased bone - marrow and blood eosinophil and neutrophil counts , and colony formation in response to gm - csf , relative to wild - type controls of comparable age and/or weight . 
 by contrast , peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency . 
 dexamethasone enhanced colony formation by gm - csf - stimulated progenitors from wild - type controls , but not pfp mice . 
 dexamethasone injection increased bone - marrow eosinophil and neutrophil counts in wild - type controls , but not pfp mice . 
 because perforin is expressed in effector lymphocytes , we examined whether this defect would be corrected by transferring wild - type lymphocytes into perforin - deficient recipients . 
 short - term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild - type donors . 
 transfer of the same amount of splenic lymphocytes from perforin - deficient donors was ineffective . 
 this demonstrates that the perforin - dependent , granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations .",pubmed
"62.4 million indians were reported to have type 2 diabetes mellitus ( t2 dm ) putting india on the forefront of diabetic epidemic across globe . 
 fear of hypoglycaemia and gain in body weight are barriers for initiation of insulin therapy . 
 modern insulin analogues are a convenient new approach or tool to glycaemic control , associated with low number of hypoglycaemia and favourable weight change . 
 a1chieve , a multinational , 24-week , non - interventional study , assessed the safety and effectiveness of insulin analogues in people with t2 dm ( n = 66,726 ) in routine clinical care . 
 please refer to editorial titled : the a1chieve study : mapping the ibn battuta trail . 
 the patient characteristics for the entire cohort divided as insulin - nave and insulin users is shown in the table 1 . 
 the majority of patients ( 73.9% ) started on or switched to biphasic insulin aspart . 
 other groups were insulin detemir ( n = 313 ) , insulin aspart ( n = 144 ) , basal insulin plus insulin aspart ( n = 53 ) and other insulin combinations ( n = 41 ) . overall demographic data after 24 weeks of treatment , overall hypoglycaemic events reduced from 1.5 events / patient - year to zero events in insulin user group while hypoglycaemia remained nil in insulin nave group similar to that of baseline . 
 no hypoglycaemic episode in insulin naive group even at 24 weeks suggests low event rate than insulin users at baseline . 
 blood pressure decreased whereas overall lipid profile and quality of life improved at week 24 in the cohort [ tables 2 and 3 ] . 
 all parameters of glycaemic control improved from baseline to study end in the total cohort [ table 4 ] . 
 overall efficacy data of the total cohort , 1561 patients started on biphasic insulin aspart  ogld , of which 1471 ( 94.2% ) were insulin nave and 90 ( 5.8% ) were insulin users . 
 after 24 weeks of starting or switching to biphasic insulin aspart , hypoglycaemic events reduced from 1.2 events / patient - year to 0.0 events / patient - year in insulin user group , whereas hypoglycaemia was nil in insulin naive group similar to baseline . 
 quality of life improved after 24 weeks of treatment [ tables 5 and 6 ] . 
 biphasic insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for both insulin nave and insulin user groups [ table 7 ] . 
 biphasic insulin aspartoral glucose - lowering drug efficacy data of the total cohort , 53 patients started on basal + insulin aspart  ogld , of which 27 ( 50.9% ) were insulin nave and 26 ( 49.1% ) were insulin users . 
 after 24 weeks of starting or switching to basal + insulin aspart , hypoglycaemic events reduced from 1.0 events / patient - year to 0.0 events / patient - year in insulin user group , while hypoglycaemia was nil in insulin naive group similar to baseline . 
 quality of life improved at the end of the study [ tables 8 and 9 ] . 
 basal+insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to basal + insulin aspart  oglds for both insulin nave and insulin user groups [ table 10 ] . 
 basal+insulin aspartoral glucose - lowering drug efficacy data of the total cohort , 313 patients started on insulin detemir  ogld was 313 , of which 302 ( 96.5% ) were insulin nave and 11 ( 3.5% ) were insulin users . 
 after 24 weeks of starting or switching to insulin detemir , hypoglycaemic events was nil in both insulin nave and user groups similar to baseline . 
 body weight decreased and quality of life improved at 24 weeks [ tables 11 and 12 ] . 
 insulin detemiroral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin detemir  oglds for both insulin - nave and insulin user groups [ table 13 ] . 
 insulin detemiroral glucose - lowering drug efficacy data of the total cohort , 144 patients started on insulin aspart  ogld , of which 131 ( 91.0% ) were insulin nave and 13 ( 9.0% ) were insulin users . 
 after 24 weeks of starting or switching to insulin aspart , hypoglycaemic events reduced from 2.0 events / patient - year to 0.0 events / patient - year in insulin user group , whereas hypoglycaemia remained nil in insulin nave group similar to baseline . 
 quality of life improved at the end of the study [ tables 14 and 15 ] . 
 insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin aspart  oglds for both insulin nave and insulin user groups [ table 16 ] . 
 of the total cohort , 1561 patients started on biphasic insulin aspart  ogld , of which 1471 ( 94.2% ) were insulin nave and 90 ( 5.8% ) were insulin users . 
 after 24 weeks of starting or switching to biphasic insulin aspart , hypoglycaemic events reduced from 1.2 events / patient - year to 0.0 events / patient - year in insulin user group , whereas hypoglycaemia was nil in insulin naive group similar to baseline . 
 quality of life improved after 24 weeks of treatment [ tables 5 and 6 ] . 
 biphasic insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for both insulin nave and insulin user groups [ table 7 ] . 
 of the total cohort , 53 patients started on basal + insulin aspart  ogld , of which 27 ( 50.9% ) were insulin nave and 26 ( 49.1% ) were insulin users . 
 after 24 weeks of starting or switching to basal + insulin aspart , hypoglycaemic events reduced from 1.0 events / patient - year to 0.0 events / patient - year in insulin user group , while hypoglycaemia was nil in insulin naive group similar to baseline . 
 quality of life improved at the end of the study [ tables 8 and 9 ] . 
 basal+insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to basal + insulin aspart  oglds for both insulin nave and insulin user groups [ table 10 ] . 
 of the total cohort , 313 patients started on insulin detemir  ogld was 313 , of which 302 ( 96.5% ) were insulin nave and 11 ( 3.5% ) were insulin users . 
 after 24 weeks of starting or switching to insulin detemir , hypoglycaemic events was nil in both insulin nave and user groups similar to baseline . 
 body weight decreased and quality of life improved at 24 weeks [ tables 11 and 12 ] . 
 insulin detemiroral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin detemir  oglds for both insulin - nave and insulin user groups [ table 13 ] . 
 of the total cohort , 144 patients started on insulin aspart  ogld , of which 131 ( 91.0% ) were insulin nave and 13 ( 9.0% ) were insulin users . 
 after 24 weeks of starting or switching to insulin aspart , hypoglycaemic events reduced from 2.0 events / patient - year to 0.0 events / patient - year in insulin user group , whereas hypoglycaemia remained nil in insulin nave group similar to baseline . 
 quality of life improved at the end of the study [ tables 14 and 15 ] . 
 insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin aspart  oglds for both insulin nave and insulin user groups [ table 16 ] . 
 our study reports improved glycaemic control and quality of life following 24 weeks of treatment with any of the insulin analogues ( biphasic insulin aspart ; basal + insulin aspart ; insulin detemir ; insulin aspart ) with or without ogld . 
 sadrs including major hypoglycaemic events or episodes did not occur in any of the study patients . 
 overall , body weight increased in insulin nave group while there was no change in body weight for insulin user group . though the findings are limited by number of patients , still the trend indicates that insulin analogues can be considered effective and possess a safe profile for treating type 2 diabetes in mumbai , india .","background : the a1chieve , a multicentric ( 28 countries ) , 24-week , non - interventional study evaluated the safety and effectiveness of insulin detemir , biphasic insulin aspart and insulin aspart in people with t2 dm ( n = 66,726 ) in routine clinical care across four continents.materials and methods : data was collected at baseline , at 12 weeks and at 24 weeks . this short communication presents the results for patients enrolled from mumbai , india.results:a total of 2112 patients were enrolled in the study . 
 four different insulin analogue regimens were used in the study . 
 patients had started on or were switched to biphasic insulin aspart ( n = 1561 ) , insulin detemir ( n = 313 ) , insulin aspart ( n = 144 ) , basal insulin plus insulin aspart ( n = 53 ) and other insulin combinations ( n = 41 ) . at baseline 
 glycaemic control was poor for both insulin nave ( mean hba1c : 8.7% ) and insulin user ( mean hba1c : 9.2% ) groups . 
 after 24 weeks of treatment , both the groups showed improvement in hba1c ( insulin nave : 1.4% , insulin users : 1.8% ) . 
 sadrs including major hypoglycaemic events or episodes did not occur in any of the study patients.conclusion:starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia .",pubmed
"over the past decade , many cell types have been evaluated in an effort to find the best source for cardiac regeneration . of these , 
 the most extensively studied have been embryonic stem cells , mesenchymal stem cells , skeletal myoblasts , and bone marrow - derived progenitor cells , while more recent possibilities have included induced pluripotent stem cells and resident cardiac stem cells . 
 embryonic stem cells ( escs ) can be obtained from the inner cell mass of a pre - implantation blastocyst and expanded in vitro almost indefinitely without loss of pluripotency [ 13 ] . when allowed to differentiate as embryoid bodies , escs give rise to most somatic lineages , including cardiovascular lineages [ 48 ] . 
 culture of embryoid bodies with specific growth factors or small molecules can drive differentiation toward cardiovascular phenotypes , thereby enriching the pool of esc - derived cardiac cells available for transplant [ 813 ] . 
 both pluripotent escs and committed esc - derived cardiac cells have been tested in rodent models with encouraging results in terms of engraftment , survival , and improvement in ventricular function [ 1416 ] . 
 however , no clinical trials have been attempted , for concerns of teratoma formation [ 16 , 17 ] , graft - versus - host disease , and bioethics . while improved in vitro differentiation of escs 
 may eliminate the first of these concerns , recent discoveries indicating that adult somatic cells can be reprogrammed to yield esc - like cells may eliminate the second and third of these issues . 
 induced pluripotent stem cells ( ipscs ) can be generated from murine and human adult somatic cells by overexpression of transcription factors critical for maintenance of esc pluripotency . while initial ipsc protocols required retro- or lentiviral transfer of four factors , namely octamer - binding transcription factor 3/4 ( oct 3/4 ) , sry - related hmg - box transcription factor ( sox2 ) , krppel - like factor 4 , and cellular myelocytomatosis oncogene [ 19 , 20 ] or oct 3/4 , sox2 , nanog , and lin28 , subsequent reports have reduced the risk of tumorigenesis introduced during the de - differentiation process by utilizing non - integrating technologies [ 2225 ] or by transferring fewer transcription factors [ 2628 ] while still achieving pluripotency . 
 once established , ipscs differentiate much like escs , yielding functional cardiovascular lineages from embryoid bodies in vitro [ 2832 ] and contributing to normal cardiovascular development from chimeric blastocysts in vivo [ 28 , 31 , 33 ] . in an initial proof - of - principle study , 
 four - factor ipscs successfully restored myocardial structure and function after coronary artery ligation in mice . 
 thus , ipscs may provide an unlimited supply of autologous donor cells , once technological advancements eliminate the risk of teratoma or other tumor formation . 
 mesenchymal stem cells ( mscs ) can be separated from bone marrow and adipose tissues based on their adherence to a culture dish . 
 mscs most readily differentiate into osteoblasts , chondrocytes , and adipocytes [ 35 , 36 ] but can be induced to differentiate into cardiomyocytes under special conditions [ 3739 ] . 
 they can be rapidly expanded in culture , thereby allowing for autologous transplantation , and they may be less immunogenic than other cell populations , thereby allowing for allogenic transplantation [ 40 , 41 ] . 
 but , their propensity to differentiate into non - cardiac tissues ( e.g. , heterotopic ossification ) currently limits their use [ 42 , 43 ] . 
 nonetheless , at least one clinical trial has shown improved ventricular function at 3 and 6  month follow - up after intracoronary infusion of autologous mscs postinfarction . 
 skeletal myoblasts , also known as satellite cells , can be harvested from muscle biopsies , expanded in culture , and autologously reimplanted , albeit on a timescale more appropriate for chronic heart failure therapy than for early postinfarction management . as these cells are further differentiated than escs , 
 they are less prone to teratoma formation ; they are also more resistant to ongoing ischemia and more apt to function in a contractile capacity postengraftment . however 
 , true cardiomyocyte differentiation has not been observed in vivo . as a result , although skeletal myoblasts may be incorporated into an infarct site , their contractions are dyssynchronous due to failure of electromechanical coupling with the surrounding myocardium [ 45 , 46 ] . 
 thus , no significant benefit has been seen in large - scale clinical trials , while a trend toward more frequent arrhythmias has been observed . 
 bone marrow - derived mononuclear cells , including hematopoietic and endothelial progenitor cells , can be mobilized with cytokine therapy or harvested by standard collection techniques . in culture 
 , they can differentiate into cardiomyocytes , endothelial cells , and smooth muscle cells [ 4851 ] , and after transplant , they can supply a broad range of paracrine factors with proangiogenic , positive remodeling , and antiapoptotic properties . a meta - analysis of 18 randomized and non - randomized trials involving 999 patients with acute myocardial infarction or chronic ischemic cardiomyopathy found that intracoronary infusion of adult bone marrow - derived stem cells improved left ventricular ejection fraction by 3.7% , decreased infarct scar size by 5.5% , and 
 lowered left ventricular end - systolic volume by 4.8  ml ( p  <  0.01 for each index ) . among these pooled studies , the largest randomized trial , 
 repair - ami , was particularly notable for not only demonstrating improvement in ventricular function but also for reducing the combined endpoint of death , myocardial infarction , or revascularization at 12  months when compared with placebo . 
 nonetheless , controversy remains regarding the fates of bone marrow - derived cells used for cardiac regeneration . 
 identification of the most safe and efficacious means of stem cell delivery remains an important challenge , both in terms of route and timing of administration . 
 current methods of stem cell delivery include non - invasive mobilization from resident niches ( especially bone marrow ) with cytokine therapy , non - selective intravenous or intracavitary injection , selective intracoronary infusion , and intramyocardial injection from either surgical or percutaneous access . 
 although some intravenously injected bone marrow - derived stem cells do home to the infarct border zone , only a small fraction ( <  5% ) of intravenously injected cells is retained in this intended destination . 
 rather , the majority of intravenously injected cells are trapped by the lungs , liver , kidneys , and spleen [ 56 , 57 ] . 
 more proximal but still non - selective delivery via intracavitary or intra - aortic ( with the distal aorta occluded ) injection marginally improves cardiac uptake [ 56 , 57 ] , suggesting that closer may be better . 
 indeed , intracoronary delivery appears superior to intravenous delivery in man [ 58 , 59 ] , although overall cell retention remains low as rapid washout follows balloon deflation . 
 clinical trials assessing the biodistribution of infused cells have recently been reviewed . in animal studies , 
 intramyocardial delivery yields better retention than intracoronary delivery [ 57 , 62 , 63 ] . 
 but so far , no clinical trial has directly compared these two modalities , although such results should be forthcoming . in theory , 
 intramyocardial delivery may provide more precisely targeted therapy without need for patent coronary arteries , while intracoronary delivery may benefit from operator familiarity and lower cost . 
 since intramyocardial injections carry risk of myocardial perforation and electrical instability shortly after infarction , these two strategies in fact may be complementary , with intracoronary cell delivery provided early and intramyocardial cell delivery provided late . despite the potential importance of correctly timing stem cell administration , few studies have directly addressed this issue . on one hand , stem cell survival may be compromised shortly after infarction by transient microvascular obstruction , recurrent ischemia , or reperfusion injury . on the other hand , 
 stem cell homing to the infarct border zone may be enhanced shortly after injury by transient upregulation of cytokines such as stromal cell - derived factor 1 ( sdf-1 ) , vascular endothelial growth factor ( vegf ) , hepatocyte growth factor ( hgf ) , and monocyte chemotactic protein-3 ( mcp-3 ) [ 6568 ] . in rodents , this latter phenomenon appears to have the upper hand , as the benefits of msc therapy are seen only when administered within 4  days postmyocardial infarction , paralleling myocardial sdf-1 expression . in man , 
 so far , stem cell administration within 1  day of reperfusion therapy has not shown clinical benefit . 
 therefore , the nih cardiovascular cell therapy research network 's time trial will compare an early time point of 3  days postmyocardial infarction to an intermediate time point of 7  days postmyocardial infarction for intracoronary stem cell infusion [ ; http://ccct.sph.uth.tmc.edu/cctrn/public/publichome.aspx ] . 
 this trial will run in parallel with a related study called late - time , which will focus on a late time point of 23  weeks postmyocardial infarction for intracoronary cell infusion . beyond that time point , 
 a recent substudy of the mystar trial found no significant differences in the long - term improvements in infarct size and ventricular function observed after bone marrow mononuclear cell delivery at 36  weeks versus 34  months . 
 treatments designed to improve stem cell homing to diseased myocardium have the potential to markedly enhance both the safety and efficacy of cardiovascular cell therapy . by increasing cardiac retention , stem cell capture by non - cardiac tissues may be reduced , thereby limiting potential side effects . 
 this may allow stem cell dosages to be increased , even as outcomes on a per cell basis are improved . 
 many current strategies to improve cardiovascular homing aim to enhance adhesion protein and cytokine interactions between donor cells and host tissues . 
 ex vivo conditioning of donor cells , for example , by incubation of endothelial progenitor cells with a 2-integrin activating antibody , can improve retention and transcapillary migration at sites of injury , where intracellular adhesion molecule-1 ( icam-1 ) and fibrinogen are upregulated . alternatively , in vivo conditioning of the host vasculature , for example , by infusion of adenosine , can promote cardiac retention of endothelial progenitor cells by upregulating coronary artery expression of p - selectin . 
 increased display of this cell adhesion protein captures endothelial progenitor cells that constitutively express p - selectin glycoprotein ligand-1 [ 73 , 74 ] . 
 adenosine can be safely infused into the human coronary circulation ; thus , it could be an adjuvant to many existing cell therapy protocols . 
 these include master regulators of ischemic responsiveness such as integrin - linked kinase ( ilk ) and hypoxia inducible factor-1 ( hif-1 ) , as well as secreted homing factors such as sdf-1 , mcp-3 , and vegf . in response to hypoxia 
 increased ilk activity then leads to increased expression of sdf-1 and icam-1 through hif-1- and nuclear factor b - mediated signaling . 
 while this beneficial property of ilk has been demonstrated only in a hindlimb ischemia model thus far , its cardiac application is likely not far off . 
 already , it is known that sdf-1 recruits endothelial progenitor cells expressing cxcr4 to injured myocardium , and icam-1 binding to its receptor , 2 integrin , on endothelial progenitor cells is required for engraftment after myocardial infarction . 
 therefore , given the functional improvements seen with ilk gene therapy after coronary artery ligation in rats , utilization of ilk in stem cell therapy would seem a logical step . 
 indeed , encouraging results have been obtained when local myocardial therapy has been combined with stem cell transplantation . 
 downstream of ilk , hif-1 regulates both sdf-1 and its receptor cxcr4 , as well as icam-1 [ 76 , 79 , 80 ] . 
 combining intramyocardial injection of adenovirus - encoded hif-1 with transplantation of skeletal myoblasts results in improved cell engraftment and cell survival after coronary artery ligation in rats . 
 this results in marked improvement in ejection fraction ( by 27% ) at 1  month posttherapy over baseline at 1  week postinfarct . in comparison , 
 monotherapy with hif-1 or myoblasts alone serves only to prevent further decline of the ejection fraction over this interval . 
 further down this ischemic response pathway , both sdf-1 and its receptor cxcr4 have been used successfully as adjuncts to cardiac cell therapy . in mice 
 , endogenous sdf-1 is upregulated for less than 1  week after myocardial infarction [ 6567 ] . 
 re - establishment of sdf-1 expression at later time points can enhance stem cell recruitment and retention when the ischemic border zone might be less hostile to cell engraftment and survival . 
 indeed , intramyocardial delivery of scaffold - bound sdf-1 after coronary artery ligation in rats and mice results in enhanced c - kit+ cell homing and improved ventricular function [ 82 , 83 ] . 
 while it may seem counterintuitive for a stem cell to deliver its own homing factor , considerable benefit has been obtained when genetically modified stem cells recruit additional native and transplanted cells to injured myocardium . 
 for example , improved cell engraftment and survival , as well as improved ventricular function , has been observed after transplantation of sdf-1expressing skeletal myoblasts ( as opposed to unmodified skeletal myoblasts ) at both early and late time points after myocardial infarction in rats [ 84 , 85 ] . 
 similarly , transplantation of sdf-1-expressing cardiac fibroblasts promotes homing of endogenous cd117 + stem cells to injured myocardium . 
 this results in improved ventricular function even without cardiomyocyte regeneration . rather , improved engraftment and differentiation of transplanted sdf-1-expressing mscs ( as opposed to unmodified mscs ) into myofibroblasts , but not true cardiomyocytes , results in improved ventricular function by providing trophic support for cardiomyocyte preservation  but not regeneration  after infarction . 
 similar results are obtained after transplantation of cxcr4-overexpressing mscs , indicating that improved stem cell homing , engraftment , and paracrine activity can be obtained via cell - based gene therapy with both stem cell homing factors and their receptors . other growth factor  chemokine - receptor pairs , which may be utilized to improve outcomes of cardiac cell therapy , include vegfflk-1 , hgfc - met , mcp-3ccr1/2 , and growth - related oncogene-1cxcr1/2 [ 66 , 68 , 8891 ] . of these , the most extensively studied factor is vegf . 
 vegf is transiently upregulated after myocardial infarction   and serves to mobilize endothelial progenitor cells while promoting neovascularization [ 92 , 93 ] . 
 vegf is both induced by sdf-1 and an inducer of sdf-1 's receptor cxcr4 [ 9496 ] . 
 early clinical trials demonstrated that intramyocardial delivery of naked plasmid - encoded vegf gene therapy is safe but results in only marginal clinical benefit [ 97 , 98 ] . 
 subsequent human and large animal studies of combination intramyocardial vegf gene delivery plus stem cell therapy have yielded mixed results [ 99101 ] . 
 meanwhile , vegf - transfected stem cells have consistently outperformed untransfected control cells after coronary artery ligation in rodents [ 102107 ] . and in those studies that have compared cell - based gene therapy to direct gene delivery , vegf - transfected skeletal myoblasts and vegf - transfected mscs 
 have improved ventricular function to a greater degree than either direct adenoviral injection or liposomal plasmid delivery , respectively [ 106 , 107 ] . taken together , these results suggest that cell - based gene therapy may be more effective than either unmodified stem cell therapy or acellular gene therapy alone . the degree 
 despite the structural and functional benefits of stem cell therapy in animal and human studies of acute myocardial infarction and chronic heart failure , limited transplanted cell survival has been observed , especially in the long - term . 
 less than 1% of transplanted cells survive in patient hearts , and even in the idealized scenario of syngeneic neonatal cardiomyocyte intramyocardial injection in healthy rats , only 15% of transplanted cells survive for 12  weeks . nonetheless , recent discoveries demonstrative of the heart 's capacity for regeneration and repair suggest that these challenges are not insurmountable . 
 measurement of c in the dna of individuals born prior to cold war era nuclear testing estimates that nearly half of all cardiomyocytes are exchanged during a normal life span . since c is incorporated equally by the division of resident cardiomyocytes and circulating progenitor cells , carbon dating can not distinguish the cellular origins of such impressive turnover . 
 rather , the phenomenon of cardiac chimerism seen after sex - mismatched heart or bone marrow transplant confirms that extra - cardiac progenitor cells are capable of long - term engraftment and survival in the healthy human heart . 
 thus , the challenge for cardiac cell therapy is to strengthen donor cells for the harsh microenvironment of the infarcted heart . 
 in addition to activating survival pathways in cardiomyocytes , ischemic preconditioning recruits endothelial progenitor cells to serve as mobile reservoirs of cardioprotective cytokines . 
 proponents of cardiac cell therapy now seek to harness this property to enhance donor cell survival . 
 hypoxic preconditioning of mesenchymal stem cells and cardiac progenitor cells prior to transplant improves not only their recruitment , survival , and differentiation at the infarct border zone but also overall ventricular structure and function [ 114 , 115 ] . 
 similar benefits might be obtained by hypoxic preconditioning of endothelial progenitor cells , based on results from an ischemic hindlimb model . 
 this is because , in each of these cell types , hypoxic preconditioning upregulates a diverse array of survival , angiogenic , and migratory proteins , including hif-1 , akt-1 , bcl-2 , ang-1 , vegf and its receptor flk-1 , sdf-1 's receptor cxcr4 , and hgf 's receptor c - met [ 114 , 115 , 117 ] . 
 together , these factors allow donor cells not only to endure but also to heal the injured myocardium . extending this concept , 
 hypoxic preconditioning can be mimicked by genetic modification of donor cells to overexpress hypoxia - responsive survival factors . 
 akt-1 is activated by hypoxia and other stimuli , including cytokines . as a general mediator of survival signaling , 
 blockade of akt-1 activation with the phosphoinositol 3-kinase ( pi3k ) inhibitor wortmannin attenuates the cardioprotective effects of endothelial progenitor cell therapy after ischemic injury in pigs . on the other hand , genetic modification of mscs to overexpress akt-1 results in marked improvement in intracardiac msc retention and survival in rat and pig infarct models [ 120 , 121 ] . in both species , transplantation of akt-1-overexpressing mscs 
 follow - up studies have demonstrated that akt-1-overexpressing mscs release paracrine factors that promote myocardial survival and recovery after infarction [ 122 , 123 ] . by means of functional genomics , 
 secreted frizzled related protein 2 ( sfrp2 ) was identified as the key akt-1msc - expressed paracrine factor mediating myocardial survival and repair after ischemic injury . when sfrp2 is suppressed in akt-1mscs , 
 alternatively , genetic modification of mscs to specifically overexpress sfrp2 promotes their retention and survival within the infarct border zone , as well as facilitates restoration of cardiac structure and function after infarction . 
 these benefits may be attributable to sfrp2-mediated inhibition of wnt binding to the frizzled receptor , which results in accumulation of -catenin and upregulation of antiapoptotic and proangiogenic signaling . 
 other paracrine factors upregulated in akt-1mscs include thymosin 4 ( t4 ) and insulin - like growth factor-1 ( igf-1 ) . 
 t4 is a secreted , g - actin sequestering , 43 amino - acid peptide that stimulates the migration and survival of cardiomyocytes and endothelial cells , as well as the migration and differentiation of epicardial coronary vascular progenitors . after coronary artery ligation in mice , intramyocardial injection of t4 protein 
 exemplary of t4 's importance in cell therapy , shrna - mediated knockdown of t4 in endothelial progenitor cells blocks the cardioprotective actions of these cells after ischemia - reperfusion in mice . 
 in addition to stimulating myocardial growth and contractility , igf-1 activates the pi3k / akt-1 pathway to promote cardiomyocyte survival and proliferation in response to injury [ 128 , 129 ] . 
 igf-1 protein has been combined with cell therapy to improve donor cell survival and differentiation in host myocardium [ 130 , 131 ] . extending these benefits , 
 igf-1 cell - based gene therapy has improved donor cell engraftment and proliferation and enhanced host cell recruitment to ischemic myocardium [ 132 , 133 ] . 
 this has been accompanied by decreased infarct size , increased angiogenesis , and improved ventricular function . 
 molecular studies have attributed igf-1 's therapeutic benefit , in part , to activation of pi3k / akt-1 and bcl-2 and to release of sdf-1 and vegf [ 132 , 133 ] . 
 downstream of igf-1 and pi3k / akt-1 , but not sfrp2 , bcl-2 has been identified as a key antiapoptotic protein with potential utility in cardiac cell therapy . 
 cell lines derived from escs which overexpress bcl-2 self - renew continuously , even in the absence of serum and feeder cells . 
 indeed , mscs , smooth muscle cells , and cardiomyoblasts genetically modified to overexpress bcl-2 have all demonstrated reduced cell loss and improved ventricular function after transplant into ischemic myocardium [ 136138 ] . 
 but while such results may be encouraging , cell - based gene therapy utilizing cell survival factors is not without its challenges , as these proteins may predispose to tumorigenesis . 
 much work still lies ahead to regulate the expression of these factors before their clinical application . 
 however , recent experience has shown that cell therapy is capable of providing significant structural and functional benefit with only minimal cardiomyocyte regeneration . 
 as we have discussed , donor cells can recruit host cells to the site of injury , especially if they are modified to overexpress stem and progenitor cell homing factors . 
 donor cells can secrete paracrine factors to promote host tissue preservation and repair , especially if they are modified to overexpress survival or growth factors [ 52 , 122 , 123 , 133 ] , and donor muscle cells can provide contractile reserve , especially if they are modified to overexpress connexins essential for electromechanical coupling . given these capabilities , one might legitimately ask if the last piece of the cardiac cell therapy puzzle  cardiomyocyte regeneration  even needs to be solved . 
 as we have discussed , recent measurements of c incorporation into the heart indicate that nature provides for cardiac regeneration throughout a normal human lifetime . 
 genetic fate - mapping studies have shown that nearly 20% of cardiomyocytes bordering a murine myocardial infarction are derived from endogenous stem or precursor cell differentiation . 
 this may be accomplished by utilization of more committed cardiac progenitor cells isolated from the adult heart or derived in culture from pluripotent stem cells . 
 the adult heart is home to a side population ( sp ) of stem cells identified by exclusion of the vital dyes hoechst 33342 and rhodamine 123 [ 141 , 142 ] . 
 isolated cardiac sp cells that express stem cell antigen-1 ( sca-1 + ) , but are negative for the endothelial marker cd31 , are capable of differentiation into functional cardiomyocytes after co - culture with adult cardiomyocytes   or treatment with a cocktail of bone morphogenetic protein 2 , fibroblast growth factors 4 and 8 , the wnt antagonist dickkoff-1 , and 5-azacytidine . 
 transplantation of both pre - differentiated and undifferentiated sca-1+/cd31 cells after myocardial infarction in mice results in efficient homing , engraftment , and differentiation into cardiomyocytes and endothelial cells , as well as improved ventricular function [ 144146 ] . 
 a second resident progenitor population comprises c - kit ( also known as cd117 ) positive cells , which are located in small clusters within the adult heart . 
 isolated c - kit+ cells do not differentiate into cardiovascular cell types in culture but showed impressive regenerative potential in some studies after transplantation into the injured rat heart , where they gave rise to cardiomyocytes , endothelial cells , and smooth muscle cells , while also improving ventricular function [ 147150 ] . 
 nonetheless , a rare sca-1+/c - kit+ cardiac cell type that differentiates into cardiomyocytes upon oxytocin treatment has been identified in mice . from a technical standpoint 
 , cardiac progenitor cells can be isolated from mouse hearts and human endomyocardial biopsies by enzymatic digestion to release round cells that form so - called cardiospheres in suspension [ 152 , 153 ] . 
 cardiosphere - derived cells express endothelial and stem cell markers , show contractile activity in culture , and can differentiate into cardiomyocytes , endothelial cells , and smooth muscle cells . 
 transplantation of these cells improves ventricular function after myocardial infarction in mice and swine , due to both direct cardiomyocyte regeneration and indirect paracrine action [ 153155 ] . to test whether these presumably committed cardiac progenitor cells have benefit in man , enrollment in the first clinical trial of  cardiosphere - derived autologous stem cells to reverse ventricular dysfunction 
 an alternative to laborious isolation of committed cardiac progenitor cells from the adult heart is differentiation of committed cardiac progenitor cells from embryonic stem cells , induced pluripotent stem cells , or other multipotent cellular resources such as adipose tissue - derived mscs . 
 after study of many different growth factor cocktails , a growing consensus now agrees that modulation of bone morphogenetic protein ( bmp ) signaling may be the key to reliable ex vivo cardiomyocyte differentiation . 
 however , consensus is lacking with regard to identification of the critical bmp agonists and antagonists , as well as their appropriate dosing . 
 treatment of murine escs or mscs with bmp2 in the presence of a second growth factor such as transforming growth factor-1 , fibroblast growth factor 2 or 4 , or leukemia inhibitory factor promotes cardiomyocyte differentiation and improves cardiac structure and function following transplantation into infarcted myocardium [ 8 , 9 , 11 , 157 ] . 
 however , it is also known that the endogenous bmp antagonist noggin and the synthetic bmp inhibitor dorsomorphin also increase cardiomyocyte differentiation [ 1012 ] , in apparent contrast to studies that blocked the inductive properties of bmp2 and bmp4 with noggin [ 8 , 13 ] . 
 a unifying hypothesis for these observations suggests that bmp signaling is necessary for at least two steps in cardiomyocyte development : mesodermal induction and cardiomyocyte differentiation [ 158 , 159 ] , between which transient block of bmp signaling is essential . 
 thus , the optimal timing and dosage of bmp agonist and antagonist treatments may be critical . 
 finally , recent evidence that adult cardiomyocytes may be induced to proliferate could provide a complementary strategy for cardiac regeneration . 
 ongoing research at the bench and the bedside will surely continue to perfect these pieces of the cardiac cell therapy puzzle : comparing sources of donor cells , testing methods of cell delivery , augmenting myocardial homing , bolstering cell survival , and promoting cardiomyocyte differentiation , and while much more work needs to be done , we have confidence that stem cell therapies in conjunction with current treatment modalities will ultimately reduce the mortality and improve the quality of life of patients with cardiovascular disease .","acute ischemic injury and chronic cardiomyopathies can cause irreversible loss of cardiac tissue leading to heart failure . 
 cellular therapy offers a new paradigm for treatment of heart disease . 
 stem cell therapies in animal models show that transplantation of various cell preparations improves ventricular function after injury . 
 the first clinical trials in patients produced some encouraging results , despite limited evidence for the long - term survival of transplanted cells . 
 ongoing research at the bench and the bedside aims to compare sources of donor cells , test methods of cell delivery , improve myocardial homing , bolster cell survival , and promote cardiomyocyte differentiation . 
 this article reviews progress toward these goals .",pubmed
"the mineral polyhalite ( chemical formula [ k2ca2mg(so4)4.2h2o ] ) commonly occurs in sedimentary evaporite successions and in rare cases as a sublimate in volcanic successions ( e.g. vesuvius , campania , italy ) ( anthony et  al . , 1996 ) . 
 polyhalite was first described from the salt mine bad ischl - perneck in austria ( province upper austria ) by the german chemist stromeyer ( 1818 ) and has since been known from many salt deposits ( e.g. warren , 2006 ) . in evaporites , 
 polyhalite is commonly associated with halite , anhydrite , glauberite , carnallite , kieserite , langbeinite , vanthoffite , chlorides and sulphides . 
 the crystal structure was first determined in samples from altaussee in austria ( schlatti et  al . , 1970 ) and later confirmed ( bindi , 2005 ) . 
 polyhalite can be synthesised under laboratory conditions by a reaction  of gypsum with appropriate solutions in the ternary system k2so4mgso4h2o at temperatures above 70  c ( freyer and voigt , 2003 ) . at lower temperatures 
 ( 2008 ) found under laboratory conditions the dehydration temperature of polyhalite to start at 255  c to a maximum of 343  c . 
 substitution of divalent cations by other elements like zn , cu , cu , ni significantly changes dehydration reactions to higher or lower temperatures ( wollmann et  al . , 2008 ) . 
 further chemical data on the conditions of formation and stability of polyhalite were presented inter alia by krupp ( 2005 ) and wollmann et  al . 
 polyhalite most commonly forms early - diagenetically ( e.g. holser , 1966 ; busson and perthuisot , 1977 ; pierre , 1985 ; khn and mller - schmitz , 1987 ) by brine - back reactions of k  mg 
 so4 brine with earlier formed gypsum , anhydrite or glauberite ( e.g. braitsch , 1962 ; schauberger , 1986 ; warren , 2006 ) after a prolonged contact ( over months or years ) of the brine with the precursor rock ( hardie , 1984 ) . 
 for example , all alpine polyhalites are of secondary origin ( leitner , 2011 ; leitner et  al . 
 a formation of polyhalite in marginal marine shallow water sedimentary environments is only suspected ( hryniv et  al . , 2007 ) . 
 polyhalite is stable between room temperature ( 025  c ) and 255343  c ( wollmann et  al . , 2008 ) or 285  c ( fischer et  al . , 1996 ) and 
 , therefore , within nearly the whole temperature range , the haselgebirge sulphates have experienced ( 200300  c , table  1 , see below ) . 
 polyhalite contains potassium in stochiometric amounts rendering the mineral interesting for absolute age dating  essential for deciphering the geological history of a region . 
 since polyhalite was considered to be rare in most salt deposits , published ages are scarce and dating was carried out with other methods . in austria and southern germany , 
 ( in the following named as haselgebirge mlange ) , a tectonically mixed evaporite succession at the stratigraphic base of the northern calcareous alps ( in the following abbreviated as nca ) ( fig .  1 ) . there , polyhalite is a minor , but widespread and important constituent and is accessible in several salt mines including the type locality of bad ischl , hallstatt , berchtesgaden  drrnberg and hall in tirol ( schauberger , 1986 ) . at the localities berchtesgaden and altaussee , polyhalite mainly occurs intergrown with anhydrite or as vein fillings and 
 , in press a , b ) , for example intergrown with fine - grained anhydrite , in veins , in nodules , and recrystallised in foliated polyhalite rocks along shear zones . 
 grgey ( 1910 ) distinguished three different types of polyhalite occurrences : ( 1 ) fine - fibrous , acicular or lamellar , yellowish - red to reddish polyhalite layers , which form 120  cm thick layers within the haselgebirge , often with an extension of several square metres ; ( 2 ) compact fine - to - medium - grained yellowish - brown to dark red coloured polyhalite layers associated with anhydrite and ( 3 ) fibrous - acicular or granular polyhalite areas , which form seams around anhydrite crystals ( like in hall in tirol ) . 
 schindl - neumayer ( 1984 ) examined the grain structures of anhydrites , polyhalites and halites of the alpine salt deposits . she  distinguished five different polyhalite types : ( 1 ) fine - felty polyhalite aggregates , ( 2 ) fine - grained polyhalite groundmasses , ( 3 ) polyhalite  twinnings in a polyhalite groundmass and in anhydrite porphyroblasts , ( 4 ) polyhalites as felty conversion seams round anhydrite and ( 5 ) polyhalite and clay / mudrock . the most recent work on polyhalite fabrics from altaussee and berchtesgaden 
 ( in press b ) distinguished three main polyhalite microfabric types : ( 1 ) fibrous polyhalite in veins , ( 2 ) porphyroblasts of polyhalite in anhydrite with subdivisions ( 2a ) ( round polyhalite blasts ) and ( 2b ) ( fibrous polyhalite blasts ) and ( 3 ) dense polyhalite comprising corroded large anhydrite grains with subdivisions ( 3a ) ( fine - grained , mylonitic polyhalite rock ) , ( 3b ) ( non - foliated polyhalite rock with local zones ) and ( 3c ) ( non - foliated polyhalite rock ) . 
 ( in press a ) described a diagenetic reaction after halite hopper crystals , which forms anhydrite and polyhalite and added another polyhalite microfabric type : polyhalite in pseudomorphic anhydrite cubes after halite hopper crystals and polyhalite from within the retaining shape of deformed halite hopper shaped cubes . 
 ( in press b ) demonstrated , on a limited number of examples , that polyhalite occurs in polyhalite rocks and as vein filling with various fabric types , which also gave distinct ar / ar ages . 
 one of the most important results of these papers is that polyhalites of the alpine haselgebirge formed at ca . 
 235210  ma ( leitner et  al . , in press b ) , ca . 
 2025  ma after the sedimentation of the haselgebirge evaporites ( leitner , 2011 ) and with a maximum estimated overburden of approximately 800  m ( e.g. rantitsch and russegger , 2005 ) . with this study 
 , we further explore the variety of microfabric types of mono- and polymineralic polyhalite rocks and vein and cavity fills from the hallstatt mine as we found that the variety is significantly larger than elsewhere in the nca . 
 the hallstatt salt mine is within the upper permian  lower triassic haselgebirge fm .. the classic division within the nca defines the bajuvaric , tirolic and juvavic nappe complexes ( tollmann , 1985 , 1987 and references therein ; mandl , 2000 ) ( fig .  1 ) . 
 the permian to lower triassic haselgebirge occurs mainly in juvavic units of the central and eastern nca ( schauberger , 1986 ; leitner and neubauer , 2011 and references therein ) and subordinately in tirolic units . 
 the age of formations in the nca ranges from late carboniferous ( ? ) or early permian to eocene . during the middle and late triassic , 
 the upper juvavic nappes comprise mostly reefs and deposits next to reefs , the tirolic and bajuvaric nappes comprise lagoonal facies types and subordinate reefs . 
 the lower juvavic nappe unit represents solely an outer shelf or a deep - sea facies type ( mandl , 2000 ) . 
 sedimentation of the lower juvavic unit occurred in basins with basinal limestones ( ptschen limestone ) and on intrabasinal ridges , with reduced sedimentary thickness ( the pelagic hallstatt limestone ) . 
 the ridges were suggested to relate to salt diapirism in triassic times ( mandl , 1982 , 2000 ; plchinger , 1984 ) . 
 the westernmost part of the expanding triassic tethys ocean is called hallstatt - meliata ocean , which comprises rare deep - sea ( ophiolitic ? ) sequences in the eastern parts of the nca ( faupl and wagreich , 2000 ; neubauer et  al . , 2000 and references therein ) . 
 most authors interpret the hallstatt limestone as an outer shelf ( tollmann , 1985 , 1987 ; mandl , 2000 ) . 
 others propose a position of the hallstatt - meliata relics between the upper juvavic and tirolic units ( schweigl and neubauer , 1997 ) . 
 the permian / middle triassic to lower upper triassic rift stage and passive margin formation was associated with widespread synsedimentary and diagenetic pb 
 the hallstatt - meliata ocean was being closed during the late jurassic ( dallmeyer et  al . , 2008 and references therein ) . 
 coevally , the sea floor dropped and reached maximum water depths with the formation of radiolarites . 
 gravitational sliding is reported from different places ( e.g. mandl , 1982 ; plchinger , 1990 ) , and this concept was developed until recent years ( missoni and gawlick , 2011 and references therein ) . 
 these authors argue for a late jurassic age of shortening . during early cretaceous , nappe 
 thrusting prograded from south to north , respectively from ese to wnw ( ratschbacher , 1986 ; linzer et  al . 
 , 1995 ; mandl , 2000 ; neubauer et  al . , 2000 ) . 
 the mechanism and the time of emplacement of the juvavic units are still a matter of controversy . 
 the classic hypothesis assumes that both juvavic nappes took their position during the eo - alpine deformational event ( late early to early late cretaceous ) by means of thrust tectonics ( kober , 1955 ; pichler , 1963 ; schweigl and neubauer , 1997 ; mandl , 2000 ) . 
 a further model explains the emplacement of all juvavic units by gravity sliding since late jurassic times , as haselgebirge clasts of various sizes have been found in the upper jurassic oberalm and lower cretaceous rossfeld fms . 
 this concept was extended to large mountain - like blocks also explained by emplacement by simple gravity sliding ( gawlick and lein , 2000 ; missoni and gawlick , 2011 and references therein ) . 
 frank and schlager ( 2006 ) propose the emplacement as a consequence of late jurassic strike - slip movements related to the opening of the penninic ocean . 
 schorn and neubauer ( 2011 ) proposed an early cretaceous emplacement of the haselgebirge - bearing nappe over tirolic units . in the eocene , the second paroxysm of the alpine orogeny occurred , when continental basement slices (  middle penninic  ) and parts of the north penninic ocean (  rhenodanubian flysch  ) 
 adriatic microcontinent ( faupl and wagreich , 2000 ; linzer et  al . , 2002 and references therein ) . 
 the present nca were partly detached from their austroalpine basement ( e.g. greywacke zone ) and thrusted over the rhenodanubian flysch and helvetic domain resulting in a wide thin - skinned tectonic nappe complex ( linzer et  al . 
 , 1995 ; mandl , 2000 ; neubauer et  al . , 2000 ) . the familiar rocksalt deposits , which are all located in the interior of the nca mostly within the lower juvavic unit , were considered to be only slightly affected by these cenozoic deformation stages , since the detachment of the nca domain occurred beneath the lowermost unit , the bajuvaric nappe ( fig .  1 ) . 
 deformation of upper cretaceous to eocene gosau basins deposited on uppermost nappes ( tirolic and juvavic nappes ) suggests significant deformation in late eocene to early miocene ( linzer et  al . , 1995 , 1997 ; 
 the salt body of hallstatt ( figs .  2 and 3 ) , which is part of the hallstatt nappe north of the echern valley ( i.a . 
 habermller , 2005 ) , was described recently by leitner and neubauer ( 2011 ) and we follow their description . 
 w direction and is around 600  m wide ( maps and sections : schauberger , 1955 ; schffer , 1982 ; scheidleder et  al . , 2001 ; habermller , 2005 and references therein ) . 
 the highest point is 1350  m above sea level , the lowest erbstollen level is at around 500  m above sea level ( section of schauberger in habermller , 2005 ) . 
 the salt body suitable for mining diminishes with depth , mostly because of incorporated country rocks . 
 the base has not yet been reached at 100  m below the level of lake hallstatt ( pers . 
 is around 55  wt ( weight ) % ( schauberger , 1931 , 1949 , 1955 ) . 
 the mining started in celtic times , as indicated by a bronze - age staircase found in the mine , which was dated accurately by dendrochronology to 1344 bc ( grabner et  al . , 2007 ) . 
 the sedimentary succession of the lower juvavic unit is nearly complete and comprises , beside the basal upper permian  lower triassic haselgebirge fm . 
 contains blocks of meta - basalts ( melaphyre ) ( e.g. schauberger , 1960b ; zirkl , 1957 and references therein ) within the  buntes salztongebirge 
 ( = colored salt - bearing claystone ) ( pak and schauberger , 1981 ; schauberger , 1986 ) which are also assumed to be of upper permian age ( e.g. klaus and pak , 1974 ) , but no reliable dating was performed . based on palynological evidence and a sulphur isotopic signature , the sediments of the haselgebirge were deposited during late permian and early triassic times ( klaus , 1955 , 1965 ; klaus and pak , 1974 ; pak , 1974 , 1978 , 1981 ; pak and schauberger , 1981 ; gtzinger and pak , 1983 ; sptl , 1987 , 1988a , 1989 ; sptl and pak , 1996 ) . 
 sptl ( 1988a ) carried out detailed studies on relict sedimentary structures in the drrnberg and hallstatt mines , and interpreted the evaporites to have formed in a sabkha - like environment , situated in an aborted rift of the tethys ocean ( sptl , 1989 ) . 
 pak and schauberger ( 1981 ) postulated , based on s - isotopy , that the rock types  rotsalzgebirge 
 ( = red salt rock ) and  grntongebirge  ( = green salt - bearing clay ) ( following the nomenclature of schauberger , 1986 ) of an older phase ( schauberger , 1986 ) were deposited during late permian ( probably until earliest triassic times ) , while the  stinkdolomitisch / anhydritische grausalzgebirge  ( bituminous dolomitic / anhydritic grey salt rock ) and probably also the  bunte salztongebirge  ( = colored salt - bearing claystone ) of a younger phase ( schauberger , 1986 ) were sedimented during the early triassic and earliest anisian times . for hallstatt , 
 pak and schauberger ( 1981 ) and pak ( 1981 ) assumed a continuous saliniferous sedimentation from the upper permian to the lower triassic / anisian boundary . 
 ( krystyn , 2008 ) are often associated with jurassic rocks ( sandlingalm fm . ) . 
 , are covered by an undeformed lid of upper jurassic rocks ( gawlick and schlagintweit , 2006 ; suzuki and gawlick , 2009 ) . a sedimentary contact between the sandlingalm fm . and the overlying plassenkalk fm . 
 is missing ( suzuki and gawlick , 2009 ) . on a map view of hallstatt , the lower juvavic unit , the rocksalt and the jurassic cover 
 are found within the area of the upper juvavic nappe ( fig .  2 ) . 
 based on vitrinite reflectance studies , maximum temperatures for the central sectors of the nca in the surroundings of the hallstatt mine are estimated between 160 and 180  c , and with 200300  c in the surroundings ( see table  1 , gtzinger and grum , 1992 ; gawlick et  al . 
 , 1994 ; wiesheu and grundmann , 1994 ; wiesheu , 1997 ; sptl and hasenhttl , 1998 and references therein ) . 
 proposed by schauberger ( 1986 ) , polyhalite is mainly part of the  rotsalzgebirge  (  red salt rock  ) , which is composed of a mixture of reddish - grey salt , anhydrite , polyhalite , glauberite , na  mg sulphates ( like bldite / simonyit ) , red and black claystones and grey - brown sandstones . 
 there , polyhalite ( and bldite [ na2mg(so4)2.4h2o ] ) are very common but in low proportions ( schauberger , 1986 ) . 
 but polyhalite is also reported from the rock types  buntes salztongebirge  (  colored salt - bearing clay  ) ,  grausalzgebirge  (  grey salt - bearing claystone  ) and  rot - grnes bergangsgebirge  (  reddish - green salt rock  ) ( nomenclature after schauberger , 1986 ) . 
 most of the described 27 samples were taken from the lower and middle levels of the hallstatt salt mine : kaiser franz josef , nusko , kaiserin elisabeth , kaiserin maria theresia and kaiserin christina . 
 for an overview of the sample locations see supplementary material , table  sm 1 and fig .  2 , fig .  3 , fig .  4 . 
 electron microprobe analysis empa on polyhalite , anhydrite , bassanite and bldite was carried out on a jeol electron microprobe ( jxa-8600 ) at the department geography and geology , university of salzburg , using a wavelength dispersive system . 
 because sulphates are unstable under the electron beam , we used an acceleration voltage of 15  kv and a low sample current of 20  na . 
 natural and synthetic mineral standards were used to calibrate the microprobe and raw data was reduced using standard zaf correction . 
 the detection limits ( 3 ) for the elements na , k , mg , mn , ca , fe and s are 0.025  wt ( weight ) % and 0.045  wt% for sr and ba . after being covered with carbon 
 the thin section blocks of the three samples ht-8b , ht-10 and ht-16 were examined by scanning electron microscopy ( sem ) and qualitative energy dispersive x - ray analysis ( edx ) with the leica stereoscan 430 equipped with rntec - edwin 98 winshell / wintools at the department geography and geology , university of salzburg . 
 the x - ray diffraction analysis was carried out with the automatic diffractometer siemens d500 using a semi - automatic divergence slit and a graphite - based secondary monochromator using cu k-radiation ( 40  kv , 35  ma ) . 
 the measurements were performed in the step - scan mode in an angular range of 375 2 , step length 0.03 with a measurement time of 12  s per step . 
 the program eva 3.0 ( bruker axs ) and the pdf-2-database ( international centre for diffraction data ) were used for evaluation of the x - ray diffraction results . 
 in the hallstatt mine , polyhalite rocks occur in tectonic lenses within the protocataclasite to protomylonite matrix . 
 thin section analysis of hallstatt samples reveals many different fabric types ( table  2 , supplementary material , table  sm 1 ) . very often , one rock sample exposes more than one polyhalite microfabric type . 
 ( in press b ) , although their polyhalite rock type 2a ( porphyroblasts of polyhalite in anhydrite , round blasts ) only occurs subordinately . in the following , we distinguish between mono- and polymineralic polyhalite rocks , and furthermore , vein and cavity - filling polyhalite ( table  2 , supplementary material , table  sm 1 ) . the thin sections of monomineralic polyhalite mylonites ( fig .  5 , fig . 
 6a and b ) show the following main features and are often characterised by large grain size variations ( from 0.05 to 0.5  mm long ) : ( 1 ) fine - grained ( 0.05    0.1  mm on average ) , well - recrystallised elongated ( long axis parallel to the foliation ) polyhalite grains with a shape preferred orientation ( spo ) ; ( 2 ) fibrous polyhalite pressure fringes ( 0.05    0.5  mm on average ) around large , rigid subeuhedral opaque mineral grains ( 0.5    0.5  mm up to maximally 20    20  mm ) within a fine - grained mylonitic fabric ( average grain sizes of 0.02    0.1  mm ) ( fig . 
 6a ) and ( 3 ) fine - grained ( 0.03    0.1  mm on average ) grain boundary migration ( gbm ) with amoeboid grain boundaries ( fig . 
 crenulation cleavage usually shows a two - phase fabric indicating two different deformation stages and these structures can be found in fine - grained polyhalite rocks ( fig . 
 6c and d and 7a ) of mono- und polymineralic polyhalite rocks show a secondary overprint by a second deformation stage . 
 such features include : ( 1 ) a refolded foliation in a fine - to - medium - grained ( ca . 
 0.1    0.3  mm long ) polyhalite rock with a crenulation cleavage ; ( 2 ) microfolds folding the foliation s1 of polyhalite grains ( fig . 
 6c ) and ( 3 ) a crenulation cleavage and partly folded clay - rich layers , coarse - grained ( 0.1    0.2  mm long on average ) polygonal anhydrite fabric and fine - grained ( 0.02    0.07  mm long on average ) polyhalite ( fig . 
 5b ) from hallstatt show various effects of recrystallisation , respectively annealing ( fig . 
 7a  c ) : fine - grained ( 0.02    0.1  mm long on average ) , fibrous polyhalite grains partly show crenulation cleavage (= microfabric type a-2 ) and overgrow coarse - grained ( 0.5    1.54  mm long on average ) polyhalite crystals typical for microfabric type a-3 ( fig . 
 coarse - grained , recrystallised polyhalite aggregates ( 0.7    0.9  mm up to 1    2.5  mm long ; single fibres on average 0.2    0.6  mm long ) are in contact with coarse - grained anhydrite ( 0.1    0.2  mm on average ) ( fig . 
 7b ) and well - recrystallised polyhalite grains still preserve a shape preferred orientation ( spo ) ( fig . 
 5c ) a number of effects related to pressure solution can be observed ( fig . 
 7d  f ) : folded dark  clay - rich layers and very fine - grained polyhalite grains ( 0.02    0.05  mm on average ) , showing pressure solution and grain boundary migration ( = gbm ) . 
 pressure solution foliation of very fine - grained ( 0.01    0.05  mm on average ) polyhalite grains surrounding coarse - grained ( 0.1    0.1/0.2  mm on average ) polyhalite crystals ( fig . 
 7d ) . pressure solution of extremely fine - grained ( 0.001    0.1  mm representing an ultramylonite ) polyhalites and more coarse - grained polyhalite grains ( 0.05    0.1/0.2  mm long on average ) of sample ht-16 ; the layer in the center represents a mylonite ( microfabric type a-1 ) with a  polyhalite fish  ( 0.05    0.3  mm ) ( fig . 
 in other cases , pressure solution of extremely fine - grained ( 0.01    0.1  mm on average ) polyhalites around a coarse - grained polyhalite twin ( 0.2    0.4  mm long ) is observed ( fig . 
 cataclastic fabrics of angular fine and coarse ( 0.0050.08  mm ) polyhalite grains with serrated grain boundaries and many grain size classes ( fig . 
 8a ) of microfabric type a-5 are only observed in sample ht-6 ( fig . 
 generally , the presence of other mineral phases inhibits the mineral growths , resulting in large grain size variations ( if no phases are removed ) . 
 some samples from hallstatt show fine - grained polyhalite ( 0.003/0.004  mm thick and up to 0.2  mm long ) with shape preferred orientation ( spo ) overgrowing older , large anhydrite porphyroblasts ( fig . 
 the anhydrite porphyroblasts ( 0.4    0.6  mm ) show a sieve structure with polyhalite inclusions ( with no shaped preferred orientation ) and crystallographic preferred orientation ( cpo ) within the anhydrite grain . 
 this indicates that the fabric formed prior to the growth of anhydrite porphyroblasts did not show an spo or cpo ( pre - tectonic porphyroblast ) ( fig . 
 folded clay - rich layers and an extremely fine - grained ( 0.001    0.01  mm ) polyhalite - rich matrix are observed in some polyhalite rocks . 
 fabric type b-1 shows a typically mylonitic fabric of polyhalite layers interbedded with clay , carbonate or anhydrite layers . 
 8c  e ) of polymineralic mylonites show a number of distinct features , which include inter alia coarse - grained ( 0.1    0.4  mm on average ) anhydrite layers with grain boundary migration ( gbm ) , which are embedded in fine - grained ( 0.003  mm thick and up to 0.2  mm long ) mylonitic polyhalite layers of microfabric type a-1 ( whole sample : microfabric type b-1 ) ( fig . 
 fine- ( 0.1    0.15/0.3  mm on average ) and coarse - grained ( 0.2    0.5  mm on average ) polyhalite layers of another sample show a foliation and amoeboid grain boundaries as well as a pressure solution , probably aligned to microfabric type b-1 ( polymineralic mylonite ) ( fig . 
 clay - rich layers are embedded in a fine - grained ( 0.01    0.05  mm on average ) mylonitic polyhalite fabric with shape preferred orientation ( spo ) , together constituting microfabric type b-1 ( fig . 
 reaction fabrics are commonly characterised by the control of the orientation of one mineral by another . 
 they might have been formed by exsolution , ( re-)crystallisation , parallel growth or replacement ( lauder , 1961 ) . 
 the yellow - coloured mineral aggregates are composed of bldite [ na2mg(so4)2.4h2o ] , the red ones of polyhalite and large , red anhydrite ( fig . 
 fine - grained ( 0.05    0.1/0.2  mm on average ) , fibrous polyhalites ( fig . 
 8f ) probably grew in 0.5    1.2  mm large areas as a reaction product between coarse - grained bldite and a large ( 2    2.5  mm ) anhydrite ( single crystal ) , indicating static p 
 bldite constitutes large areas ( 17    20  mm ) , with single grains measuring between 1    1  mm and up to 4    8  mm . 
 mainly polyhalite or anhydrite , subordinate clay and carbonate minerals , which are growing into cavities , represent cavity infilling . 
 commonly , large polyhalite fibres ( fibrous aggregates : 0.1/0.5    2.5/3  mm ; single fibres are only 0.020.1  mm thick ) grew into cavities , which were filled afterwards by halite ( see also figs . 
 in other cases , a cavity filled with halite and coarse - grained ( 0.1    0.2/0.4  mm on average ) , polygonal anhydrite grains occurs in a matrix of fine - grained ( 0.03/0.04    0.2  mm ) , folded polyhalite grains ( fig . 
 furthermore , polyhalite and anhydrite ( 0.05    0.1/0.2  mm on average ) grains grew together into halite - filled cavities . 
 9d ) and is consistent with polyhalite type 1 ( fibrous polyhalite in veins ) described by leitner et  al . 
 the vein - filling polyhalite samples of altaussee and berchtesgaden described by leitner ( 2011 ) and leitner et  al . 
 sample hst-21 does not show a well - developed median suture line , which means that the large ( 0.2    1  mm on average , up to 0.2    1.5  mm ) , in part sheaf - shaped polyhalite fibres ( following the description of leitner , 2011 ) , which are orientated about perpendicular to the vein walls of clay and mudstone , have grown in only one direction ( fig . 
 three fine - grained samples ( ht-8b , ht-10 and ht-16 ) were examined by sem and qualitative edx measurements in order to check the nature of fine - grained minerals . 
 polyhalite ultramylonite  between coarse - grained magnesite breccia fragments , with halite cavity - filling and fine - grained dark carbonate or clay schlieren / layers ( fig . 
 sample ht-8b represents a recrystallised polyhalite rock with many questionable clay schlieren and layers and salt cavity filling . 
 rock sample ht-10 contains polyhalite mylonites , rare anhydrite , questionable fine - grained polyhalite  clay layers / schlieren and halite cavity filling . 
 similar to samples ht-8b and ht-16 the dark , fine - grained schlieren of sample ht-10 ( fig . 
 10b ) contain a polyhalite  clay mineral mixture composed of s , mg , k , ca , si , al and fe . 
 the elements si and al argue particularly for the presence of a clay mineral ( fig . 
 measurements the light - grey breccia fragments of sample ht-16 are composed of the elements mg , c , o and traces of fe , constituting the mineral magnesite ( fig . 
 magnesite grains are isometric with an average size of 0.005  mm ( fig . 
 furthermore , the sem - measurements clearly show that the fine - grained polyhalite fibres and coarse - grained polyhalites and anhydrites ( at least of sample ht-16 ) partly overgrow halite ( microfabric type c-1 ) indicating an older relative age compared to halite ( fig . 
 electron microprobe ( emp ) measurements were performed on some polyhalite types ( samples ht-2 , ht-3 , ht-12 , ht-16 , ht-27 ( 2 ) and ht-30 ) , associated anhydrite and on bldite in order to characterize compositional variations during various stages of mineral growth ( see table  3 ) . 
 polyhalite grains of the microfabric types a-1 , a-4 , b and c-1 show only minor variation in the polyhalite composition ( see also supplementary material , table  sm 2 ) . only the sro abundances exhibit some minor variation which is usually between 0.05 and 0.28  wt ( weight ) % . in samples 
 ht-12 and ht-16 , the sro content is slightly elevated ranging from 0.28 to 0.74  wt% . 
 the na2o content ranges between 0.025 and 0.48  wt% , and is increased up to 1.80 respectively to 4.1  wt% in sample ht-12 ( although a measurement error is conceivable as well ) . a reaction fabric of bldite 
 , a polyhalite seam and associated anhydrite is observed in sample ht-12 , representing a metamorphic reaction fabric ( fig . 
 the presence of bldite was found by both emp analysis ( table  3 ) and x - ray diffraction ( supplementary material , fig . 
 bldite of the haselgebirge fm . may occur primarily as nodules or more common intergrown with the mineral lweite ( schauberger , 1986 ) , which has a nearly identical chemical composition and is stable at higher temperatures . in sample ht-3 , fibrous bassanite ( caso4.0.5h2o ) 
 partial exsolution from an unidentified small mineral of round shape within polyhalite is observed in sample ht-16 ( fig . 
 microprobe measurements demonstrate a nearly polyhalite - like chemistry ( supplementary material , fig . 
 in this chapter , we first highlight the significance of various polyhalite fabrics and try to establish the timing of tectonic events by comparison with dated polyhalite fabrics from other alpine salt deposits ( leitner et  al . 
 , in press a , b ) as unfortunately no ar / ar ages are available from hallstatt . then , we compare these fabrics with such from calcite and quartz , and , finally , we discuss the regional implications of the new observations . finally , we debate the formation conditions of some of the observed particular mineral compositions , which in part allow us to deduce formation temperatures and the secondary origin of polyhalite 
 . the examined polyhalite samples of hallstatt show a wide variety of microfabrics and many of them , the mylonitic ones , resemble closely to such observed from rock - forming constituents of continental crust , like quartz , feldspar and calcite . 
 similarly , the microfabrics may have resulted from a wide range of external conditions like temperature , strain rate and differential stresses ( passchier and trouw , 2005 ) . 
 the aim is now also to deduce conditions of formations of these microfabrics within the temperature stability field of polyhalite . 
 we combine here all observations and show models for the suggested mode of origin in fig . 
 the vein - filling , fibrous polyhalite samples of microfabric type c-2 ( hst-21 ) and probably some of microfabric type c-1 ( e.g. ht-2 , ht-3 ) are expected to show the oldest ages . 
 these rocks can be compared with the fibrous polyhalites in veins ( polyhalite rock type 1 ) of leitner et  al . 
 ar / ar age dating of similar types mainly yielded ages between 208 and 235  ma . 
 thus , 2025 million years after the deposition of the haselgebirge fm . during the main extensional stages of the nca and a maximum overburden of 800  m ( rantitsch and russegger , 2005 ) migrating brines of likely hydrothermal origin were reacting with the haselgebirge sulphates to form polyhalite . following the interpretation of leitner 
 ( in press b ) and leitner ( 2011 ) , these circulating brines and heating were probably generated by the opening of the meliata ocean ( rifting ) , which lasted until 225  ma ago . during recrystallisation , temperatures of 6090  c might have been reached as authigenic quartz originating from a transformation of mudrock minerals indicates ( sptl et  al . 
 , 1998a , b ) . some of the fibrous polyhalites of altaussee and berchtesgaden yield ar / ar age steps of 204209  ma , which can probably be explained by a separate polyhalite growing event during norian times , when subsidence took place in the sedimentation area ( and the 1.5  km thick dachstein limestone was deposited over the haselgebirge fm . 
 the veins might have formed due to extension during middle triassic times . during evaporation of seawater the polyhalite fibres probably formed by back reactions of the k  mg  cl 
 so4 brine ( the k and mg might originate from pre - existing clay minerals or clay stones ) with earlier formed gypsum , anhydrite or glauberite ( inter alia warren , 2006 ) on the vein walls ( fig . 
 the polyhalite mylonites ( e.g. samples ht-1 , ht-11 , ht-30 ) of microfabric types a-1 and b-1 were most likely formed along shear zones . these foliated and lineated rocks ( fig . 
 5a and b ) can be characterised by their comparatively small grain sizes ( varying from very fine - grained 
 ultramylonitic  to medium - grained fabrics ) and distinct penetrative or closely spaced and planar foliation with a shape preferred orientation ( spo ) . 
 porphyroclasts , derived from a relatively  rigid  material , are quite common for mylonites , which usually indicate one deformation stage . 
 so4 brines infiltrated ( upwards and downwards ) the evaporite rock along opened faults and joints and polyhalite - forming brine - back reactions took place . 
 the mylonites were formed at high strain rates due to intracrystalline deformation , may have developed from a more coarse - grained precursor metamorphic rock and probably suffered several stages of deformation . the low crystallographic preferred orientation ( cpo ) of polyhalite may indicate some recrystallisation postdating mylonitisation . 
 furthermore , the mylonites are partly overprinted by recrystallised polyhalite grains ( microfabric type a-3 ) , which probably indicate a static growth . 
 these polyhalite mylonites can probably be compared to polyhalite rock type 3a ( fine - grained , mylonitic polyhalite rock ) and 3b ( non - foliated polyhalite rock with local shear zones ) described by leitner et  al . 
 ar / ar age dating shows two stages of growth of fine - grained polyhalite 
 152158  ma , the later between 105 and 118  ma ( leitner et  al . 
 ages between 152 and 158  ma may be related to the closure of the meliata ocean and the initiation of alpine nappe stacking and accompanying ductile deformation ( leitner et  al . 
 furthermore , these ages correlate well with the 145154  ma old authigenic feldspar reported from moosegg quarry by sptl et  al . 
 ( 1996 , 1998a , b ) . a second age of 105118  ma of fine - grained polyhalites in shear zones is related to cretaceous - aged deformation ( low - grade metamorphic ductile overprint during eo - alpine nappe stacking ) and corresponds well to ar / ar white mica ages of 114120  ma reported from the southern margin of the nca ( frank and schlager , 2006 ) . 
 the overburden must have been relatively high , which contradicts the small overburden deduced by fluid inclusion studies . 
 the argon loss of the fine grains of mylonitic polyhalites at about 44  ma is explained by leitner et  al . 
 ( in press b ) by a low temperature thermal overprint in the eocene , which was postulated inter alia by hejl and grundmann ( 1989 ) and pueyo et  al . 
 ( 2007 ) due to a regional thrusting processes in the nca ( for example , linzer et  al . , 1995 ) . 
 ( in press b ) published a wide range of ar / ar age data ( 15844  ma ) for this rock type the mylonitisation was most likely induced by well reported early cretaceous thrusting events ( mandl , 2000 ; schorn and neubauer , 2011 and references therein ) ( fig . 
 12 ) because of nearly complete resetting of the ar isotopic system at 113  ma ( leitner et  al . 
 the polyhalite samples showing crenulation cleavage and folds ( microfabric type a-2 ) , experienced a microfolding of the existing foliation and , therefore , at least two different deformation stages . 
 the recrystallised polyhalites of microfabric type a-3 were formed due to healing processes of the fabric after the main deformation process . 
 crenulation cleavage is commonly induced by microfolding of an existing foliation ( e.g. a slaty or phyllitic cleavage ) , typically due to shortening of foliation about parallel to its layering . 
 many samples , especially polyhalite mylonites ( of microfabric type a-1 ) , also show pressure solution of microfabric type a-4 , which indicates high strain rates , the presence of intergranular fluids and a second deformation stage under lower p  t - conditions . 
 so4 brines infiltrated polyhalite rocks ( upwards and downwards ) along joints and faults , which were opened during several tectonic deformation stages ( some of them were probably the same events which were also responsible for the mylonitisation and cataclastic fabrics ) and induced the polyhalite recrystallisation ( microfabric types a-2 , a-3 and a-4 ) at elevated temperatures ( fig . 
 the microfabric type a-5 ( sample ht-6 ) has to be relatively young ( compared with the other microfabric types ) , otherwise its cataclastic fabric would not have been preserved . in the course of the mylonitisation during early cretaceous thrusting , or in younger geological times , a cataclastic deformation took place , which is still preserved in the boudinaged polyhalite  anhydrite layer and the cataclastic polyhalite microfabric ( type a-5 ) of sample ht-6 . 
 it is characterised by equidimensional angular grains of variably size ( 0.0050.08  mm ) and areas with secondary grain size reduction . 
 this fabric was formed due to relatively high strain rates , high fluid pressures at non - metamorphic to very low - grade metamorphic conditions . 
 so4 brines were circulating in the cataclastically opened voids , inducing / generating the polyhalite precipitation ( fig . 
 the polyhalite grains , which marginally grow into halite - filled cavities ( microfabric type c-1 , e.g. samples ht-2 , ht-3 , ht-16 , fig . 
 9a ) , are definitely younger than the microfabric types c-2 , a-1 and b-1 and probably also younger than a-2 , a-3 and a-4 . 
 otherwise , the salt filling could not have been preserved until now . the k  mg  cl 
 so4 brines may have infiltrated the rock ( upwards and downwards ) after the main alpine deformation stages along already opened cracks or joints ( fig . 
 the metamorphic reaction fabric ( microfabric type b-2 ) of sample ht-12 is constituted by bldite , a fine - grained polyhalite seam and large anhydrite crystals . 
 the decomposition reaction of bldite might have taken place at 61.4  c ( if a saturation in halite is assumed or 71  c without nacl - saturation ) or slightly above ( for a detailed discussion see chapter 7.3 ) . 
 it is not clear whether this polyhalite type is younger or older than microfabric type c-1 or a-3 . 
 however , as this decomposition reaction took place at relatively low temperatures , a large overburden can be excluded . 
 therefore , this microfabric type is certainly younger than the microfabric types c-2 , a-1 and b-1 and probably also younger than a-2 , a-3 and a-4 types . as no ar 
 however , microprobe measurements and photos show isomorphic polyhalite fibres and corroded anhydrite and bldite crystals ( fig . 
 11c and d ) , which strongly argue for a very young ( probably cenozoic ) polyhalite age ( in comparison with the other minerals ) and a progressive metamorphism at low temperatures and elevated pressures . 
 so4 brines might have infiltrated the host rock along already existing cracks or faults ( of a former tectonic event ) . 
 the polyhalite fibres were precipitated by brine - back - reactions with earlier formed gypsum or anhydrite ( fig . 
 at least the microfabric types a-1 ( and b-1 , probably a-5 ) , b-2 , c-1 and c-2 formed during different geological events , which were accompanied by fluid migration . 
 each of these brines , which infiltrated the haselgebirge evaporites showed a different chemical composition , which , as we expected at the beginning of our study , would result in the precipitation or formation of polyhalite rocks with an appropriate and varying chemical composition . 
 but microprobe measurements only showed a very small chemical variation ( supplementary material , fig . 
 sm 2 ) of polyhalite samples from microfabric types a-1 , a-3 , a-4 , b , b-2 , c-1 and c-2 ( table  3 , supplementary material , table  sm 2 ) . obviously 
 , brines of different chemical compositions within the haselgebirge precipited polyhalites of nearly the same mineral chemistry . 
 the examined polyhalite samples of hallstatt show a wide variety of microfabrics and many of them , the mylonitic ones , resemble closely to such observed from rock - forming constituents of continental crust , like quartz ( stipp et  al . , 2002 ) , 
 similarly , the microfabrics may have resulted from a wide range of external conditions like temperature , strain rate and differential stresses ( passchier and trouw , 2005 ) and the temperatures at hallstatt remained low ( < 250  c ) . 
 we also note that recrystallisation and annealing plays an important role veiling preferred crystal lattice patterns , which could not observed in or mylonitic samples . 
 the influence of migrating brines to various microfabrics ( see discussion in following sections ) is more obvious in polyhalite rocks than in usual continental rocks with their predominance of quartzite , feldspar and calcite . in oceanic salt deposits , 
 bldite is mainly formed secondarily from kieserite due to solution metamorphism and often observed in kainite hats or associated with halite , lweite and mg sulphates ( braitsch , 1962 ) and may also precipitate primarily from normal marine water ( braitsch , 1962 ; zayani et  al . , 1999 ) , 
 na2so4-rich salt lakes or nitric acid deserts . in the haselgebirge fm . of the eastern alps , polyhalite intergrown with bldite ( samples ht-12 and ht-15 , ht-27 ) was , inter alia , described by grgey ( 1909 ) from hall in tirol 
 . the lower crystallisation temperature of bldite is given as 6.26.4  c ( under laboratory conditions ) in the presence of halite ( autenrieth and braune , 1960a ; charykova et  al . 
 , 1992 ) ( 4.5  c van't hoff , 1912 ) or 20.0  c in the ternary system na2so4mgso4h2o ( charykova et  al . , 1992 ) . 
 the upper formation temperature ranges between 61.4  c ( with nacl - saturation ) ( autenrieth and braune , 1960b ) and 71  c ( without nacl - saturation ) ( van't hoff , 1912 ) . 
 this decomposition reaction of bldite and probably anhydrite , which was observed in sample ht-12 ( microfabric type b-2 ) might have taken place , therefore , at 61.4  c  the upper formation temperature of bldite , if a halite saturation is assumed ( or 71  c without nacl - saturation )  or slightly above . 
 the polyhalite fibres of this metamorphic reaction fabric might have formed after the partly decomposition of the low temperature salt bldite . 
 as bldite has formed at relatively low temperatures , this decomposition reaction might also indicate a weak static reheating of the rock , during tectonic processes in young geological ( probably cenozoic ) times and the age of ca . 
 44  ma found in the altaussee deposit may represent this event ( leitner et  al . 
 furthermore , this microfabric type might indicate , that the polyhalite rocks of the alpine haselgebirge fm . have been infiltrated by extraordinary na - rich brines , which induced the bldite formation at low temperatures , during late stages of the alpine deformation  probably during cenozoic times . 
 bldite sample ht-12 were extraordinarily rich in na ( table  3 , supplementary material , fig . 
 autenrieth ( 1958 ) , who examined the six component system k  na  mg  ca  so4cl 
 h2o , reported the formation of a new , metastable sediment  na - polyhalite ( formula : 25caso4.2k2so4.3na2so4.15h2o ; autenrieth , 1958 , 1959 ) during polyhalite precipitation . 
 after a while , these prismatic to rod - shaped , na - polyhalites are converted to fine - grained , milky to dumbbell - shaped polyhalite . in most cases 
 na - polyhalite may be intermediary formed at temperatures above 50  c from gypsum or brines with high caso4-concentrations within nearly the entire stability field of polyhalite ( autenrieth , 1958 ) . 
 polyhalite may only convert directly ( and quite fastly ) to na - polyhalite at temperatures above 50  c in the presence of mg - free or very mg - poor brines , which are saturated with respect to kcl and nacl ( autenrieth , 1958 ) or above 90  c in the presence of cacl2 and low mgcl2-concentrations ( autenrieth , 1959 ) . 
 the tendency of na - polyhalite formation might increase with rising temperatures and is relatively high at 90  c ( autenrieth , 1959 ) . 
 according to gudowius and hodenberg ( 1979 ) na - polyhalite shows structural similarities with the minerals bassanite ( caso4.0.5h2o ) and -caso4 , which was later verified by reisdorf and abriel ( 1987 ) . for further information on the stability of na - polyhalite , 
 see supplementary material sm-4 . therefore , we conclude tentatively , that na - polyhalite was most likely formed at temperatures above 5090  c , most likely from gypsum or at very low mg - concentrations from polyhalite . 
 metamorphic reaction fabric b-2 and might indicate another , probably older stage of polyhalite formation . 
 as it forms no oceanic salt minerals it is very suitable as index element ( khn , 1968 ) . 
 therefore , khn ( 1972 ) examined the rubidium contents of sulphate salts like sylvite , carnallite , langbeinite , leonite and polyhalite of several salt deposits . 
 primary polyhalites usually show low rubidium contents of < 0.0001  wt ( weight ) % ( khn , 1972 ) , while secondary polyhalites are rubidium - rich and this observation may potentially allow distinction between primary and secondary polyhalite . 
 the polyhalites of hallstatt constitute 0.0012  wt% rb ( khn , 1972 ; schauberger , 1986 ) , implying the reworking of rb - richer salts , and strongly arguing for a secondary origin of the polyhalites within the haselgebirge fm .. our examined hallstatt samples did not show high enough rubidium concentrations for microprobe measurements . due to the lack of sedimentary structures during thin section analysis leitner et  al . 
 ( in press b ) also proposed a clearly secondary origin of the polyhalites of the alpine haselgebirge . according to our microprobe measurements 
 the sro contents of the polyhalite of hallstatt are slightly elevated and range from 0.28 to 0.74  wt ( weight ) % . 
 the variation likely originates from exchange with anhydrite , which has a higher sro content ( schauberger , 1986 ) . 
 the sr content of all examined anhydrite samples of the alpine salt deposits mainly ranges between 0.1 and 0.2% ( ruscha , 1976 , unpublished data quoted from schauberger , 1986 ) or 0.305% for hall in tirol ( sptl , 1988b ) or even lower , strongly arguing for a diagenetic and secondary formation of the alpine anhydrites ( due to conversion of gypsum , which was formed in the caso4-phase ) ( schauberger , 1986 ) . 
 strontium ions of the seawater may isomorphously replace ca and sr ( due to a linear function ) in the crystal lattice of aragonite , calcite , anhydrite and gypsum ( usdowski , 1973 ) . 
 according to usdowski ( 1973 ) anhydrite , which was formed by conversion of gypsum , shows average sr - contents of 0.2  wt% , while primary anhydrite constitutes sr - concentrations as high as 2.9  wt% . 
 we conclude , therefore , that the relatively low sr content of polyhalite is of secondary origin . 
 the magnesites of the haselgebirge fm . from the eastern alps are of secondary origin and may have formed by exchange reactions between mg - bearing solutions and primary low mg - carbonates during several stages of diagenesis or metamorphism ( niedermayr et  al . , 1989 ) . within the haselgebirge fm . 
 , fine - grained magnesite is one of the major minerals of the carbonate fraction ( niedermayr et  al . , 1989 ) . 
 magnesite ( mgco3 ) is inter alia reported from the evaporitic claystone of hallstatt salt mine ( schauberger , 1986 ) . 
 sptl ( 1988a ) described traces of magnesite ( by x - ray diffraction ( xrd ) 
 measurements ) in dolomitic limestones of a lower triassic bituminous dolomitic / anhydritic grey salt rock profile in the level kaiser franz josef . within the alpine haselgebirge fm . 
 , magnesite may have formed by epigenetic saliniferous processes ( which means post - sedimentary - diagenetic recrystallisation ) ( schroll , 1961 ; schauberger , 1986 ) . 
 sptl ( 1988a ) suggested that the examined succession of fossil - free , bituminous , laminated siltstones , anhydrites and carbonates of the level kaiser franz josef ( hallstatt ) might have been deposited in a subtidal , hypersaline and anaerobe basin milieu ( sptl , 1988a ) . 
 ( during ca - sulphates and salt precipitation ) by the infiltration and percolation of mg - bearing brines in the basin center , which induced the dolomite and even magnesite formation of carbonate sediments . 
 salt diapirism and several alpine metamorphic stages may have led to mobilisation and recrystallisation of predominantly coarse - grained magnesite ( of the nca ; see also ebner et  al . , 2000 ) . 
 we conclude therefore , that the fine - grained magnesite associated with polyhalite is of secondary origin . 
 in this paper , we present for the first time a polyhalite microfabric classification particularly to the hallstatt salt mine , which shows a much wider variety of fabrics as reported in recent works from other salt mines in the eastern alps ( leitner et  al . , 
 in press a , b ; leitner , 2011).(1)polyhalite microfabrics from hallstatt include : ( 1 ) vein - filling , fibrous polyhalite of likely middle to late triassic age , ( 2 ) polyhalite mylonites of early cretaceous age , ( 3 ) metamorphic reaction fabrics of a post - early cretaceous age and ( 4 ) cavity - filling polyhalite of uncertain age.(2)the polyhalite mylonites contain a wide range of shear fabrics commonly known in mylonitic quartzo 
 feldspathic shear zones within the ductile crust and developed from a more coarse - grained precursor rock.(3)the mylonites are partly overprinted by recrystallised , statically grown polyhalite grains.(4)metamorphic reaction fabrics ( microfabric type b-2 ) of  fine - grained polyhalite seams , which have grown due to  the  decomposition of bldite ( or astrakhanite ) [ na2mg(so4)2.4h2o ] and anhydrite have also been found . according to previous reports 
 , bldite may occur primarily as nodules or , more commonly , intergrown with lweite , which has a nearly identical chemical composition and is stable at higher temperatures . 
 reaction fabrics may have formed by exsolution , ( re-)crystallisation , parallel growth or replacement . 
 this fabric type was only found in one sample in relation with bldite , and has a formation temperature of 6.261.4  c in the presence of halite.(5)for the first time , we demonstrate that microfabrics like mylonitic shear fabrics , porphyroclast systems , annealing etc . , which are described for other minerals like quartz , etc . 
 , are also preserved in relatively rare sulphates like polyhalite , which is stable between low temperatures and ca . 
 255  c.(6)in general , coarse - grained polyhalite grains or twins in a fine - grained polyhalite matrix ( microfabric type a-4 ) were only observed in samples from hallstatt . 
 the cataclastic polyhalite microfabric a-5 was also only found in one hallstatt sample ( ht-6 ) and the metamorphic reaction fabric b-2 was also reported from one polyhalite 
 polyhalite microfabrics from hallstatt include : ( 1 ) vein - filling , fibrous polyhalite of likely middle to late triassic age , ( 2 ) polyhalite mylonites of early cretaceous age , ( 3 ) metamorphic reaction fabrics of a post - early cretaceous age and ( 4 ) cavity - filling polyhalite of uncertain age . 
 the polyhalite mylonites contain a wide range of shear fabrics commonly known in mylonitic quartzo 
 feldspathic shear zones within the ductile crust and developed from a more coarse - grained precursor rock . 
 metamorphic reaction fabrics ( microfabric type b-2 ) of  fine - grained polyhalite seams , which have grown due to  the  decomposition of bldite ( or astrakhanite ) [ na2mg(so4)2.4h2o ] and anhydrite have also been found . according to previous reports 
 , bldite may occur primarily as nodules or , more commonly , intergrown with lweite , which has a nearly identical chemical composition and is stable at higher temperatures . 
 reaction fabrics may have formed by exsolution , ( re-)crystallisation , parallel growth or replacement . 
 this fabric type was only found in one sample in relation with bldite , and has a formation temperature of 6.261.4  c in the presence of halite . 
 for the first time , we demonstrate that microfabrics like mylonitic shear fabrics , porphyroclast systems , annealing etc . 
 , are also preserved in relatively rare sulphates like polyhalite , which is stable between low temperatures and ca . 
 255  c . in general , coarse - grained polyhalite grains or twins in a fine - grained polyhalite matrix ( microfabric type a-4 ) 
 the cataclastic polyhalite microfabric a-5 was also only found in one hallstatt sample ( ht-6 ) and the metamorphic reaction fabric b-2 was also reported from one polyhalite","in the hallstatt salt mine ( austria ) , polyhalite rocks occur in 0.51  m thick and several metre long tectonic lenses within the protocataclasite to protomylonite matrix of the alpine haselgebirge fm ..  thin section analysis of hallstatt polyhalites reveals various fabric types similar to metamorphic rocks of crust - forming minerals , e.g. quartz and feldspar . 
 polyhalite microfabrics from hallstatt include : ( 1 ) polyhalite mylonites , ( 2 ) metamorphic reaction fabrics , ( 3 ) vein - filling , fibrous polyhalite and ( 4 ) cavity - filling polyhalite . 
 the polyhalite mylonites contain a wide range of shear fabrics commonly known in mylonitic quartzo 
 feldspathic shear zones within the ductile crust and developed from a more coarse - grained precursor rock . 
 the mylonites are partly overprinted by recrystallised , statically grown polyhalite grains . 
 metamorphic reaction fabrics of polyhalite fibres between bldite ( or astrakhanite ) [ na2mg(so4)2.4h2o ] and anhydrite have also been found . according to previous reports 
 , bldite may occur primarily as nodules or intergrown with lweite . 
 reaction fabrics may have formed by exsolution , ( re-)crystallisation , parallel growth or replacement . 
 this fabric type was only found in one sample in relation with the decomposition of bldite at ca . 
 61  c in the presence of halite or slightly above , testifying , therefore , a late stage prograde fabric significantly younger than the main polyhalite formation .",pubmed
"oxidative stress can be described as an imbalance between the production of free radicals and antioxidant defence . over the last decades , 
 it has become amply evident that oxidative stress , usually in the form of reactive oxygen species ( ros ) , is a critical pathogenic factor in the development of several systemic diseases [ 1 , 2 ] . 
 therefore , inhibition of ros formation , scavenging of ros , or interfering with ros pathogenic signaling pathways might be the potential ways to protect against oxidative stress - induced ailments . 
 propofol ( 2,6-diisopropylphenol ) ( figure 1 ) , a highly lipid - soluble anaesthetic , is widely used for induction and maintenance of general anaesthesia . 
 propofol ameliorates oxidative injury in several organs , including the heart , lungs , brain , liver , and testis . 
 propofol is chemically similar to the endogenous antioxidant -tocopherol ( vitamin e ) and , theoretically , it should have similar properties . 
 it is not surprising therefore that many studies have demonstrated antioxidant effects of propofol in vitro [ 1012 ] and in vivo . 
 propofol has also been shown to elicit antiapoptotic effect in human umbilical vein endothelial cells by acting as an antioxidant . to the best of our knowledge , 
 the effects of propofol at high doses on the lipid profile , cardiovascular marker enzymes , and enzymatic and nonenzymatic antioxidant indices in rodents have not been determined . 
 propofol also called disoprivan by astrazeneca ( switzerland ) used for this study was obtained from one of the coauthors who is an anaesthetist ( dr . olugbenga akinwonmi of the department of anaesthesia , university college hospital , ibadan , nigeria ) . 
 glutathione , hydrogen peroxide , 5 ,  5-dithios - bis-2-nitrobenzoic acid ( dntb ) , and epinephrine were purchased from sigma chemical co. , saint louis , mo , usa . other chemicals were of analytical grade and purest quality available . 
 inbred male wistar rats weighing between 200 and 220  g were purchased from the central animal house located in the department of physiology , university of ibadan , nigeria . 
 the animals were kept in well - ventilated cages at room temperature ( 2830c ) , and maintained on laboratory chow ( ladokun feeds , ibadan , nigeria ) and water ad libitum . 
 rats handling and treatments protocol conforms to the guidelines of the faculty of basic medical sciences , university of ibadan animals ' ethical committee as well as the national institute of health ( nih publication 85 - 23 , 1985 ) for laboratory animals ' care and use . 
 twenty - one male albino rats ( wistar strain ) were randomly distributed into three groups of seven animals each . 
 the first group served as the control and was given corn oil ( vehicle for the drug ) . 
 the second group received propofol at a dose of 2  mg / kg body weight ( equivalent of human therapeutic dose ) , and the third group received propofol at a dose of 4  mg / kg body weight ( twice therapeutic dose ) . 
 propofol was prepared with corn oil and was administered intraperitoneally three times in a week for four consecutive weeks . 
 twenty - four hours after the last dose of the drug , the animals in each group were sacrificed by cervical dislocation , and blood was collected from the inferior vena cava of the heart into plain centrifuge tube . 
 blood was allowed to stand for 1  h and was then centrifuged at 3000  g for 15  min in a bench centrifuge to obtain serum . 
 the serum samples were used for the analysis of biochemical indices and lipid profile of the animals .   liver and kidney from the animals were quickly removed and washed in ice - cold 1.15% kcl solution , dried , and weighed . 
 the tissues were homogenized in 4 volumes of 50  mm phosphate buffer , ph 7.4 , and centrifuged at 10,000  g for 15  min to obtain postmitochondrial fraction ( pmf ) of the liver and kidney , which were used for assay of the antioxidant parameters . 
 protein levels in the samples were assayed by the method of lowry et al .   using bovine serum albumin as standard . 
 the activities of serum alanine and aspartate aminotransferases ( alt and ast ) were assayed by the combined methods of mohun and cook   and , reitman and frankel . 
 pmf lipid peroxidation levels were assayed by the reaction between 2-thiobarbituric acid and malondialdehyde , an end product of lipid peroxides , as described by walls et al . . 
 pmf - reduced glutathione ( gsh ) level was assayed by measuring the rate of formation of chromophoric product in a reaction between 5,5-dinitrobis-2-nitrobenzoic acid and free sulphydryl groups at 412  nm as described by moron et al . . 
 pmf superoxide dismutase ( sod ) activity was measured by the nitro blue tetrazolium reduction method of mccord and fridovich . 
 pmf catalase ( cat ) activity was assayed spectrophotometrically by measuring the rate of decomposition of hydrogen peroxide at 240  nm as described by aebi   while glutathione - s - transferase ( gst ) activity was determined by the method of habig et al . . 
 the activities of serum creatinine phosphokinase ( ck - mb ) were estimated using immune - inhibition kinetic assay according to the method of okinaka et al . . 
 lactate dehydrogenase ( ldh ) activities in the serum were determined according to the method of zimmerman and weinstein . 
 the total bilirubin levels were assayed by the method of rutkowski and debaare   while serum creatinine and urea were estimated by the methods of  jaffe   and talke and schubert , respectively . 
 serumtriglyceride ( tg ) and total cholesterol ( tc ) levels were assayed using commercial diagnostic kits ( randox ) . for the determination of hdl level , vldl and 
 after centrifugation , the supernatant containing the hdl fraction was assayed for cholesterol using randox diagnostic kit . 
 ldl - cholesterol ( ldl - c ) was calculated using the formula of friedewald et al . . 
 data were analyzed using one - way anova followed by the post - hoc duncan 's multiple - range test for analysis of biochemical data using spss version 10.0 ( spss inc . 
 reactive oxygen species ( ros ) attack cellular components containing polyunsaturated fatty acid residues of phospholipids , which are sensitive to oxidation . 
 the peroxyl radicals formed by the attack can be rearranged via a cyclization reaction to endoperoxides which are precursors of mda , which subsequently lead to lipid peroxidation . 
 lipid peroxidation ( lpo ) has been implicated in the pathogenesis of several injuries by many toxicants and may be responsible for cell membrane alterations which can result into enzymes leakage in animals . in this study , 
 administration of propofol did not significantly ( p > 0.05 ) affect the levels of serum and renal and hepatic mda in the rats ( table 3 ) , indicating that the metabolism of propofol may not be linked to the generation of free radicals within the animal tissues . this observation is not strange since propofol has a very active phenolic ring within its structure which resembles -tocopherol and may release electron to terminate free radical reactions during its metabolism in the rats . 
 however , table 2 shows that the levels of serum total cholesterol , triglycerides , and ldl - cholesterol were significantly ( p < 0.05 ) increased following administration of propofol . 
 precisely , propofol at 2 and 4  mg / kg increased the levels of serum total cholesterol by 74% and 55% , triglycerides by 97% and 115% , and ldl - cholesterol by 45% and 73% , respectively . 
 in addition , propofol at both doses significantly ( p < 0.05 ) decreased the levels of serum hdl - cholesterol in the rats by 41% and 54% . 
 lipids are a heterogeneous group containing active metabolic substances that play an important role in the pathogenesis of drugs - induced liver disease . 
 the most common lipid abnormalities during drug toxicity are hypercholesterolemia and hypertriglyceridemia , which was confirmed in this study . 
 also , in propofol - treated rats , ldl - c was remarkably increased in the serum , while hdl - c was found to be reduced . 
 the increased cholesterol level during propofol administration may be linked to increase in alpha - hydroxyl methyl glutaryl coa reductase activity , which is the rate limiting step in cholesterol biosynthesis . 
 likewise , the increased triglycerides levels may be due to the increased availability of free fatty acid , glycerophosphates , decreased triglycerides lipase activity , or decreased fatty oxidation . in the present study 
 propofol at 2 and 4  mg / kg increased the levels of hepatic gsh by 42% and 37% , respectively , when compared to the control ( table 3 ) . 
 in addition , propofol at 2 and 4  mg / kg dose dependently and significantly ( p < 0.05 ) increased the activities of hepatic glutathione peroxidase by 1.9 and 2.1 folds and glutathione - s - transferase by 2.3 and 2.4 folds , respectively ( figure 3 ) . 
 however , there were no significant ( p > 0.05 ) differences in the activities of renal and hepatic sod and cat of propofol - treated rats relative to controls ( figure 2 ) . 
 reduced glutathione acts as a free radical scavenger and regenerator of alpha - tocopherol , and it plays a significant role in sustaining protein sulfhydryl groups in proteins and other key molecules . 
 in addition , gsh may act as an essential cofactor for antioxidant enzymes , especially gpx and gst . 
 the increase in the levels of hepatic gsh observed in this study may result in increased detoxifying and antioxidant capability of the liver . 
 the gst and gpx are groups of multifunctional proteins that play a central role in the detoxification of electrophilic chemicals and the hepatic removal of potentially harmful hydrophobic compounds from blood 
 . the increased activities of hepatic gst and gpx observed may partly be due to increase in their substrate or induction of de novo synthesis of the enzymes . 
 importantly , propofol increased the levels of gsh and activities of gst and gpx in the liver and thus enhancing the detoxifying potential of the animals . 
 the cardiotoxicity of xenobiotics can be evaluated using the serum activities of marker enzymes especially ldh and ck - mb , which are distributed throughout the body and have isoenzymes that are recognized as markers for muscle and heart lesion . 
 results from this study showed that propofol at 2 and 4  mg / kg significantly ( p < 0.05 ) increased the activities of serum ldh by 1.7 and 1.8 folds and ck - mb by 2.0 and 2.1 folds , respectively ( figure 4 ) . in their studies , hayden and tyagi   linked the increase in serum ck - mb and ldh of diabetic rats to cardiac muscular damage caused by the disease . 
 similarly , wiernsperger   stated that the activities of serum ck - mb and ldh are a measure of the state of necrosis in cardiac tissues . in view of the observed increase in ck - mb and ldh activities in propofol - treated rats 
 administration of propofol at 2 and 4  mg / kg significantly ( p < 0.05 ) increased the level of total bilirubin by 52% and 53% , respectively , while serum urea , alanine and aspartate aminotransferases ( alt and ast ) , and creatinine levels were insignificantly ( p > 0.05 ) affected ( table 1 ) . 
 it is established that ast can be found in the liver , cardiac muscle , skeletal muscle , kidney , brain , pancreas , lungs , leukocytes , and erythrocytes , whereas alt is predominantly present in the liver . 
 also , serum creatinine and urea levels are the sensitive and reliable biochemical indices for evaluation of renal function in animal models . 
 the insignificant effects of propofol on the levels of ast , alt , and creatinine indicate that the drug has no adverse effect on the hepatic and renal tissues of the rats . 
 our data showed that propofol promotes the antioxidant status of the rats by increasing the levels of reduced glutathione and gsh - dependent enzymes . 
 however , propofol elicited a detrimental effect on the lipid profile resulting in hypercholesterolemia which subsequently leads to abnormally high activities of serum creatinine phosphokinase and lactate dehydrogenase in the rats . 
 the study , therefore , suggests that the use of propofol in patients with cardiovascular or lipid disorders should be carefully reviewed .","in recent years , the activity of anaesthetic propofol on biological processes has been attracting attention . 
 the effect of propofol on biochemical indices in animals is unknown . in this study 
 , we examined the effects of propofol on lipid profile , antioxidant indices , and cardiovascular marker ( cvm ) enzymes in rats . 
 the study consists of three groups of seven rats each . 
 group one received corn oil ( control ) while groups two and three received propofol ( doses of 2 and 4  mg / kg body weight , resp . ) . 
 results showed that administration of propofol caused a significant ( p < 0.05 ) and dose - dependent increase in the levels of total bilirubin . 
 propofol at 2 and 4  mg / kg increased the levels of serum total cholesterol by 74% and 55% , triglycerides by 97% and 115% , and ldl - c ( low - density lipoprotein - cholesterol ) by 45% and 73% , respectively , while hdl - c ( high - density lipoprotein - cholesterol ) decreased by 41% and 54% , respectively . 
 propofol significantly ( p < 0.05 ) increased the levels of the hepatic reduced glutathione ( gsh ) and activities of gsh - dependent enzymes . 
 propofol at 2 and 4  mg / kg increased the activities of cvm enzymes : lactate dehydrogenase by 1.7 and 1.8 folds and creatinine phosphokinase by 2.0 and 2.1 folds , respectively . 
 taken together , propofol increased the levels of gsh and gsh - dependent enzymes but adversely affected the lipid profile of the rats .",pubmed
"idiopathic hypogonadotropic hypogonadism ( ihh ) refers to gonadal dysgenesis due to deficiency of gonadotropin - releasing hormone ( gnrh ) release from the hypothalamus . 
 the prevalence of ihh is about 1/10,000 in men and 1/50,000 in women with a m / f ratio of 4 - 5  :  1 . 
 since ihh is rare , the number of ihh patients is small in any one geographical area . 
 it is often difficult for a single clinical center to perform comprehensive assessment of the disease due to the limited number of cases . 
 internationally , large - scale clinical studies on this rare disease are usually conducted in the form of multicenter trials . however , these kinds of studies are time consuming and require large amount of resources as well as close coordination among study groups . with the advances in information technology in the recent years , people have utilized networks for different research purposes . by december 2011 , 
 the number of internet users in china had exceeded five hundred million . with more wide - spread availability of networks and the dramatic increase in the number of internet users , 
 they form disease - specific social groups to share their knowledge and experience spontaneously , which makes it possible for medical researchers to conduct medical research through networks [ 3 , 5 ] . 
 network investigation is a new modality of means of medical research that can cover large populations with low investigation costs , especially for investigation of a rare disease in a particular network social group 
 . it can collect large numbers of clinical cases within a short period for the research purpose . in the present study , we collected the clinical data of ihh patients in china by interviewing ihh patients through network questionnaire and their medical records . 
 we compared the obtained data with those reported in previous studies in the literature for the purpose of improving our understanding , diagnosis , and treatment of ihh . 
 the kallmann liangjian social group is the largest ihh social networking group in china ( qq as the real - time network communication tool , id7685615 ) with 103 members . 
 the items of the questionnaire investigation were designed by endocrinologists on the basis of the data obtained from literatures of ihh in china and abroad . 
 the information obtained through the questionnaire survey includes the following : demographic characteristics , family history , age , clinical symptoms and laboratory tests at onset of disease , particular therapeutic protocols ( testosterone ( t ) , human chorionic gonadotropin ( hcg ) , human menopausal gonadotropin ( hmg ) , luteinizing - hormone - releasing hormone ( lhrh ) , or random combination of them ) , developmental stage of the testis and secondary sex characteristics , sexual function , level of sexual hormones , and quality - of - life ( qol ) rating . using the tanner classification pictures 
 , patients made a self - assessment of the development of the external genital organs , pubic hair , and breasts . 
 self - reported data was referred to the same standard , while the laboratory examination results and disease diagnosis were actually obtained from medical records . 
 online questionnaire survey submission included electronic consent as part of the website log - on process . 
 this study was approved by the institutional review board of huashan hospital in shanghai , china . 
 a total of 75 questionnaires answering all of the 64 questions were obtained online between october 2011 and march 2012 . 
 all of the participants were asked to provide copies of their medical records ; then , the records were examined by one endocrinologist , and only ihh patients were allowed to join the qq group . all activities and exchanges involved in the investigation were voluntary and free of charge . 
 participants can submit the questionnaire survey only after all of the 64 questions were answered ; thus , there was no missing data for the investigation . 
 all participants had to hand in their medical records for ihh and provide their contact means such as the e - mail address and telephone number , so that the investigators could check whether or not the information that the participants provided was authentic . 
 finally , the computer system generated tables automatically , and the investigated data were stored in the data bank directly . 
 differences between categorical variable groups were compared by chi - square test or bilateral probability of fisher 's exact test . 
 scores for overall qol and correlations of clinical parameters were analyzed by spearman 's correlation analysis . 
 all of the 74 patients participating in the ihh investigation were males with the mean age being 27.3  3.4 years ( ranging from 20 to 35 years ) . 
 the mean age at disease onset was 15.2  6.6 years , and the duration of the disease ranged from 1 to 25 years with a mean of 12.1  7.2 years . 
 the height of the 74 patients ranged from 157 to 188  cm with a mean of 174.4  6.9  cm . 
 ( mean : 24.27  3.73  kg / m ) , belonging to overweight ( bmi of 24  kg / m is used as cutoff point for the chinese adults to define overweight ) . 
 of the 74 patients , 28 ( 37.8% ) patients had a history of smoking ( 2 cigarettes per day on average ) . 
 all patients had junior middle school or higher education level , 25 ( 33.8% ) patients had senior middle school education , and 37 ( 50% ) patients had college education . 
 all the 74 patients were diagnosed with ihh , and 84% of them were diagnosed in general hospitals , 13.5% in district hospitals , and 2.7% in community hospitals . 
 42 ( 56.8% ) patients were diagnosed and treated in endocrinology unit , 25 ( 33.5% ) patients in urinary surgery unit , and 6 ( 8.1% ) patients in pediatrics unit . 
 of the 74 patients , 38 ( 51.4% ) patients had hyposmia or anosmia , and 69 ( 93.2% ) patients presented with obvious pubertal delay . 
 the serum testosterone levels at disease onset were decreased with varying degrees in all of the 74 patients , as the medical reference ranges in different laboratories are different . 
 serum follicle - stimulating hormone ( fsh ) levels were decreased in 71 ( 95.9% ) patients , and serum luteinizing hormone ( lh ) levels were decreased in 69 ( 93.2% ) patients . of the 74 patients , 68 patients received hormone replacement therapy , and the therapeutic protocols are shown in table 1 . 
 20 ( 27.0% ) patients had cryptorchidism , and 11 patients received  cryptorchidism surgery . 
 except for the family history , there was no significant difference in gender composition , age , bmi , testosterone level at disease onset , pubertal development , small penis , testicular volume , breast enlargement , cryptorchidism , and number of patients receiving  cryptorchidism surgery  between the populations in our study and the study reported by the reproductive endocrine unit of massachusetts general hospital   ( table 2 ) , while , in one multicenter study from england , more patients had family history of ihh , and the testicular volumes of ihh patients are smaller than those in our study . in the 74 patients , the mean length of the penis before erection was 3.98  1.73  cm , and the mean testicular volume was 4.11  2.12  ml . 
 based on the tanner classification criteria , there are 38 patients at genital development stage i and 36 patients at stage ii , and nobody was at stage iii , iv , or v. there are 46 patients at pubic hair development stage i and 28 patients at stage ii , and nobody was at stage iii , iv , or v. all of the 74 patients had no or small laryngeal prominence and no or sparse facial hair including 12 ( 16.2% ) patients with small laryngeal prominence and 9 ( 12.2% ) with sparse beard . 
 of the 74 patients , only 27 ( 36.5% ) patients had the male erection function , and 8 ( 10.8% ) patients had physiological nocturnal spermatorrhea . among the 68 patients who received hormone replacement therapy , 
 for example , they had more masculine body , more facial hair and pubic hair , larger laryngeal prominence , more developed external genital organs , larger penis and scrotum , stronger erections , and nocturnal spermatorrhea . 
 there was significant difference in penis length , testicular volume , development of external genital organs , pubic hair , beard , laryngeal prominence , penis erection , and spermatorrhea before and after treatment ( p < 0.001 , table 3 ) . 
 the normalization of testosterone level rose from 0% before treatment to 66.2% after treatment , excluding 3 patients who are now receiving exogenous testosterone therapy . 
 of the 74 patients , 38 ( 51.4% ) patients now have sexual activities , including 14 ( 18.9% ) patients whose spouses became pregnant . 
 thirty - seven patients who received hcg or hcg + hmg treatment were included into a single gonadotropin group ( g group ) , and 25 patients who received hcg + t or hcg + hmg + t treatment were included into a gonadotropin + androgen replacement group ( g + a group ) . 
 those who were still receiving testosterone replacement therapy were excluded from the g + a group , and the remaining 22 patients who once received testosterone replacement therapy discontinued its use for 18 years ( mean : 4.5  2.6 years ) . 
 there was no significant difference in age at the time of investigation , age at disease onset , testosterone level before treatment , and decrease rate of fsh and lh between g group and g + a group . 
 it was found that g + a group was significantly superior to g group in terms of penis length , pubic hair , laryngeal prominence development , and normalization of testosterone level ( p < 0.05 , table 3 ) . compared with g group , 
 g + a group also exhibited more improvement in testicular volume , genital organs , beard , and spermatorrhea , though the difference was not statistically significant . 
 qol of the ihh patients was assessed with respect to physical strength , appetite , sleep , libido , and sense of happiness . 
 the mean score of physical strength was 5.1  1.7 , that of appetite was 7.3  1.8 , that of sleep was 6.8    2.3 , that of libido was 7.2  2.0 , and that of sense of happiness was 5.7  2.6 . 
 the overall qol scored was 1045 , and the mean value was 32.1  7.5 . 
 the ihh patients reported that they still felt poor in the sense of happiness and physical strength after treatment . 
 there was a significant difference in the physical strength score between g group and g + a group ( 5.40  0.36 versus 4.65  0.30 , p = 0.025 ) , while there was no significant difference in appetite , sleep , libido , and sense of happiness . 
 the overall qol score of the ihh patients was positively correlated with the level of testicular volume ( r = 0.324 ;  p = 0.005 ) and the developmental state of the external genital organs ( r = 0.281 ; p = 0.015 ) after treatment . 
 the most unique characteristic of the present study is the utilization of a web - based disease - specific social networking group for our investigation . to the best of our knowledge , 
 the continuous expansion of internet users provides a good foundation for the development of network investigation , a new approach for medical research in china and elsewhere . 
 there had been multiple studies comparing network studies with conventional studies , and they found that there was no significant difference in the study population characteristics and statistical results [ 11 , 12 ] . 
 table 2 shows that the results obtained in the present network investigation are similar to those reported in the literature   with respect to sex , age , bmi , and clinical characteristics of the ihh patients , which further confirms that group data obtained from network investigation are representative . however , ihh patients in the study reported no family history , which may be related to the age and sporadic characteristics of the patients . 
 the patients ' parents did not have ihh , or else they would not have offspring as there was no domestic treatment method for ihh several decades ago . 
 this finding is consistent with those of the previous large - sample studies on ihh in china . 
 ( 1 ) it can include more participants , especially when network investigation is directed at a particular disease - specific social group . 
 in addition , it is not restricted by time , space , or location ( i.e. , a single medical center ) , so that more patients with different severities and courses of the disease distributed in different regions of china or around the world can be investigated , and , therefore , the data obtained are more representative . 
 ( 2 ) information can be obtained more quickly and conveniently , because network investigation saves amount of time in patient recruitment . in this study 
 , we only spent five months to collect the data of the 74 ihh patients compared with 20 years for the massachusetts general hospital . 
 in addition , network questionnaire investigation has a quick response rate , and the data obtained can be imported into the data bank directly , thus saving time , material , and manpower and reducing the cost of research effectively as compared with the conventional research methods . ( 
 network investigation can be completed in a relatively independent situation , devoid of external interferences , so that the patients are able to express freely their sexual function and other private information [ 3 , 15 ] . 
 network investigation can be carried out by a relatively small and independent research team , and any clinician can conduct the research as long as he / she has a reasonable study design . 
 although network investigation is a new method for medical research and carries many advantages , it has some disadvantages . 
 ( 1 ) as only network users can participate in the study , there may be some bias in patients selection . 
 the participants need to be able to get on networks and have a certain education level . 
 ( 2 ) network investigation has to use the layman 's language for communication , and , therefore , it is not suitable for studies that strongly depend on medical terminology . 
 prolonged sex hormone deficiency may otherwise produce adverse effects on the development , metabolism , and psychology . 
 all treated patients reported their improvements in the penis , testis , external genital organs , pubic hair , beard , laryngeal prominence development , erection , spermatorrhea , and testosterone normalization . 
 these findings are basically consistent with those of the previous studies of us massachusetts general hospital and uk manchester royal infirmary [ 8 , 19 ] . 
 more than 50% of the patients in this study have regained their sexual function after treatment , and part of the patients ' spouses have got pregnant . 
 the possible reason is that most of the ihh patients in our study were overweight ; with physical development of the body and accumulation of the adipose tissue , the breasts became larger , which made self - assessment of the mammary development according to the tanner classification criteria difficult . 
 for this reason , ultrasonography should be used to evaluate mammary gland development more accurately . 
 in addition , as the physical strength of ihh patients is generally weak and the sense of happiness is usually low , it is necessary to provide psychosocial support to ihh patients . 
 unfortunately , there is a lack of such studies in both china and other countries . in the present study 
 , we interestingly found that the therapeutic effect in g + a group was superior to that in g group with respect to the penis length , pubic hair growth , testosterone normalization rate , and self - assessment scores of physical strength . 
 the testosterone normalization rate in the g + a group ( excluding the three patients who were still receiving testosterone replacement therapy in the recent three months ) was significantly higher than that in g group ( 90.9% versus 51.7% ) , indicating that improvement of the clinical symptoms in g + a group was not due to the immediate exogenous testosterone treatment but due to the therapeutic effect of the testosterone treatment that they previously received . whether exogenous testosterone replacement therapy is beneficial to 
 the response of ihh patients to gonadotropin therapy remains inconclusive , but the report from harvard reproductive endocrine sciences center seems to support this . 
 the conditions of ihh can be reversed in about 10% of the patients , and all of these reversed cases received androgen replacement therapy , although the course of treatment may vary from individual to individual . 
 therefore , they postulated that androgen may be able to improve the plasticity of the brain nerve network in generating gnrh , thus promoting reversal of the ihh condition . 
 androgen has been found in regulating the generation of neurons in human olfactory epithelium , olfactory bulb , and hippocampal dentate gyrus and maintaining a normal number of neuronal synapses . in our investigation 
 , 62/68 patients received exogenous hcg and/or hmg therapy , and the feedback inhibition of exogenous testosterone does not exist , while only 6 patients receiving lhrh may be mildly influenced . 
 another simple reason is that the hcg dose may not have been adequately titrated up , so androgen was being used as a kind of  top - up  therapy to increase spermatogenesis efficiency and fertility in the males . 
 similar to a recent study , recombinant fsh pretreatment followed by gnrh is successful in inducing testicular growth and fertility in men with congenital hypogonadotropic hypogonadism with prepubertal testes . 
 the mechanism(s ) for the more favorable clinical outcome of g + a treatment over the simple g treatment can not be elucidated in the present study . 
 however , the phenomenon of  ihh reversal  as observed by harvard reproductive endocrine sciences center suggests that testosterone treatment may improve the therapeutic effect of gonadotropin through an unknown pathway or mechanism in a particular stage . 
 future randomized controlled clinical trials and mechanistic studies are needed before any definitive conclusions can be made . in conclusion 
 , disease - specific network investigation can be used as an alternative method of medical research , although its effectiveness needs to be further verified . in the present study , we made use of the network information technology to conduct an investigation on ihh and found that hormone replacement therapy could improve the conditions of ihh patients effectively . 
 in addition , the commonly used gonadotropin + androgen therapy appears to be superior to androgen replacement alone , although the actual clinical significance needs to be confirmed in prospective randomized control studies .","idiopathic hypogonadotropic hypogonadism ( ihh ) is a rare condition in which puberty does not take place naturally . 
 we aimed to develop and follow an internet - based cohort and to improve our understanding of the disease . 
 we established an internet - based questionnaire survey . 
 a total of 74 male ihh patients were recruited from the chinese largest ihh network social group . 
 the clinical symptoms before treatment mainly included small testis , underdeveloped secondary sexual characteristics , and sexual dysfunction . after treatment , 
 the penis length , testicular volume , external genital organ development , pubic hair , beard , laryngeal prominence , erection , and spermatorrhea were improved significantly ( p < 0.001 ) . 
 18.9% of the patients completed fertility ; however , more than half of the patients still complained of poor happiness and low physical strength . 
 in addition , improvements in penis and pubic hair development , testosterone normalization and the physical strength in ihh patients who received gonadotropin and androgen replacement therapy were better than in those who received single gonadotropin therapy ( p < 0.05 for all ) . in conclusion 
 , disease - specific network investigation can be used as an alternative method of medical research for rare diseases . 
 the results of our cross - sectional study showed the effectiveness of hormone replacement therapy for ihh and implied that gonadotropin and androgen replacement therapy may be superior to gonadotropin treatment alone .",pubmed
"nonsurgical endodontic retreatment is indicated as the primary choice to eliminate or substantially reduce the microbial content from endodontically failed teeth . 
 nevertheless , well - compacted root fillings offer resistance to the instruments used for retreatment , leaving root filling residues . 
 this restricts access to the apical foramen and impairs root canal disinfection and complete reshaping . 
 these include hand files , engine - driven rotary retreatment files ( rfs ) , ultrasonic tips / files , and heat - carrying instruments . 
 the use of rotary files successfully facilitates an operative removal of the bulk of gutta - percha - based fillings from the root canal system . however , many studies have reported that complete removal is not gained . 
 endodontic retreatment might result in pushing the irritants in the form of root canal filling materials , necrotic pulp tissues , bacteria , or root canal irrigants into the periapical region . 
 these extruded irritants are responsible for postoperative inflammation , flare - ups , and even impaired healing . according to the literature , all instrumentation techniques exhibit some degree of apical extrusion of debris , the amount varying with different instrumentation techniques . 
 recent studies have reported the inability of the universal protaper rfs ( d1-d3 ) to entirely remove the root canal fillings and proposed the use of supplementary files to aid removal of the root filling residues . 
 hence , the current study was formulated to evaluate the extrusion of debris apically after the use of protaper universal rfs and different finishing files for removing the residual root canal fillings . 
 the finishing files used in the current study were protaper next ( ptn ) , waveone ( wo ) , and self - adjusting file ( saf ) . 
 the null hypothesis tested was that the three different supplementary files used would exhibit no significant differences in the amount of debris extruded . 
 standard oval access was gained with access cavity burs ( endo z access kit , dentsply tulsa , oklahoma , usa ) . 
 the canal patency was accomplished and working length ( wl ) was determined as 1 mm short of the length of a size 15 k - file that was visible beyond the apical foramen . 
 the teeth were cleaned and shaped using protaper universal rotary files ( sx - f3 ; dentsply maillefer , ballaigues , switzerland ) . 
 irrigation was performed using a 27-gauge side - vented needle placed 2 mm short of the apex between each file with sodium hypochlorite ( naocl ; 5.25% ) . 
 the shaping procedure was continued till size f3 reached wl , followed by a final irrigation with 1 ml naocl . 
 ethylenediaminetetraacetic acid ( edta ) gel ( rc help , prime dental products , mumbai , india ) served as a lubricant for the file with every reinsertion . 
 the canals were then irrigated with 3 ml of 17% aqueous edta solution , which was followed by flushing the canal with distilled water and drying the canal with paper points . 
 the root canal obturation was done by standardized f3 master cones ( dentsply maillefer ) and ah - plus sealer ( dentsply - detrey , konstanz , germany ) by warm lateral compaction , adding accessory cones . 
 the excess gutta - percha was sheared by hot hand plugger and the access cavities were sealed using composite resin . 
 the teeth were then kept for 30 days at 37c and 100% humidity to allow complete setting of the sealer . 
 following this , these 80 samples were randomly divided into four experimental groups for retreatment . 
 the present study used the model described by myers and montgomery for evaluating apical extrusion of debris . 
 an analytical balance ( single pan k-15 , k roy instruments pvt . ltd . , 
 varanasi , india ) with an accuracy of 10 g was used to help determine the initial weights of the tubes . 
 three consecutive weights were obtained for each tube ( pre- and postinstrumentation ) , and the mean was computed . the root fillings in this group 
 were removed using protaper universal rfs : d1 , d2 , and d3 at 2-ncm torque and 500-rpm speed using x - smart plus endomotor ( dentsply maillefer ) . 
 the files were used in a brushing motion resting against the walls in a crown - down direction until it reached the wl . 
 d1 was used only for the cervical third , d2 only for the middle third , and d3 through the entire wl . 
 the rfs were used for removing the bulk of the root canal fillings as described in group 1 . 
 then , ptn files ( x2 , x3 , and x4 ) were used to remove the root filling residues that remained . 
 these files were used in an x - smart plus endomotor ( dentsply maillefer ) at 300 rpm and 3-ncm torque . whenever the file met resistance the file was withdrawn , the canal was irrigated , the canal was dried , and the procedure was continued . 
 the above procedure was performed till no root filling material debris was seen on the files after the x4 reached the wl . 
 the initial retreatment protocol was performed in a similar way to that in group 1 . following the removal of bulk , a wo large file 
 this file was used in a preprogrammed endomotor x - smart plus ( dentsply maillfer ) . 
 after every three pecks the file was withdrawn , the canal was irrigated , and the file was reintroduced into the canal . 
 the above procedure was performed till no root filling debris was seen on the file after the file reached the wl , as in group 2 . 
 after the removal of the bulk of root canal filling by rfs , the residual filling was removed using saf ( 2.0 mm ) in this group . 
 this file instrumentation was accompanied by continuous irrigation ( bidistilled water ) provided by a peristaltic in - built pump in the endostation ( redent nova , raanana , israel ) for 1 min , removing the coarse root fillings . following this , 
 the pump was turned off , the canal was filled with endosolv r ( septodont , paris , france ) , and saf was operated for 1 min . 
 again the canal was irrigated by bidistilled water , and dried and filled with drops of solvent ; saf was operated for additional 1 min . 
 next , the pump was turned on and the file was operated for the last 1 min . 
 the file was used in the canal till it reached the wl . after each instrument ( rotary ) or after three pecks ( reciprocating ) , 
 the irrigation needle was placed as deep as possible into the canal but not deeper as the predetermined wl minus 1 mm . 
 for all the groups , endosolv r was used as a gutta - percha solvent . the debris extruded by groups 2 , 3 , and 4 
 the teeth were removed from the tube and the debris adhering to the root surface was collected by washing off the apical area of the tooth with 1 ml of distilled water into the centrifuge tube . 
 the centrifuge tube was stored in an incubator at 70c for 5 days to allow the moisture to evaporate , before weighing the dry debris , using an electronic balance [ figure 1 ] . 
 three consecutive weight measurements were then taken for each collection assembly , with the mean value recorded . 
 collected debris after retreatment procedure the one - way analysis of variance ( anova ) and post hoc test were used to determine the significant group ( spss 20 , spss inc , chicago , il , usa ) with the alpha - type error set at 0.05 . 
 the weights obtained after the use of three experimental supplementary files ( ptn , wo , and saf ) were compared to the control group to find out the percentage ( % ) of the debris extruded . 
  
 the root fillings in this group were removed using protaper universal rfs : d1 , d2 , and d3 at 2-ncm torque and 500-rpm speed using x - smart plus endomotor ( dentsply maillefer ) . 
 the files were used in a brushing motion resting against the walls in a crown - down direction until it reached the wl . 
 d1 was used only for the cervical third , d2 only for the middle third , and d3 through the entire wl . 
 the rfs were used for removing the bulk of the root canal fillings as described in group 1 . 
 then , ptn files ( x2 , x3 , and x4 ) were used to remove the root filling residues that remained . 
 these files were used in an x - smart plus endomotor ( dentsply maillefer ) at 300 rpm and 3-ncm torque . whenever the file met resistance the file was withdrawn , the canal was irrigated , the canal was dried , and the procedure was continued . 
 the above procedure was performed till no root filling material debris was seen on the files after the x4 reached the wl . 
 the initial retreatment protocol was performed in a similar way to that in group 1 . following the removal of bulk , 
 this file was used in a preprogrammed endomotor x - smart plus ( dentsply maillfer ) . 
 after every three pecks the file was withdrawn , the canal was irrigated , and the file was reintroduced into the canal . 
 the above procedure was performed till no root filling debris was seen on the file after the file reached the wl , as in group 2 . 
 after the removal of the bulk of root canal filling by rfs , the residual filling was removed using saf ( 2.0 mm ) in this group . 
 this file instrumentation was accompanied by continuous irrigation ( bidistilled water ) provided by a peristaltic in - built pump in the endostation ( redent nova , raanana , israel ) for 1 min , removing the coarse root fillings . following this , 
 the pump was turned off , the canal was filled with endosolv r ( septodont , paris , france ) , and saf was operated for 1 min . again the canal was irrigated by bidistilled water , and dried and filled with drops of solvent ; saf was operated for additional 1 min . 
 next , the pump was turned on and the file was operated for the last 1 min . 
 after each instrument ( rotary ) or after three pecks ( reciprocating ) , 2 ml of bidistilled water was used as irrigant . the irrigation needle was placed as deep as possible into the canal but not deeper as the predetermined wl minus 1 mm . 
 for all the groups , endosolv r was used as a gutta - percha solvent . the debris extruded by groups 2 , 3 , and 4 
 following the completion of the retreatment procedure , the teeth were removed from the tube and the debris adhering to the root surface was collected by washing off the apical area of the tooth with 1 ml of distilled water into the centrifuge tube . 
 the centrifuge tube was stored in an incubator at 70c for 5 days to allow the moisture to evaporate , before weighing the dry debris , using an electronic balance [ figure 1 ] . 
 three consecutive weight measurements were then taken for each collection assembly , with the mean value recorded . collected debris after retreatment procedure 
 the one - way analysis of variance ( anova ) and post hoc test were used to determine the significant group ( spss 20 , spss inc , chicago , il , usa ) with the alpha - type error set at 0.05 . 
 the weights obtained after the use of three experimental supplementary files ( ptn , wo , and saf ) were compared to the control group to find out the percentage ( % ) of the debris extruded . 
 the median and standard deviation of debris extrusion by the groups is shown in table 1 . 
 the weights obtained after the supplementary use of ptn , wo , and saf were subtracted from the control group , giving as the result the actual amount of debris extruded by the supplementary files used for retreatment [ table 2 ] . according to the statistical results of the twofold retreatment protocols , 
 the wo file used after rfs extruded significantly more debris , followed by ptn and saf ( p < 0.01 ) . 
 saf was associated with the least extrusion of debris among the three experimental groups ( p < 0.01 ) [ graph 1 ] . when compared to the control group , wo was associated with 27% increase in the extrusion of debris , ptn with 19% increase , and saf with 12% increase [ graph 2 ] . 
 in addition , the debris extruded by twofold retreatment protocol using three investigational files after the rfs median and standard deviation of debris extruded by the twofold retreatment procedure subtracted from the control group . 
 all the groups were statistically significant ( p < 0.01 ) meanwise distribution of the weights of apically extruded debris by the three experimental files ( ptn , wo , and saf ) after the use of universal protaper rfs . 
 statistically significant difference seen in all the three groups with waveone ( * ) exhibiting the highest and saf ( # ) exhibiting the lowest extrusion of debris when used in retreatment percentage of increased debris extruded by the experimental files after subtracting from the weights obtained from the control group . 
 wo was associated with 27% , ptn with 19% , and saf with 12% increase in debris when used as supplementary files after using the pt rfs 
 the results of the current study exhibited that debris extruded apically regardless of the retreatment protocol . 
 the current study aimed to conclude the debris extrusion using supplementary files after using protaper universal rfs . 
 this twofold retreatment protocol was formulated , as previous studies stated that the rfs alone fail to render the canals free of root fillings . 
 using wo after rfs resulted in more extrusion of the debris apically when compared to ptn and saf ( p < 0.01 ) . 
 after the use of rfs , it is important to use supplementary files , in order refine the apical preparation because of the diameter of a d3 file being equal to size 20 , impairing any reasonable cleaning of the apical portion . 
 using supplementary files for finishing provides a cleaner root canal , facilitating removal of the residual root fillings that cause periapical extrusion of debris . 
 hence , the current study compared debris extrusion by supplementary files to the control group . 
 the results obtained for the control group are in accordance with earlier studies ; however , to the best of the authors knowledge , there is no report on apical extrusion of debris using supplementary files ptn , wo , or saf . 
 additionally , in the present study bidistilled water was used as an irrigation solution to prevent possible crystallization of naocl . 
 the saf group was associated with the lowest percentage of debris extruded apically in the current study ( 12% ) . 
 it is a single - file system , devoid of a central metal core and any cutting edge or flutes , and instead has an abrasive surface . 
 this hollow file adapts to the canal irregularities and the abrasive surface helps in creating fine dust while shaping , which is easily removed by associated , continuous irrigation . 
 the continuous flow of irrigant does not build up any pressure in the canal as the metal meshwork allows free escape of the irrigant . in the narrowest apical part , the saf leaves more than 38% of the canal cross section free for backflow of fluid and dentin debris . 
 de melo ribeiro et al . stated that in the apical third , the saf created cleaner canal walls when compared to the rotary system . 
 the high amount of debris formed in the apical third may also cause its extrusion periapically . 
 higher extrusion of debris by wo has been reported in the literature by various studies on primary endodontic in vitro studies , which can be attributed to the results of the present retreatment study as well . 
 the wo files are characterized by a modified triangular cross section , which results in decreased cutting efficacy and smaller chip space , resulting in auguring the formed debris after instrumentation , periapically . 
 the wo files also exhibit a larger taper of 0.08 at the apical 3 mm , which can be attributed to excessive debris formation apically , and extrusion periapically . 
 the ptn file system has very rare reports on the apical extrusion of debris with its instrumentation . 
 this design provides more cross - sectional space for enhanced cutting , loading , and successfully allowing the debris to travel out of a canal ( coronally ) . 
 in addition , the swaggering motion of the file while in rotation may help the file to touch a greater root canal area . 
 this may aid in better removal of the root canal filling residue and hence was used in one of the experimental groups in the current study . 
 the samples in the group instrumented by ptn resulted in less extrusion of the debris when compared to the wo group . 
 the current study was an attempt to find the amount of debris extruded by the use of supplementary files after using protaper universal rfs . under the experimental conditions of the present study 
 , it can be possible to conclude that all retreatment systems caused apical debris extrusion . 
 saf instrumentation after the rfs results in less extrusion than ptn and wo . there are no conflicts of interest . 
  
 ","aim : this study evaluated whether using supplementary files for removing root canal filling residues after protaper universal retreatment files ( rfs ) increased the debris extrusion apically.materials and methods : eighty mandibular premolars with single root and canal were instrumented with protaper universal rotary system ( sx - f3 ) and obturated . 
 the samples were divided randomly into four groups ( n = 20 ) . 
 group 1 served as a control ; only protaper universal rfs d1d3 were used , and the extruded debris was weighed . 
 groups 2 , 3 , and 4 were the experimental groups , receiving a twofold retreatment protocol : removal of the bulk , followed by the use of supplementary files . 
 the bulk was removed by rfs , followed by the use of protaper next ( ptn ) , waveone ( wo ) , and self - adjusting file ( saf ) for removal of the remaining root filling residues . 
 debris extruded apically were weighed and compared to the control group . 
 statistical analysis was performed using one - way analysis of variance ( anova ) and post hoc tukey 's test.results:all the three experimental groups presented significant difference ( p < .01 ) . 
 the post hoc tukey 's test confirmed that group 4 ( saf ) exhibited significantly less ( p < .01 ) debris extrusion between the three groups tested.conclusion:saf results in less extrusion of debris when used as supplementary file to remove root - filling residues , compared to wo and ptn .",pubmed
"traditionally , maxillary teeth have been anesthetized by administering an injection in the mucobuccal supraperiosteal fold in the proximity to the apices of teeth to be anesthetized . 
 it is a convenient , safe , and effective way to achieve pulpal anesthesia in maxillary teeth and associated tissues . 
 the disadvantage is that it requires multiple injections to cover more than one tooth and causing concomitant anesthesia of the lip and facial muscles . 
 however , the maxillary teeth can also be anesthetized with troncular techniques ( eg , infraorbital , maxillary ) or intraosseous and intraligamentary injection . 
 the literature did not describe a technique that is produced with a single injection , multiple teeth anesthesia in maxilla , and without causing collateral anesthesia of the face . 
 therefore , friedman and hochman ( 1998 ) proposed an anesthetic technique in maxilla , the anterior and middle superior alveolar ( amsa ) nerve block . 
 the authors state effective pulpal anesthesia from the central incisor to second premolar through palatal approach with a single injection and an expected anesthesia duration of 45 minutes to an hour [ figure 1 ] , using a computer - controlled injection system : the wand plus ( milestone scientific , deerfield ) . 
 furthermore , the authors affirm that the anesthesia is achieved without causing lip numbness and no interference with the muscles of the facial expression . 
 anesthetic area of amsa injection : buccal gingiva , teeth ( central incisor , lateral incisor , canine , first premolar , second premolar and mesiobuccal root of first molar ) , palatine gingiva and mucosa to midline amsa nerve block derives its name from the supposedly anesthesia of the anterior and middle superior alveolar nerves due to diffusion of the anesthetic solution via numerous nutrient channels on the palatal process of the maxillary bone [ figure 2 ] . 
 both nerves are collateral branches of the infraorbital nerve in the homonymous canal and part of the maxillary nerve.[1710 ] the anterior superior alveolar ( asa ) nerve originates approximately 5 - 8 mm posterior to the infraorbital foramen . 
 it is responsible for the pulpal innervation of the central incisor , lateral incisor , and canine.[1710 ] meanwhile , the middle superior alveolar ( msa ) nerve originates approximately 10 mm posterior to the infraorbital foramen . 
 it is responsible for pulpal innervation of the premolars and the mesiobuccal root of the first molar.[1710 ] however , the msa nerve is not always present in several human dissection studies , which shows their presence in a range of 30 - 72%.[710 ] when the msa nerve is absent , a plexus is formed between the posterior superior alveolar nerve and the asa nerve to supply its innervation.[710 ] palatal nutrient channels ( arrows ) the amsa nerve block injection site is located palatally at a point that bisects the premolars and is approximately halfway between the midpalatine raphe and the crest of the free gingival margin [ figure 3 ] . 
 this injection site is the confluence area of the asa and msa nerves , or the plexus when the last nerve is absent . 
 theoretically , this technique would be beneficial , because a bilateral amsa nerve block technique supposedly anesthetizes 10 maxillary teeth extending from the second premolar of one side to the opposite side without causing anesthesia of the facial muscles , making it a great advantage in restorative dentistry . 
 injection site ( arrow ) , halfway along an imaginary line ( a ) connecting the mid - palatal suture ( b ) to the free gingival margin ( c ) fukayama et al . and lee et al . 
 evaluated the efficacy of this technique using the computer - assisted injection system ( wand ) , but the percentages of pulpal anesthesia obtained were questionable . 
 other studies have reported its application in periodontal surgery but using the conventional syringe instead of the computer - assisted injection system . 
 the benefits are excellent hemostatic control of palatal soft tissue , avoiding collateral facial anesthesia and fewer cumulative injections . due to few studies about this technique in literature , 
 the aim of our study is to determine the success rates of pulpal anesthesia with the amsa nerve block technique using the conventional syringe ( carpule type ) . 
 in this controlled clinical study , 30 caucasians patients ( 16 men and 14 women ) , with an average age of 22 years - old ( range , 21 - 25 years - old ) , belonging to the school of dentistry of los andes university ( santiago , chile ) , were selected . during the selection of patients , 
 the following exclusion criteria were established : presence of systemic pathologies that contraindicate local anesthetics with vasoconstrictors , pregnancy , use of medications that alter pain perception , active orthodontic treatment , extractions , fixed prostheses , extensive fillings , and/or endodontic treatment in the experimental teeth ( maxillary central incisor , lateral incisor , canine , and first and second premolars ) . 
 this study was approved by the ethics committee of the school of dentistry ( los andes university ) , and patients voluntarily decided to participate in the study by signing the written informed consent . 
 the 30 patients received an amsa nerve block on one side of the maxilla randomly selected using the conventional syringe ( carpule type , hu - friedly ) , 1 ml of lidocaine 2% with epinephrine 1:100,000 ( octocaine100 , novocol pharmaceutical ) was injected to all patients . 
 the teeth that were tested are , the maxillary central incisor , lateral incisor , canine , and first and second premolars of the side previously chosen . 
 the mandibular canine was used as the non - anesthetized control tooth to ensure that the pulp tester was operating adequately during the study . at the beginning of each appointment and 
 before any injections were given , the experimental teeth and control canine were tested with the pulp tester ( sybron endo , vitality scanner ) to record baseline vitality . 
 after isolation with cotton rolls , the topical anesthetic ( benzocaine 20% ; alpha dental ) was applied to the tip of the pulp tester , which was placed midway between the gingival margin and the incisal or occlusal border of the tooth to be tested . 
 the current rate was set on the pulp tester at 25 seconds to increase from no output ( 0 ) to the maximum output ( 80 ) . to establish the injection site on the palate , 
 the amsa injection site was centered halfway between the midpalatine raphe and the gingival margin of the first and second premolars [ figure 3 ] . 
 a cotton - tip applicator was used to apply the topical anesthetic ( benzocaine 20% ; alpha dental ) at the injection site for a minute . 
 the injection was given with a 30-gauge needle , 22 mm ( terumo dental needle ) . 
 the needle with the bevel against the bone was oriented at an angle of 45. then slowly the needle penetrated the palatal mucosa to establish contact with the palatal bone , to deposit 1 ml of anesthetic solution ( lidocaine 2% with epinephrine 1:100,000 ; octocaine100 , pharmaceutical novocol ) in three minutes [ figure 4 ] . 
 execution of the technique the anesthetic success was monitored with the electric pulp tester ( sybron endo , vitality scanner ) , and we followed the protocol established by lee et al . in their study . 
 a minute after the amsa injection , pulp test readings were obtained for the first and second premolars . at two minutes 
 the testing continued in four - minute cycles for a period of an hour . no response from the subject to the maximum output ( 
 no response at the 80 reading ) of the pulp tester was used as the criterion for pulpal anesthesia . 
 anesthetic success only considers when two consecutive 80 readings occur at some point over an hour . 
 the induction time ( time it takes to succeed anesthetic ) and the duration of anesthetic success was also registered . 
 additionally , we verify that there was anesthesia in the palatal soft tissue , upper lip , and the surface of the face . 
 thirty adult patients participated in this study , 16 men and 14 women with an average age of 22 years- old ( range , 21 - 25 years- old ) . all the patients received an amsa nerve block on one side of the maxilla randomly selected using the conventional syringe ( carpule type , hu - friedly ) , 1 ml of lidocaine 2% with epinephrine 1:100,000 ( octocaine100 , novocol pharmaceutical ) was injected to all patients . 
 the anesthetic success of the amsa nerve block technique using conventional syringe is presented in table 1 . 
 the anesthetic induction times ranged from 6 to 12 minutes and the duration of pulpal anesthesia ranged between 23 - 40 min [ table 1 ] . 
 percentages of anesthetic success ( two consecutive no response at the 80 reading of the pulpal tester ) , mean anesthetic induction time ( min ) and mean anesthetic duration time ( min ) for every experimental tooth with amsa nerve block all the subjects obtained palatal soft tissue anesthesia , extending from the central incisor to the mesial of the first molar , never crossing the midline . 
 no patient obtained pulpal anesthesia from the second premolar to the central incisor and facial anesthesia . while , in eight patients ( 26.7% ) had no anesthetic success in any of the experimental teeth . 
 no severe pain was recorded during the administration of the local anesthetic in the palate . any complications or adverse reactions during and/or after completion of the anesthetic technique 
 the main theoretical advantage of this amsa nerve block is that it reduces the number of injections and the quantity of anesthetic solution administered in comparison with the conventional supraperiosteal infiltrative anesthesia applied in multiple injections for each tooth . 
 in addition , it would be ideal to use in cosmetic dentistry as it does not cause numbness of the lip and face . 
 however , our results show pulpal anesthetic success rates ( two readings in 80 of the pulp tester ) ranging from 16.7 to 66.6% . 
 failure to respond to 80 ( maximum output of the pulp tester ) was the criterion for pulpal anesthesia based on clinical studies by dreven et al . and certosimo et al . 
 , because their studies showed that the readings below 80 resulted in pain during operatory procedures . in our study , 
 the anesthetic blockade of the five experimental teeth was not obtained in any of our patients as expounded by friedman and hochman . 
 the duration of pulpal anesthesia was gradually declining during the 60 minutes ; we can not confirm the clinical impression of the authors that there is duration of pulpal anesthesia for 60 minutes . on the other hand 
 , we can say that there is no anesthesia of the lips and facial muscles of expression . 
 studies of this technique using a computer - controlled system , which was made by fukayama et al . 
 , obtained anesthetic success in the range of 42 - 86% and 35 - 58% by lee et al . in our study , the success rates were significantly low with the use of conventional syringe ( 17 - 66% ) . 
 the low success rates of conventional syringe system could be due to the superiority of the computer - controlled system to inject the anesthetic solution with a controlled and continuous flow . with the conventional syringe system , 
 the flow pressure depends on the operator although all injections were performed by the same investigator . 
 the anesthetic flow can not be controlled as precisely as with the computer - controlled system . 
 speculated that the injection with the computer - controlled system creates an improved pressure gradient environment for diffusion of the anesthetic solution through the numerous nutrient channels of the palatal vault . in the case of the low anesthesia percentages , 
 the central and lateral incisor could be attributed to an increased presence of the msa nerve in the patients of the study . 
 thus , in such cases , the msa nerve is anesthetized , and not the asa nerve , due to its distance from the puncture site . 
 anatomical studies on dissection of cadavers have found the presence of the msa nerve in the range of 30 - 72% , and when it is absent , its innervation is supplied from a plexus formed by the asa and the posterior superior alveolar nerves.[1810 ] the exact role of the absence of the msa nerve in the amsa nerve block success is not known . 
 malamed defines a nerve block when the solution is deposited in the vicinity of the main nerve trunk , so that the anesthetic should be placed close to the asa or msa nerves , resulting in high rates of anesthetic success , but we saw that this did not happen because the solution is deposited on the palatine process of the maxilla for its diffusion , searching the terminal branches of the alveolar nerves . 
 so based on our clinical observations , it is more of an infiltrative technique than a nerve block . on the other hand , the reduction of multiple injections reduces the total amount of delivered vasoconstrictor and may be useful for cardiovascular - compromised patients that required maxillary anesthesia . 
 for maxillary mucogingival procedures , the amsa nerve block with palatal delivery of anesthetic with vasoconstrictor provides excellent hemostasis and reduces the need for multiple re - injections to obtain hemostatic control during periodontal procedures or graft harvest . 
 one of the main disadvantages of the amsa nerve block is that palatal injections are generally considered the most painful injections . 
 wahl et al . showed that palatal injections caused significantly more pain than other intraoral injections , probably due to the result of pressure . 
 however , in our study , severe pain during the technique was not reported , possibly due to the prior application of topical anesthetic and the slow and controlled injection of the anesthetic solution . other problem of this technique is that it is restricted only for the five anterior maxillary teeth and procedures such as periodontal surgeries require anesthesia of the full maxillary arch that may complement with another technique . 
 a second technique that would produce anesthesia from the central incisor to second premolar with a single injection is the infraorbital nerve block ( ionb ) . 
 having the advantage that the injection site is not in the palate , it is therefore less painful , but with the inconvenience of causing facial numbness . 
 karkut et al . in their study found that ionbs were ineffective in providing profound pulpal anesthesia of the maxillary central incisor ( 15% success rate ) and lateral incisor ( 22% success rate ) . 
 the pulpal anesthesia success rate was 92% for the canine and 80% for first and second premolars . 
 moreover , berbeich et al . obtained similar results in 40 patients with ionb where pulpal anesthesia was ineffective in providing profound anesthesia of maxillary central and lateral incisors . 
 successful pulpal anesthesia of the canine and first and second premolars ranged from 75 - 92% . 
 these previously discussed studies occupy anesthetic evaluation protocols similar to those in our study , but getting better rates of success with the ionb . 
 although corbett et al . conducted a study to compare the efficacy of the amsa nerve block with that of the ionb in achieving pulpal anesthesia in the anterior maxilla , anesthetic success was significantly greater with the amsa nerve block than with the ionb in central and lateral incisors ; however , anesthesia was achieved in only 42.9% of central incisors with the amsa nerve block . 
 the authors observed a significantly greater number of anesthetic success episodes in the premolar and canine teeth after ionb . 
 the incidence of subjective lip numbness was 100% after ionb and 14.3% after amsa nerve block . 
 there was no significant difference in the rates of injection discomfort between the two techniques . 
 the authors conclude that the ionb has better success rates of pulpal anesthetic , but amsa has the advantage that the same injection has the same potential to provide pulpal anesthesia from central incisor to second premolar . in addition , the amsa nerve block anesthetize palatine tissue without causing facial numbness . 
 the shape of the palate was not recorded but we believe that there was a homogeneous distribution between the deep and shallow palate in this study . because this anesthetic technique was applied to young population , the results may not be applied to children or the elderly . 
 because of the unpredictable anesthetic success of the experimental teeth and variable anesthesia duration , the technique is disadvantageous for clinical application as the first choice , counting with other techniques that have greater efficacy in the maxilla 
 . however , it may be clinically useful in restorative dentistry ( by not anesthetizing the facial muscles and not affecting the smile line ) and in periodontal surgery due to the excellent hemostatic control in palatal soft tissues .","background : dental procedures in the maxilla typically require multiple injections and may inadvertently anesthetize facial structures and affect the smile line . to minimize these inconveniences and reduce the number of total injections , a relatively new injection technique has been proposed for maxillary procedures , the anterior and middle superior alveolar ( amsa ) nerve block , which achieves pulpal anesthesia from the central incisor to second premolar through palatal approach with a single injection . 
 the purpose of this article is to provide background information on the anterior and middle superior alveolar nerve block and demonstrate its success rates of pulpal anesthesia using the conventional syringe.materials and methods : thirty caucasian patients ( 16 men and 14 women ) with an average age of 22 years - old , belonging to the school of dentistry of los andes university , were selected . 
 all the patients received an amsa nerve block on one side of the maxilla using the conventional syringe , 1 ml of lidocaine 2% with epinephrine 1:100.000 was injected to all the patients.results:the amsa nerve block obtained a 66% anesthetic success in the second premolar , 40% in the first premolar , 60% in the canine , 23.3% in the lateral incisor , and 16.7% in the central incisor.conclusions:because of the unpredictable anesthetic success of the experimental teeth and variable anesthesia duration , the technique is disadvantageous for clinical application as the first choice , counting with other techniques that have greater efficacy in the maxilla . 
 although , anesthetizing the teeth without numbing the facial muscles may be useful in restorative dentistry .",pubmed
"malaria continues to be a serious public health problem in south - east asia including india . about 36% of the world 's population ( 2020 million ) 
 the south - east asian region contributes 2.5 million cases to the global burden of malaria , of which india 's contribution is 76% . according to the world malaria report 2014 , 22% ( 275.5 m ) of india 's population live in high transmission ( > 1 case/1000 population ) areas , 67% ( 838.9 m ) live in low transmission ( 01 cases/1000 population ) areas , and 11% ( 137.7 m ) live in malaria - free ( 0 cases ) areas . in 2013 , 
 0.88 million cases have been recorded , with 128 million tests being conducted on the suspected cases , with plasmodium falciparum causing 53% and plasmodium vivax causing 47% of the infections . 
 the incidence of malaria in india accounted for 58% of cases in the south - east asia region of who . at present , 
 official figures for malaria in india , available at national vector borne disease control programme , indicate 0.71.6 million confirmed cases and 4001,000 deaths annually . however , a study conducted by teams from the office of the registrar general of india , centre for global health research at st . 
 michael 's hospital and university of toronto , canada , published in the lancet on november 20 , 2010 has reported that malaria causes 205,000 malaria deaths per year in india before age 70 years ( 55,000 in early childhood , 30,000 at ages 514 years , and 120,000 at ages 1569 years ) with a 1.8% cumulative probability of death from malaria before age 70 years . 
 the report says that 90% of the deaths were recorded in rural areas , of which 86% occurred at home without any medical attention . 
 beliefs and practices of malaria are often related to culture which can influence the effectiveness of control strategies . 
 thus , local knowledge and practices related to the disease are key to implementation of culturally appropriate , sustainable , and effective interventions . 
 community perception , beliefs , and attitude about malaria control , symptom identification , treatment , and prevention influence efforts to address malaria and are often overlooked in control efforts ; and vary from country to country and among individual households . failure to consider community 's knowledge , attitude , and practice ( kap ) about malaria may contribute to the inability of the program to achieve sustainable control . 
 hence , kap can be an important step in developing strategies aimed at controlling malaria . 
 extensive pubmed search reveals very little in terms of evidence regarding the current kaps of the population in general and rural population , in particular , in this part of the country . keeping this in view , 
 further , the state of jammu and kashmir falls in the low endemic zone with annual parasite incidence ( api ) of 0.11 only . 
 however , the major concentration of malaria cases in jammu and kashmir is restricted to jammu division of the state only with kashmir division recording an api of < 0.1 . in this 
 , the jammu division of the state resembles the rest of north india more than the kashmir division in the same state . 
 this differential distribution of malaria cases within a state with a low incidence of malaria is of great public health importance . 
 the importance will lie in our ability to identify the potential contributors to this differential distribution . 
 it also opens up the potential for developing preventive strategies across different parts of india . 
 a community - based descriptive cross - sectional study was carried out in rs pura health block of jammu district in jammu and kashmir state in north india . 
 the main outcome of interest was assessment of kap regarding malaria among inhabitants rs pura health block . 
 rs pura block is located in the south - west of jammu city adjacent to the indo - pak border with a total area of 273 km and average density of 658/km . 
 there are 176 villages and 1 town ( 11 wards ) in the block with an estimated population of 179,636 . 
 the methodology used for the purpose of this study has been developed by institute for research in medical statistics as an alternative to probability proportionate to size ( pps ) methodology . 
 the pps methodology although self - weighing in nature has a somewhat centrist bias which distorts this property . to obviate this bias , alternative methodology ( irms ) was developed and tested with beneficial results across different parts of india by scientists from indian council of medical research . 
 a sample size of 300 distributed as described below is in agreement with this methodology . 
 hence , no separate sampling strategy was adopted to identify the required number of households to be included in the study . 
 the same methodology has been used in the current study . for the purpose of this study , only the rural area of the block 
 the stratification was done in accordance with population size of villages located in rs pura block . 
 the villages were identified as the primary sampling units while households located within the villages were identified as the secondary sampling units . 
 the stratification was conducted as detailed below : 
 stratum 1 small villages - villages with population of < 500stratum 2 moderately small villages - villages with population of 500999stratum 3 medium size villages - villages with population of 10001999stratum 4 large size - villages with population 2000 . 
 stratum 1 small villages - villages with population of < 500 stratum 2 moderately small villages - villages with population of 500999 stratum 3 medium size villages - villages with population of 10001999 stratum 4 large size - villages with population 2000 . 
 , 15 households with at least one member above age of 18 years at the time of the study were selected . 
 the village selected was mapped , and a house - to - house survey was conducted until 15 households were completed . only one adult per household was interviewed . 
 study villages from rs pura block from these 300 households , 300 adult respondents were identified for administration of the study questionnaire . 
 the questionnaire was administered either to the head of the household or in his absence to a responsible adult above 18 years of age . 
 the questionnaire included questions on : demographic details , on knowledge and understanding of malaria transmission , on recognition of sign and symptoms , on perception of cause , treatment seeking patterns , preventive measures , and protective patterns . 
 stratum 1 small villages - villages with population of < 500stratum 2 moderately small villages - villages with population of 500999stratum 3 medium size villages - villages with population of 10001999stratum 4 large size - villages with population 2000 . 
 stratum 1 small villages - villages with population of < 500 stratum 2 moderately small villages - villages with population of 500999 stratum 3 medium size villages - villages with population of 10001999 stratum 4 large size - villages with population 2000 . 
 , 15 households with at least one member above age of 18 years at the time of the study were selected . 
 the village selected was mapped , and a house - to - house survey was conducted until 15 households were completed . 
 study villages from rs pura block from these 300 households , 300 adult respondents were identified for administration of the study questionnaire . 
 the questionnaire was administered either to the head of the household or in his absence to a responsible adult above 18 years of age . 
 the questionnaire included questions on : demographic details , on knowledge and understanding of malaria transmission , on recognition of sign and symptoms , on perception of cause , treatment seeking patterns , preventive measures , and protective patterns . 
  
 however , data on 4 households was found to be incomplete at the time of analysis and , therefore , were excluded . 
 out of 296 study participants interviewed 194 ( 65.5% ) were males , while 102 ( 34.5% ) females [ table 1 ] . 
 the demographics revealed that the maximum participants ( 33.7% ) belonged to the 4150 years age group . 
 education wise , one - third ( 29.7% ) of the participants were illiterate , and an almost equal proportion ( 29.1% ) had studied up to middle standard ( 8 grade ) . 
 maximum ( 31% ) number of study participants was agriculturists followed by homemakers ( 29.7% ) . more ( 56% ) number of study participants belonged to nuclear families . 
 the demographic details of the study participants all of the study participants had heard of malaria [ table 2 ] . 
 importantly , 97% of the study participants correctly associated malaria with mosquito bite [ table 2 ] . the majority ( 76.35% ) of the study participants had knowledge of malaria symptoms , and a majority of them had knowledge that malaria could be both prevented ( 90.5% ) as well as cured ( 93.2% ) . 
 further , a majority ( 77.7% ) of the study participants knew that malaria could be fatal if not treated . 
 an overwhelming ( 95% ) number of the study participants had knowledge regarding various modes of disease prevention . 
 knowledge about malaria among respondents the response to questions on knowledge with multiple options drew multiple responses . 
 however , the most common response ( 68.2% ) to questions on the source of information regarding malaria was television ( tv)/newspaper . 
 adequate knowledge was also found among them regarding resting habits of mosquitoes with only 2.8% of responses registering 
 the responses to other questions are detailed in table 2 . a majority ( 92.5% ) of the study participants 
 considered malaria to be a serious health problem , thus reflecting their attitude to the disease [ table 3 ] . 
 more than two - third of them would consult a doctor in case of child 's illness though the majority of them had no knowledge regarding malaria treatment . 
 the overall attitude of the study population toward the use of bed nets was positive and similar was the case with vector control program . 
 attitude of respondents towards malaria regarding practices , a majority ( 71.6% ) of the study participants preferred going to doctors at government hospitals for malaria treatment , and 56% of study participants were willing to seek medical help in < 24 h in case of a child having a febrile episode [ table 4 ] . with regard to personal protective measures , 
 regarding preventive practices , more than half of the respondents would avoid collections of stagnant water while a quarter of them would prefer indoor spraying . 
 this is of greater value in diseases such as malaria , wherein awareness about the cause and spread is a major stakeholder for prevention . 
 it assumes significance in a state like jammu and kashmir , wherein the distribution of malaria cases within this state is not geographically uniform . 
 therefore , the strength of this study lies in its ability to capture the probable contribution of disease awareness in lower local disease burden . 
 the findings of this study could shape the future discourse of research on local approaches to prevention of endemic diseases in india . 
 this is in agreement with findings of some studies but is in contrast to other studies . 
 this finding assumes importance in view of the fact that the study participants belong to a low incidence state and , therefore , have a lower exposure to cases of malaria . 
 this may be because the main source of information in the current study population was mass media ( tv / newspaper ) followed by friends / neighbors . 
 this finding is in contrast to the findings reported by singh et al . where an individual 's experience with malaria was the main source . 
 creation of awareness through mass media is an excellent model of intervention in the prevention of malaria . 
 reported health facility ( 29% ) as the main source of information among the respondents . 
 knowledge regarding causation of disease , its cure and prevention were very high in this study , though knowledge that it can be fatal was a bit on the lower side . 
 this again may be because of less number of personal interactions with patients of malaria . 
 the results from our study regarding the transmission of malaria are in contrast to a nigerian study where only a small proportion of respondents correctly answered questions about malaria transmission and its cause . 
 the study results have demonstrated that respondents had good knowledge about malaria signs and symptoms , and the results are consistent with some other studies . 
 this high level of awareness can best be explained by increased access to mass media and health education by government agencies . 
 97.2% respondents made correct association between malaria and mosquito bite which is in agreement with those reported by hlongwana et al . 
 knowledge regarding breeding and resting places of mosquitoes were high among the study population in line with the results reported by some other studies like the one by chirebuv et al . 
 similarly , knowledge regarding prevention of mosquito breeding and prevention of malaria was very high , and the results concur with those reported by other authors . 
 it must be mentioned that excellent knowledge does not guarantee better preventive measures as observed by the authors . 
 this could be explained by the low literacy levels and poor socioeconomic status of the study population and is reflective of the trend followed by health seekers in other health initiatives . 
 therefore , probably the key lies in creating awareness across all socioeconomic domains and identifying the possible bottlenecks . 
 the respondents attitude toward malaria was reflected when more than 90% labeled it as a serious health problem . the majority ( 70.9% ) 
 consulted a doctor in case the child was found to be febrile , and only 18.2% reported using a home remedy . 
 the results are in contrast to those reported by eversole et al . where treatment involved traditional medicines more frequently . 
 the reasons for better access to registered medical practitioner in our study could be attributed to the better availability of health care in rs pura block as the block is a part of medical college field practice area 
 . that may be the probable reasons for only 2.7% reporting to a traditional healer . 
 another positive finding was that the motivation to seek care for a febrile child was the condition of the child in an overwhelming 91.8% of the study population . 
 however , only 15.5% knew chloroquine to be the best treatment for malaria , and the most obvious reason could be low literacy in this rural population . 
 in contrast , khan et al . reported 90% respondents could name drugs used in malaria treatment . despite this attitude toward vector control program was very positive . 
 the practice of going to government hospital / doctor for malaria treatment was found at a healthy rate of 72% among the respondents and 56% would seeking help in < 24 h of the febrile episode . 
 the results are in agreement with those reported by hlongwana et al . though a higher rate ( 88% ) sought treatment within 24 h of onset of malaria symptoms . 
 the majority of the respondents in the current study had adequate knowledge about protective as well as preventive measures against malaria which concur with those reported by hlongwana et al . in the study conducted in south africa . 
 probably , a large study sample with distribution in rural , urban , and tribal areas of jammu and kashmir will provide us with a better understanding on the role knowledge and awareness on differential distribution malaria . 
 probably , a large study sample with distribution in rural , urban , and tribal areas of jammu and kashmir will provide us with a better understanding on the role knowledge and awareness on differential distribution malaria . 
 results revealed that kap among respondents were reasonably good and key sociocultural , and related indicators need to be identified as a part of malaria elimination strategy . 
  
 ","introduction : an extensive search on pubmed reveals very little in terms of evidence regarding the current knowledge , attitude , and practices ( kap ) of the population in general and rural population , in particular , in this part of the country . 
 therefore , a study was conducted with the aim to assess the communities knowledge of malaria transmission , recognition of signs and symptoms , treatment seeking.materials and methods : a stratified two - stage design was used to conduct a house - to - house survey using a semi - structured questionnaire in rs pura block of jammu district of jammu and kashmir state in north india.results:a total of 300 households were included in the study . 
 however , data on 4 households was found to be incomplete at the time of analysis and , therefore , were excluded . 
 out of 296 study participants interviewed 65.5% were males , while 34.5% females . 
 all of the study participants ( 100% ) had heard of malaria , and the main source of their information was television / newspaper . 
 92.5% of the study population considered malaria to be a serious health problem , thus reflecting their attitude to the disease . 
 regarding practices , 71.6% of the study participants preferred going to doctors at government hospitals for malaria treatment , and 56% were willing to seek medical help in < 24 h in case of a child has a febrile episode.conclusions:results revealed that kap among respondents were reasonably good and key sociocultural , and related indicators need to be identified as a part of malaria elimination strategy .",pubmed
"recent advances in molecular biology and microscopy technologies have enhanced our understanding about the higher - order folding of the genome in an unprecedented manner . 
 it is now widely accepted that the dynamic folding of the chromatin is fundamental in regulating gene expression and dna replication . 
 the genome is folded in a hierarchical manner into chromosome territories , genomic compartments , and topologically associating domains ( tads ) , in which specific long - range looping interactions occur . 
 each of these structures can be dynamically regulated during development , and perturbations in these folding units are associated with multiple diseases and cancer . 
 one interesting feature that was revealed upon fine mapping of the folding of the genome is the tad structures , which range from 100 kb up to 1 mb in size . 
 tads are units of chromosomes that exhibit higher frequency of physical contacts between genes and their cognate regulatory regions . 
 the tads have been shown to be stable across different species , cell types , and cellular conditions . 
 the replication timing , the presence of different histone modifications and the expression of the genes inside a tad are highly correlated . 
 however , the underlying features that generate invariant tad boundaries remain unknown . in human cells , 
 tad boundaries have been associated with the enrichment of ctcf , cohesin , dnase i hypersensitivity , certain histone marks and the timing of replication domains . 
 furthermore , in drosophila , the combinatorial binding of different types of insulators ( also referred to as architectural proteins ) , such as beaf32 and tfiiic , is associated with the strength of the tad boundary . 
 the boundaries serve as barriers to long- and short - range interactions between dna sequences , thus the most likely interaction partner for a genomic region is another region within the same tad . a strong tad boundary limits interactions between the two adjacent tad domains , whereas a weak tad boundary allows a higher frequency of inter - tad interactions . 
 although a relationship between tad boundaries and the binding of insulators has been demonstrated , the effects of enzymes that modify or remodel chromatin are largely unknown . 
 the major atpase subunit of the swi / snf chromatin remodeling complex , brg1 ( also known as smarca4 ) , is required for proper nucleosome occupancy and positioning at target regions as well as for establishing numerous long - range chromatin interactions . 
 , we highlight our recent finding that brg1 contributes to tad boundary strength and discuss the implications of brg1 loss on nucleosome occupancy and higher order chromatin organization . 
 to delineate the role of brg1 in genome architecture , we performed rna - seq and hi - c in control and brg1 knockdown mammary epithelial mcf-10a cells . moreover , to probe brg1 localization , we performed chip - seq in wild type parental mcf-10a cells . 
 we reported that brg1 regulates the expression of genes related to extracellular matrix and lipid synthesis . 
 in addition , brg1 knockdown resulted in the differential expression of many long non - coding rnas , which are thought to be involved in genome organization . 
 chip - seq analysis of brg1 demonstrated binding primarily ( 60% ) at genic regions . 
 intergenic brg1 peaks included super - enhancers , which are defined as clusters of regulatory regions with unusual enrichment of factor binding sites and/or histone modifications that are in close proximity , though this classification has recently been challenged . 
 comparison of the control and brg1 knockdown hi - c data sets revealed that the majority of the tad boundaries were largely overlapping between control and brg1 knockdown cells , consistent with prior results . 
 however , more than 10% of the tad boundaries were unique to either the control or the brg1 knockdown cells , providing evidence that brg1 may contribute to the integrity of tad boundaries . in support of this concept 
 this result may be expected since tad boundaries have been shown to be enriched for both housekeeping and developmentally regulated genes . surprisingly , the strength of the tad boundaries , as assessed using the insulation method , was significantly reduced in brg1 knockdown human mammary epithelial mcf-10a cells . in other words , upon brg1 knockdown , there was a higher frequency of inter - tad genomic interactions . 
 we further validated this result by intersecting tad boundaries with brg1 chip - seq data and categorizing the boundaries as either brg1 bound or not bound . 
 the strength of tad boundaries that were bound by brg1 was stronger than the boundaries that lacked brg1 localization . 
 an explanation for the effect of brg1 knockdown is that brg1 loss may disrupt chromatin accessibility and preclude the binding of transcription factors and/or chromatin modifiers at tad boundaries . to examine this possibility 
 , we analyzed a publicly available mnase - seq data set from wildtype and brg1 knockdown mouse embryonic fibroblast ( mef ) cells . by intersecting the nucleosome occupancy data with the encode mef ctcf chip - seq data 
 set , we showed that there was decreased nucleosome occupancy at ctcf - bound regions in brg1 knockdown cells when compared to control cells . 
 thus , our results suggested that brg1 plays a role at tad boundaries by regulating nucleosome occupancy and possibly ctcf localization . 
 dixon et  al . recently hypothesized that the orderly positioning and occupancy of nucleosomes at tad boundaries renders the local chromatin at the boundary  less flexible ,  and thus prevents long - range interactions surpassing the boundary . 
 the orderly positioning and occupancy of nucleosomes at tad boundaries may be achieved by enriched ctcf binding , which positions 20 nucleosomes around its binding sites . 
 our observation of the relationship between brg1 knockdown and the reduction of nucleosome occupancy around the ctcf sites supports such a mechanism . 
 it was recently shown that the directionality of ctcf binding is very strongly associated the formation of tads , though the relationship between brg1 binding and ctcf binding site orientation remains to be determined . 
 we propose that loss or knockdown of brg1 results in the reduction of nucleosome occupancy at ctcf sites either through loss of its atp - dependent chromatin remodeling activity or by negatively impacting ctcf binding ( fig .  1 ) . 
 such an effect of brg1 may also occur at regions where other chromatin organizers ( e.g. cohesin ) are bound . 
 figure 1.a schematic figure depicting the possible connection between brg1 , ctcf and topoisomerases . in the presence of brg1 ( top panel ) , ctcf and topoisomerases can efficiently bind to tad boundaries and promote proper nucleosome occupancy and uncoiling of the dna , resulting in a strong tad boundary . 
 therefore , we propose that upon brg1 knockdown , ctcf and topoisomerases may interact with tad boundary sequences , but the lack of atp - dependent remodeling activity may alter nucleosome occupancy and affect boundary strength ( middle panel ) . alternatively , the binding of ctcf and topoisomerases may be perturbed , resulting in altered nucleosome occupancy and reduced boundary strength ( bottom panel ) . 
 a schematic figure depicting the possible connection between brg1 , ctcf and topoisomerases . in the presence of brg1 ( top panel ) , ctcf and topoisomerases can efficiently bind to tad boundaries and promote proper nucleosome occupancy and uncoiling of the dna , resulting in a strong tad boundary . 
 therefore , we propose that upon brg1 knockdown , ctcf and topoisomerases may interact with tad boundary sequences , but the lack of atp - dependent remodeling activity may alter nucleosome occupancy and affect boundary strength ( middle panel ) . alternatively , the binding of ctcf and topoisomerases may be perturbed , resulting in altered nucleosome occupancy and reduced boundary strength ( bottom panel ) . 
 the model proposed by dixon et al . , is reinforced by accumulating evidence that highlights the role of the topoisomerase complex as a regulator of tad boundaries . 
 recent results demonstrate that topoisomerase ii beta ( top2b ) binding overlaps with almost half of ctcf / cohesin binding sites , and that top2b may facilitate supercoiling at ctcf sites in a transcription - dependent manner . 
 direct links between topoisomerases and brg1 are provided by a proteomic profiling using mass spectrometry that identified a significant interaction between brg1 and top2b . 
 consistent with this finding , brg1 also binds to topoisomerase ii alpha ( top2a ) . 
 furthermore , brg1 is required for the recruitment of topoisomerase i ( top1 ) to chromatin , and in the case of both top2a and top1 , the atpase activity of brg1 has been shown to be essential for the recruitment of the topoisomerase proteins . 
 these findings suggest an interplay and functional cooperativity between ctcf , brg1 and topoisomerases in the organization of tads via the active regulation of tad boundary regions . 
 additional evidence for engagement of topoisomerases in genome organization is provided by studies from bacteria and yeast . 
 a hi - c study in prokaryotes examined the biology of chromosomal interaction domains ( cids ) , which are prokaryotic chromatin folding structures that are analogous to the eukaryotic tads . 
 treatment of the prokaryotic cells with novobiocin , a drug that inhibits dna gyrase ( a homolog of topoisomerase ii ) and thus supercoiling , perturbed the sharpness and the positions of cid boundaries . in yeast , 
 fine resolution nucleosome mapping determined that self - associating domains similar to but smaller than tads exist and that their boundaries were bound by the rsc chromatin remodeling enzyme . 
 the rsc atpase is structurally related to brg1 , and the genetic analyses performed confirmed a role for rsc in yeast genome organization . 
 moreover , hrp1 , an atp - dependent chromatin remodeling protein from the chd subfamily , was shown to collaborate with top1 to maintain open chromatin at active gene regions in yeast . 
 taken together , these findings suggest a strong link between chromatin remodeling enzymes , including the mammalian swi / snf complex , architectural proteins , and topoisomerases in genome organization . 
 we propose that the association between brg1 knockdown and reduction in tad boundary strength may be due to perturbations in the recruitment of ctcf and topoisomerases , and may therefore affect integrity of the chromatin structure and the  stiffness  of the chromatin at tad boundaries ( fig .  1 ) . in further support of the chromatin 
 hypothesis , a recent study showed that a small deletion of a tad boundary was not sufficient to disrupt the tad domain , as it remained stable . 
 this result suggests that the boundary is not defined by the exact boundary sequence or length , but instead depends either on the supercoiling or the overall composition of the factors present at the boundary . our recent data indicating that brg1 , and hence the mammalian swi / snf chromatin remodeling enzyme , binds to tad boundaries and promotes boundary strength adds a novel biochemical activity , atp - dependent chromatin remodeling , to the complex structure that regulates tad formation and function . 
 other chromatin remodeling complexes may play similar roles , as these enzymes can function in a redundant manner . continued examination of the factors found at tad boundaries will yield important insights into the biophysical properties of tads and their boundaries , as well as into chromatin folding and overall genome organization that supports biological control . 
  
 ","abstractthe eukaryotic genome is partitioned into topologically associating domains ( tads ) . despite recent advances characterizing tads and tad boundaries , 
 the organization of these structures is an important dimension of genome architecture and function that is not well understood . 
 recently , we demonstrated that knockdown of brg1 , an atpase driving the chromatin remodeling activity of mammalian swi / snf enzymes , globally alters long - range genomic interactions and results in a reduction of tad boundary strength . 
 we provided evidence suggesting that this effect may be due to brg1 affecting nucleosome occupancy around ctcf sites present at tad boundaries . in this review 
 , we elaborate on our findings and speculate that brg1 may contribute to the regulation of the structural and functional properties of chromatin at tad boundaries by affecting the function or the recruitment of ctcf and dna topoisomerase complexes .",pubmed
"when the objective of the study is investigation of count data using some variables , statistical modeling is used . in statistics , poisson distribution is used for investigating the count data if the mean and variance of the distribution are the same . if these two measures are not the same ( variance is larger than the mean = over dispersion ) , then negative binomial distribution is preferred . 
 statistical modeling is from suitable methods to investigate the relationship between various phenomena , especially in medicine and health . 
 poisson regression is of the models which is used in count variables such as the number of blood donations , the number of stopping addiction , the number of failed courses or semester etc . and 
 the poisson regression is a subset of a large set of statistical modeling which is called generalized linear model ( glm ) . 
 sometimes the count variables which are used to build a statistical modeling , have an inflation in zero which are divided into poisson zero inflated or negative binomial zero inflated distributions according to the nature of the variables . 
 
 there are various researches that used statistical modeling on count data which applied negative binomial or poisson regressions ; zero inflated poisson or negative binomial regressions . 
 rafiee ( 1 ) used negative binomial distribution for modeling of the period of hospitalization of mothers after child birth as the best model . 
 wong and lam ( 2 ) applied poisson regression with zero inflated for modeling of dmf for the students health situation . 
 barondess et al ( 3 ) used poisson regression with zero inflated to model the estimated number of cigarettes which is used by new smokers of different races in the usa in 2010 . 
 mohammadfam et al ( 4 ) applied a model for the number of work accidents in 2009 and showed the best model is a poisson regression with zero inflation . 
 one of the applications of this method is in the modeling of educational count data such as the number of failed courses or the number of failed semesters in university students . 
 this application which is considered as a part of the students academic performance has a great efficient . 
 the academic performance has relationship with some educational and demographic factors of the students as well . 
 mlambo ( 5 ) showed that gender , age and entry qualifications have a relationship with the academic performance . 
 foster ( 6 ) proved that gender , age , motivation , prior academic performance are variables which have relationship with the academic performance . 
 kooi ( 7 ) showed that age and academic background have strong relationship with the academic performance . 
 garkaz et al ( 8) showed that the type of diploma , students interest , employment status and gender are factors which have relationship with the academic performance . 
 trockel et al ( 9 ) showed that exercise , eating and sleep habit , time management , religious habit , period of work in a week , gender and age are from the variables which affect the academic performance of the students . 
 since there are many demographic , educational , andeconomicalfactors which affect the academic performance of the students , in each research only some of them are presented . 
 also , in most recent research , descriptive statistics or ordinary or logistic regressions are used to analyze or model the educational data and there is very few work on academic performance of the university students using poisson of negative binomial regression with zero inflation . 
 unfortunately , there is nothing in the literature about the application of zero inflated poisson or negative binomial for modeling of the failed courses and semesters in university students . 
 this studyintend to apply statistical modeling , especially poisson and negative binomial regressions with zero inflated to model the number of failed courses and semesters in the students of iran university of medical sciences . 
 in these models some educational and demographic factors which affect the academic performance of the students were used as the predictor variables . 
 this cross - sectional study was performed in 2009 and 2010 in iran university of medical sciences . 
 the target population was all current students of the university in 2009 which were almost 6000 in different educational levels . 
 the sample of 670 students was selected using stratified random sampling . to choose the sample from the target population , simple random sampling in each stratum 
 the demographic and educational data was collected from the university education file and the information regarding to attendance of the student in university accommodation was collected from the deputy head of the university in students affairs . 
 the data consist of the first and surnames of the students , the student i d , discipline , faculty ( one for each faculty and zero for others ) , the educational level ( one for each level and zero for others ) , gender ( one for males and zero for females ) , the year of entrance , marital status according to year of entrance ( single=1 and married=0 ) , using university accommodation , being native , quota system ( no quota=1 , others=0 ) , university average , the number of failed courses and semesters . 
 the students score are from zero to 20 in all iranian university systems and if a student receives a score of less than 10 in undergraduate or less than 14 in postgraduate courses , he fails the course . also , the mean of less than 12 in undergraduate and less than 14 in postgraduate semesters are considered as the failed semester . the students can not pursue their study if they have more than four failed semesters . 
 the students demographic and educational data were combined to the file of the number of failed courses and semesters to produce the final data set . 
 
 for data analysis and modeling , 
 the descriptive statistics and zero inflated poisson regression and zero inflated negative binomial regression were used to analyze the final data set . 
 the number of students with no failed courses were 478 ( 71.3% ) and 192 ( 28.7% ) students had one or more failed courses . 
 the students with one , two , three , four or more failed courses were 92 ( 13.7% ) , 37 ( 5.5% ) , 22 ( 3.3% ) and 41 ( 6.2% ) , respectively . 
 the number of students with one or more failed semester were 24 ( 3.6% ) and 31 ( 4.6% ) , respectively . 
 the results of zero inflated ( zi ) poisson regression fit are shown in table 1 . 
 
 in poisson regression for the number of failed courses ( table 1 ) 
 , one unit increase in total university average resulted in 0.64 decreases in logarithm of the number of failed courses . 
 also , one unit increase in the number of failed semesters caused 2.73 decreases in the logarithm of the odd of inflated zero . 
 furthermore , the change of educational level from other levels to bachelor or master levels resulted in 0.27 and 0.96 decrease of the expected logarithm of the numbers of failed courses . 
 finally , the change of gender from male to female caused 0.24 decrease of expected logarithm of the numbers of failed courses , and the change of marital status from married to single increased 0.05 of the expected logarithm of the numbers of failed courses . 
 
 in the regression of failed semesters , the university average had negative relationship with the numbers of failed semesters and a unit increase in university average resulted in 0.24 decrease in expected logarithm of the numbers of failed semesters . 
 also , with the change of quota system from other quota system to free quota system , the expected logarithm of the numbers of failed semester decreased by 0.47 . 
 furthermore , a unit increase in the number of failed courses caused the logarithm odd of inflated zero to decrease by 1.56 . 
 * - using robust method 
 
 as one can see , in regression of the number of failed courses , a unit increase in university average resulted in 0.83 decreases in logarithm of the number of failed courses . also , with a unit increase in the number of failed semesters , the logarithm of odd of the zero inflated decreased by 35.88 . 
 the change of faculties from other to faculty of management and rehabilitation caused 1.24 and 1.07 increase in expected logarithm of the number of failed courses , respectively . 
 the change of educational level from other levels to bachelor , certificate and master levels , the expected logarithm of the number of failed courses decreased by 0.81 , 0.8847 and1.45 , respectively . 
 
 in regression of failed semesters 
 , the university average has negative relationship with the number of failed semesters and with a unit increase in university average , the expected logarithm of the number of failed semesters decreased by 0.42 . also , with the change of quota system from other system to free quota system , the expected logarithm of the numbers of failed semesters decreased by 0.47 . 
 furthermore , with a unit increase of the numbers of failed courses , the logarithm odd of inflated zero decreased by 1.56 . 
 all of these results are the same as table 1 on the regression with the response variables of the number of failed semesters . 
 the research results showed that in poisson and negative binomial regressions with zero inflated using the number of failed semesters as the response variable , the variables of the university average and quota system have inverse relationship with the response variable , so the increase of the university average and the change from other quota system to free quota system caused a decrease in failed semesters . in a research ( 10 ) it was shown that in the students with free quota system , the number of failed semesters is less than other students which is the same as this study . 
 also , we have to consider the fact that failing is less in clever students and in these students the failure is a rare outcome . 
 furthermore , the students with free system are almost clever and have very less failure . therefore , quota system is from the factors which decrease the failed semesters . on the other hand , 
 a unit increase in the number of failed courses in both models , decrease the logarithm odd of inflated zero . 
 thus , the increase in the number of failed courses resulted in the number of failed semesters . 
 this is in the same direction due to the high correlations ( 0.81 ) of the failed courses and semesters . 
 therefore , with increase of the number of failed courses , the chance of having failed semesters increase . 
 in both regression models with the response variable of the number of failed courses , the university average and the bachelor and master educational levels have negative relationship . in both models , the increase of university average resulteda decrease of the number of failed courses , and the decrease in negative binomial regression is more than poisson regression . in a research 
 it was shown that there is a negative relationship between the university average and the number of failed courses which is the same as this research ( 10 ) . also , the change from other educational levels to bachelor and master levels caused a decrease of the number of failed courses in these levels . 
 the decrease in the number of failed courses in bachelor and master levels is logical due to the little failure courses in these levels . 
 
 in poisson regression with the failed courses as the response variable , the variables of genderandmarital status have inverse and positive relationship to the number of failed courses , respectively , and the failed courses are more in males than females and in singles than married . in different research ( 5,6,8,10,11 ) 
 , it was shown that gender and marital status have effect to the failed courses or semesters . 
 research it is shown that the singles are more successful in their educations than married and have less failed courses which is opposite of this study ( 12 ) . 
 
 in the negative binomial regression , with the number of failed courses 
 , the variables of bachelor level and the faculties of management and rehabilitation caused the increase in the number of failed courses . 
 since the number of failed courses in bachelor level is more than master level , so the result makes sense . also , rehabilitation faculty has the most failure in the university which is the same as the model result . 
 finally , in seems that the entrance of the management faculty to the model , with many failures , is for adjustment of the effects of other variables in the model . on the other hand , 
 a unit increase in the failed semester , in both models , increases the logarithm odd of inflated zero and this reduction in negative binomial model is 13 times of the poisson model , and this result was expected due to the high correlation between the failed courses and semesters . 
 it is concluded from all results that in the failed courses regression model , the university average and quota system have the most roles and the increase in university average resulted in the reduction of failed semesters . also , the change from other quota system to free quota system resulted in the decrease of failed semesters . 
 furthermore , in these models , the increase in the number of failed courses cause a decrease in the logarithm odd of zero inflated . therefore , with the increase of the university average and choosing free quota system it is possible to reduce the failed semesters . 
 
 in the regressions of the number of failed courses 
 , the university average has an important role and its increase causes the decrease of the number of failed courses . 
 other affected variables to the number of failed courses are different in both models . on the other hand , the increases in the number of failed semesters , 
 decrease the logarithm of zero inflated in both models and the amounts of these changes are different . 
 therefore , the increase in university average and choosing more bachelor and master students can reduce the failed courses . 
 this work was supported by iran university of medical sciences under the grant number of 370 . 
 ","background : the number of failed courses and semesters in students are indicators of their performance . 
 these amounts have zero inflated ( zi ) distributions . using zi poisson and negative binomial 
 distributions we can model these count data to find the associated factors and estimate the parameters . 
 this study aims at to investigate the important factors related to the educational performance 
 of students . 
 methods : this cross - sectional study performed in 2008 - 2009 at iran university of medical sciences 
 ( iums ) with a population of almost 6000 students , 670 students selected using stratified random 
 sampling . 
 the educational and demographical data were collected using the university records . 
 the 
 study design was approved at iums and the students data kept confidential . 
 the descriptive statistics 
 and zi poisson and negative binomial regressions were used to analyze the data . 
 the data were 
 analyzed using stata . 
 results : in the number of failed semesters , poisson and negative binomial distributions with zi , 
 students total average and quota system had the most roles . for the number of failed courses , total 
 average , and being in undergraduate or master levels had the most effect in both models . 
 conclusion : in all models the total average have the most effect on the number of failed courses or 
 semesters . 
 the next important factor is quota system in failed semester and undergraduate and master 
 levels in failed courses . 
 therefore , average has an important inverse effect on the numbers of 
 failed courses and semester .",pubmed
"electrostatic 
 interactions have been shown to play a vital role 
 in enzyme catalysis . 
 these interactions assist in the binding of ligands 
 and facilitate the enzyme - catalyzed chemical reactions by keeping 
 the reacting species positioned properly as well as stabilizing the 
 transition state in some cases . 
 moreover , these electrostatic interactions are expected to be significantly 
 modulated by the conformational changes that occur over the catalytic 
 cycle . in particular , the electrostatic environment in the 
 active site is likely to change as the ligands bind , the chemical 
 reaction occurs , and the ligands are released . quantifying the electrostatic 
 contributions to the individual catalytic steps , as well as 
 the impact 
 of conformational changes on the electrostatic interactions , is a 
 challenging yet important goal in the field of enzyme catalysis . to gain a better understanding of the catalytic role of electrostatics 
 and conformational motions 
 , we probed the changes in the active site 
 electrostatic environment resulting from structural rearrangements 
 that occur along the catalytic cycle of escherichia 
 coli dihydrofolate reductase ( ecdhfr ) . 
 this enzyme catalyzes the nadph - dependent conversion of 7,8-dihydrofolate 
 ( dhf ) to 5,6,7,8-tetrahydrofolate ( thf ) . 
 x - ray crystallographic , nuclear 
 magnetic resonance ( nmr ) , theoretical , and photophysical studies have 
 shown that ecdhfr exhibits significant structural 
 rearrangements along the catalytic cycle . 
 thus , ecdhfr is a highly suitable 
 system for studying how conformational changes can affect the active 
 site electrostatic environment , which can then modulate enzyme catalysis . 
 recently , thiocyanate and other vibrational probes have been incorporated 
 into a number of biological systems to investigate the local electrostatic 
 environments . 
 boxer and co - workers have explored the use of nitrile ( cn ) probes 
 to detect differences in the electric field projected along the cn 
 axis in proteins upon ligand binding , protein folding , and protein  protein 
 association . using this method 
 , they showed that the electrostatic 
 environment in the active site of -3-ketosteroid 
 isomerase ( ksi ) exhibits limited rearrangement upon photoexcitation 
 of a ligand , where this excitation was proposed to mimic the movement 
 of charge during the catalyzed chemical transformation . 
 the authors concluded that the ksi active site 
 is electrostatically preorganized for catalysis in its tertiary protein 
 structure and that major rearrangement is not necessary for catalysis . 
 this observation is not unexpected due to the relatively rigid structure 
 of ksi that limits substantial conformational mobility , 
 thereby limiting electrostatic rearrangement . 
 however , calculations 
 have shown that further active site reorganization , manifesting as 
 small conformational changes that position the reacting species for 
 chemistry , is still required in ksi to enable the catalyzed proton 
 transfer reactions . 
 in contrast to ksi , 
 which exhibits limited protein motion along the catalytic cycle , ecdhfr is known to undergo significant conformational changes 
 along the catalytic cycle , suggesting that larger changes in electrostatics 
 could play a more significant role in all steps of the catalytic cycle . to examine the electrostatic interactions within the active site 
 of ecdhfr , we incorporated thiocyanate probes at 
 site - specific locations . using vibrational and c nmr 
 spectroscopic techniques , as well as computer simulations , we analyzed 
 the electrostatics and degree of hydration of the microenvironments 
 surrounding the probes . 
 the electrostatic interactions arising from 
 the active site microenvironments surrounding the small , catalytically 
 nonintrusive probes led to different observed cn vibrational stretching 
 frequencies and c nmr chemical shifts along the catalytic 
 cycle . 
 experimental data clearly show changes in the active site electrostatic 
 environment due to structural rearrangements . 
 further insights were 
 obtained through mixed quantum mechanical and molecular mechanical 
 ( qm / mm ) analyses of classical molecular dynamics ( md ) simulations . 
 these calculations were able to reproduce the experimentally measured 
 vibrational frequency shifts reported by the probes across the enzyme - catalyzed 
 hydride transfer step of the catalytic cycle . 
 moreover , 
 the electrostatic contributions from the active site microenvironments 
 surrounding the probe were decomposed into the major components ( individual 
 residues and ligands ) . 
 in addition , the experimentally validated theoretical 
 model allowed us to examine the electric field along the hydride transfer 
 pathway between the substrate and cofactor , a property that is catalytically 
 important yet unattainable experimentally . 
 this integrated approach 
 provides valuable insights into the interplay between electrostatic 
 and conformational changes along the catalytic cycle of ecdhfr . 
 7,8-dihydrofolate 
 ( dhf ) and [ ( 4r)-h]nadph 
 ( nadpd ) were prepared 
 according to previously described procedures . 
 -nicotinamide 
 adenine dinucleotide phosphate reduced tetra(cyclohexylammonium ) salt 
 ( nadph ) , -nicotinamide adenine dinucleotide phosphate hydrate 
 ( nadp ) , potassium cyanide - c ( 99 atom % c ) , methotrexate , methotrexate - agarose , dithiothreitol ( dtt ) , 
 folic acid ( 97% ; fol ) , sodium phosphate monobasic monohydrate 
 ( 98102% ) , and 5,5-dithiobis(2-nitrobenzoic acid ) ( ellman s 
 reagent ) were purchased from sigma - aldrich and used without further 
 purification . 
 sodium phosphate dibasic 
 heptahydrate and 2-nitro-5-thiocyanatobenzoic acid ( 97% ; ntcb ) were 
 purchased from emd and tci america , respectively . 
 ph values were measured 
 using an accumet model 13 - 620 - 300 standard combination electrode calibrated 
 with vwr certified standard aqueous buffers ( ph = 4 , 7 , and 10 ) . 
 all 
 of the experiments , including kinetics and spectroscopic measurements , 
 were done in ph 7.0 50 mm sodium phosphate buffer unless otherwise 
 specified . 
 first , the two native cysteines ( c85 and 
 c152 ) in wild - type ( wt ) ecdhfr were mutated to ala 
 and ser , respectively , to generate 
 cys ecdhfr using the stratagene quikchange 
 site - directed mutagenesis kit and the wt ecdhfr template 
 as described elsewhere . 
 primer sequences 
 were 5-c gaa gcc atc gcg gcg gct ggt 
 gac gta cca g-3 ( c85a ) and 5-c tcg cat agc tat tca ttc gaa atc ctc g-3 ( c152s ) . 
 the choice of 
 amino acid substitution was based on a previous report showing that 
 the c85a / c152s substitution on ecdhfr induced minimal 
 perturbation to the enzymatic activity . 
 selective incorporation of cysteine was achieved through subsequent 
 mutations using the following primers : 5-ca atc ggt agg cct tgc ccc ggc cgc aaa aat att atc-3 ( l54c ) and 
 5-g att atg ggg cgc cat tgc tgg gaa 
 tca atc g-3 ( t46c ) . 
 plasmid construction , protein expression , 
 and purification of mutant dhfrs were performed according to published 
 protocols . 
 labeling of the single - cysteine ecdhfr constructs 
 ( cys l54c and cys t46c , denoted as l54c - cn and t46c - cn , 
 respectively ) was achieved using a modified version of a published 
 procedure . inside an amicon 
 ultra-4 10k 
 nominal molecular weight limit ( nwml ) centrifugal filter tube , approximately 
 400 m of each ecdhfr construct was mixed with 
 four times excess of ntcb in 4 ml of 0.1 m sodium phosphate buffer 
 at ph 7.4 . 
 the mixture was allowed to react at room temperature for 
 10 min , and reaction progress was monitored by the appearance of the 
 phenolic product using a uv  vis spectrophotometer . 
 the protein 
 mixture was then concentrated by spinning at 5k rpm in a centrifuge 
 at 4 c . 
 the protein sample was then washed twice with 12 mm 
 kcn in ph 7.0 50 mm sodium phosphate buffer , with a concentration 
 step after each wash . 
 this was followed by washing the protein sample 
 with 50 mm ph 7.0 sodium phosphate buffer two more times before concentrating 
 the protein solution down to 0.8 ml , which was then applied 
 onto a size exclusion column ( econo - pac 10dg ; bio - rad ) that was pre - equilibrated 
 with ph 7.0 50 mm sodium phosphate buffer . 
 the isolated labeled proteins 
 were concentrated in an amicon ultra-4 10k nmwl centrifugal filter 
 tube . 
 the intact masses of scn labeled and unmodified samples were 
 confirmed by esi - ms , which yield deconvoluted m / z values of 17965 ( l54c)/17978 ( t46c ) and 17940 ( l54c)/17953 
 ( t46c ) da , respectively . 
 the theoretical molecular weights for the 
 unmodified samples are 17941 ( l54c)/17953 ( t46c ) da . 
 similar 
 preparation procedures were used for the scn labeled 
 samples . a mixture containing 10 mm ellman 
 s reagent and 0.5 
 m kcn in ph 7.0 0.1 m sodium phosphate buffer was prepared 
 at room temperature for 15 min . 
 this mixture was then allowed to react 
 with a sample of the unlabeled protein in an amicon ultra-4 10k nmwl 
 for 10 min at room temperature . 
 the final concentrations of a typical 
 reaction mixture were 500 m protein , 5 mm ellman s reagent , 
 and 0.25 m kcn . 
 the subsequent purification and isolation 
 steps were similar to those for scn labeling , except that kcn was used for washes rather than kcn . 
 successful labeling was confirmed 
 spectroscopically by the release of one equivalent of the phenolic 
 product as well as c nmr comparison with the unlabeled 
 proteins . the pre - steady - state 
 hydride transfer reaction was monitored on an applied photophysics 
 stopped - flow spectrophotometer thermostated at 25 c . 
 all of 
 the enzymatic reactions were performed in 50 mm sodium phosphate buffer 
 ph 7.0 . 
 one of the syringes in the stopped - flow analyzer was loaded 
 with 20 m enzyme , 250 m nadph , and 2 mm dtt . 
 after combining dhfr 
 and nadph as described above , the mixtures were prepared on ice for 
 5 min prior to the onset of the chemical reaction . upon mixing , 
 the 
 final concentrations of the individual species in the reaction chamber 
 were halved . 
 the conversion of dhf to thf was followed by the loss 
 of fluorescence resonance energy transfer from the enzyme to nadph . 
 the reaction mixture was excited at 290 nm , and the emission was measured 
 using a 400 nm cutoff output filter . 
 the 
 measure of absorbance vs time trace ( of burst phase ) was fit to standard 
 single exponential decay to obtain the hydride transfer rate ( khyd ) . 
 the kinetic isotope effect ( kie ) for the 
 hydride transfer rate was also determined under the same experimental 
 conditions , with the exception that nadpd was used instead of nadph 
 in comparative experiments . 
 steady - state enzyme turnover kinetics 
 ( kcat ) were studied following similar 
 experimental conditions , with the exception that the reaction progress 
 was monitored at 340 nm . under each condition , at least 5 independent 
 kinetic runs were conducted , and the average rate constants were used 
 for analysis . 
 ftir spectra were 
 obtained at room temperature using a mattson research series ftir 
 spectrometer ( madison instruments , inc . ) 
 equipped with a liquid - nitrogen 
 cooled hgcdte ( mct ) detector ( infrared associates , inc . ) . cyanylated 
 dhfr samples ( [ protein ] = 1.72.5 mm ) were mixed with appropriate 
 ligands ( 1:10 mol equiv ) in aqueous buffer and placed in a liquid 
 cell with two caf2 windows separated by a 56 m teflon 
 spacer . 
 all nitrile ( cn ) stretch absorbance spectra for cyanylated 
 dhfr samples consisted of 2000 scans and the data were collected with 
 1.0 cm resolution . 
 the cn stretch absorbance spectra 
 for cyanylated dhfr samples were obtained by subtracting a spectrum 
 of the aqueous buffer solution from the protein spectrum . 
 the baseline 
 for each spectrum was corrected by fitting a fifth - order polynomial 
 fit to the data , as done previously by fafarman et al . and mcmahon et al . 
 the fifth - order polynomial local fit functions were obtained by 
 defining roots at least 15 cm from the peak maximum , 
 meaning the experimental data and fits were forced to be equal at 
 points more than 15 cm from the central frequency . 
 all published spectra are the averages of two independently prepared 
 protein samples after the baseline has been corrected and the peak 
 height has been normalized to one . 
 the peak positions obtained from 
 independently prepared protein samples typically differed by < 0.5 
 cm , which is substantially less than the variation 
 among peak positions due to the protein binding or releasing the ligands . 
 detailed experimental setup and data analysis are provided in the supporting information . 
 c nmr spectra were 
 collected on a bruker av - iii-600 ( 150.9 
 mhz c frequency ) at 25 c . 
 samples were prepared 
 with 1.6 mm of the scn labeled protein , 8 mm ligands , 
 and 50 mm potassium phosphate ( ph 7.0 ) in water . 
 a sealed capillary 
 tube containing 1 m acetone in d2o was inserted into the 
 nmr tube that holds the protein sample . 
 the 1d c 
 nmr spectral data obtained for each sample were compared with its 
 corresponding c dept 45 ( distortionless enhancement 
 by polarization transfer ) data to confirm the identity of the assigned 
 peaks . under the same experimental conditions , we were unable to detect 
 the c nmr signals corresponding to the scn probes for 
 samples containing either the nonisotopically enriched scn label or 
 the unlabeled proteins . 
 crystals 
 of e. coli t46c - cn and l54c - cn dhfr 
 variants were obtained by the hanging - drop vapor diffusion method 
 using conditions similar to those previously described . 
 detailed methods , data collection , and refinement 
 statistics are provided in the supporting information . 
 we calculated 
 the ir spectra for the cn stretching mode of the incorporated thiocyanate 
 probes using a two - step method . 
 first we propagated classical md trajectories 
 for the entire solvated enzyme with bound ligands . 
 the configurations 
 sampled from the md trajectory were then analyzed using a series of 
 qm / mm single point energy calculations to determine the cn stretching 
 potential energy curve . 
 the qm region was treated with a semiempirical 
 method that was reparameterized for the calculation of nitrile vibrational 
 frequencies , and the mm region was represented as a collection of 
 atomic point charges . 
 the instantaneous vibrational wave functions 
 and associated frequencies from the energy level splittings were obtained 
 from the cn stretching potential energy curve using a grid - based numerical 
 approach . 
 the simulated ir spectra were calculated using the fluctuating 
 frequency approximation , which includes the dynamical contributions 
 of the protein motion to the simulated lineshapes . 
 a full description of the methodology can be found in our 
 previous application to ksi , with some 
 minor adjustments that will be discussed below . 
 we propagated classical 
 md trajectories for the cysteine - free t46c - cn and l54c - cn ecdhfr mutants using the gromacs program . 
 the first state has folate ( fol ) bound 
 in the substrate pocket and the nicotinamide adenine dinucleotide 
 phosphate cofactor bound in the oxidized form ( nadp ) . 
 in this state , 
 the met20 loop is in the closed conformation , which 
 is thought to facilitate the hydride transfer reaction by positioning 
 the ligand and cofactor while excluding solvent molecules . 
 this state has been shown to structurally mimic 
 the reactive michaelis complex ( e : dhf : nadph ) prior to hydride transfer . although we can simulate the reactive michaelis 
 complex without hydride transfer occurring , we simulated the e : fol : nadp ternary complex to compare directly with the experimentally 
 measured ir spectra . 
 the second state that we simulated has the product 
 thf and nadp bound . in this state , 
 the met20 loop is in 
 the occluded conformation , and the nicotinamide ring of nadp is excluded from the active site . 
 these two states are used to model 
 the closed to occluded transition that occurs across the hydride transfer 
 step of the ecdhfr catalytic cycle . 
 the initial 
 structures for the md simulations were obtained from crystal structures 
 solved with the nitrile probes included for both the l54c - cn and t46c - cn 
 variants ( pdb accession codes 4p68 and 4p66 , respectively ) . 
 the crystal structures 
 have methotrexate bound in the substrate binding pocket and nadph 
 bound in the active site . in both of these structures , 
 this state is thought to mimic the 
 transition state for the hydride transfer reaction . 
 for the simulations in the closed conformation ( nadp / fol ) , the folate molecule was incorporated by aligning the 
 folate substrate with the corresponding heavy atoms of the methotrexate 
 ligand from the crystal structure . for the simulations in the occluded 
 conformation ( nadp / thf ) 
 , we used the initial coordinates 
 of a wt crystal structure of ecdhfr complexed with 
 nadph and 5,10-dideazatetrahydrofolate ( ddthf ) ( pdb 1rx6 ) . 
 the thiocyanate probes were incorporated into the occluded 
 structure by aligning the backbone atoms of the five previous and 
 five subsequent residues to the cyanylated cysteine residue in the 
 new crystal structure that contains the probe to the corresponding 
 backbone atoms of the 1rx6 structure . 
 we then replaced the specific residue with 
 the aligned cyanalated cysteine and optimized the probe residue geometry 
 and position using a steepest - descent algorithm in gromacs , while 
 the ligands , the crystallographic water molecules , and the rest of 
 the enzyme were held fixed . 
 the substrate ligand ( ddthf ) was replaced 
 by thf by aligning the corresponding heavy atoms in ddthf and thf , 
 and the oxidized cofactor was modeled by aligning the heavy atoms 
 of nadp with the nadph from the crystal structure . 
 the 
 modeled ligands were also optimized using the steepest - descent algorithm 
 with the enzyme and the crystallographic water molecules held fixed . 
 for all of the md simulations , we used the amber99sb force field 
 to compute the potential energy and forces for the protein . 
 the bonded and nonbonded parameters for the nadp cofactor were consistent with those from our previous 
 work . 
 the fol and thf charges were calculated 
 using the restrained electrostatic potential ( resp ) method , and the bonded and van der waals parameters 
 were obtained from the generalized amber force field ( gaff ) . for methyl thiocyanate and the cyanylated cysteine 
 residue , the bonded parameters were obtained from cho et al . , and the nonbonded parameters were obtained from 
 lindquist and corcelli , as used in our 
 previously published work on ksi . 
 each of the enzymatic systems was solvated with tip3p water in a truncated octahedral box , where the sides 
 of the box were at least 10.0  from every atom in the enzymatic 
 complex , including the crystallographic waters . 
 after solvation , we 
 added 12 sodium atoms using the gromacs genion utility 
 to neutralize the system before simulation . 
 all bonds involving a 
 hydrogen atom were constrained to their equilibrium bond lengths using 
 the lincs algorithm as implemented in gromacs . 
 nonbonded van der waals interactions were calculated with 
 a cutoff of 1.0 nm , and electrostatic interactions were calculated 
 using the particle - mesh ewald method . after the initial preparation of each system , we followed 
 a well - defined 
 equilibration procedure . 
 the solvent configurations were minimized 
 using the steepest - descent algorithm in gromacs with the enzyme and 
 ligands held fixed to mitigate any possible bad contacts between the 
 solvent and solute molecules . 
 the solvent molecules and counterions 
 were then equilibrated for 50 ps at 300 k in the canonical ( constant 
 nvt ) ensemble with the protein and ligands still fixed . 
 after solvent 
 equilibration , the entire system was then minimized using the steepest - descent 
 algorithm . 
 subsequently , the system was equilibrated using a simulated 
 annealing procedure in which the initial velocities were generated 
 according to a boltzmann distribution at 50 k. the system was equilibrated 
 in the isothermal  isobaric ensemble ( constant npt ) at 50 k 
 for 50 ps , and then the temperature was increased up to 300 k in increments 
 of 50 k with 50 ps of equilibration at each temperature . 
 after this 
 simulated annealing procedure , an additional 1 ns of equilibration 
 in the constant npt ensemble was performed , and finally 1 ns of equilibration 
 in the constant nvt ensemble was performed . during the simulations , 
 the nos  hoover thermostat was used to maintain the temperature , and the parrinello 
 the production trajectories were propagated in 
 the canonical ensemble 
 at 300 k with a time step of 1 fs and a total simulation time of 1 
 ns for each independent trajectory . 
 configurations were saved at every 
 time step and were analyzed using the qm / mm methodology described 
 below . for each mutant and catalytic state , we propagated at least 
 two independent trajectories with different initial configurations . 
 the data for a single trajectory are presented in the main paper , 
 while data from additional trajectories are provided in the supporting information . 
 the results for independent 
 trajectories associated with the same system were found to be consistent 
 within the numerical errors of the methodology . 
 the methodology 
 used to calculate the vibrational frequency of the 
 nitrile group is similar to that used in our previous work . here 
 we highlight the differences between the 
 methods used in our previous work and the methods employed in the 
 present work . as in our previous work , we calculated the vibrational 
 frequency by generating the one - dimensional potential energy curve 
 along the cn stretching mode using a qm / mm method . the potential energy 
 is calculated along the cn vibrational coordinate by freezing all 
 of the atomic centers in the system and moving only the nitrogen atom 
 to stretch the cn distance from 1.0 to1.4  with a step size 
 of 0.05  . 
 the qm calculations were performed with the mm region 
 treated as atomic point charges that polarize the qm region . 
 these 
 atomic point charges were the same as those used in the md simulations 
 and were obtained from the amber99sb force field for the standard 
 amino acid residues , the tip3p force field for the water molecules , 
 and the resp calculations for the ligands . 
 the choice of the qm region 
 for each probe studied in this work will be discussed below . in our 
 previous work , 
 the one - dimensional potential 
 energy curve for the cn stretching mode was fit to a morse function , 
 and the energy levels were calculated analytically from the morse 
 parameters . in the present work , 
 the potential energy curve was fit 
 to a third - order b - spline potential , and 
 the vibrational wave functions and energy levels were calculated numerically 
 using the fourier grid hamiltonian ( fgh ) method . 
 an advantage of the fgh method is that it does not assume any functional 
 form of the potential energy curve and therefore avoids the bias associated 
 with fitting to the morse function , which depends on only three parameters . in principle 
 , any electronic structure method can be used to calculate 
 the vibrational stretching potential , but the level of theory must 
 be computationally tractable for the millions of configurations that 
 are required to obtain statistically converged ir spectra . as in our 
 previous work , we used a reparameterized version of the semiempirical 
 pm3 method . in this semiempirical method , 
 some of the computationally expensive integrals in hartree 
 these empirical parameters can be varied to reproduce 
 properties from either electronic structure theory calculations or 
 experimental measurements . because we altered our approach to use 
 the fgh numerical method to calculate the vibrational frequencies , 
 we reoptimized the pm3 parameters . 
 the reparameterization procedure 
 is similar to that from our previous studies , and a full description 
 is provided in the supporting information . 
 we also confirmed that the new set of parameters reproduces the 
 results for ksi from our previous work when the fgh numerical method 
 is used for calculating the vibrational frequencies . 
 table s6 provides the calculated frequencies for the two different 
 vibrational probes in ksi that were studied previously . to assist 
 in the analysis of the vibrational frequency calculations 
 , we calculated 
 the average electric field at the midpoint of the cn bond in the thiocyanate 
 group from our md simulations . 
 the vibrational stark effect between 
 two states is described by1here h is 
 planck s 
 constant , cn is the shift in vibrational 
 frequency between the two states , eenv is the difference in the electric field vector at 
 the nitrile bond as a result of the change in the environment , cn is the difference dipole moment 
 for the vibrational mode that lies along the cn bond axis , and  
 is the angle between these two vectors . the magnitude of cn is the stark tuning rate , which 
 is a measure of the susceptibility of the probe to changes in the 
 local electric field . 
 boxer and co - workers have measured the stark 
 tuning rate for thiocyanate probes in various environments , including 
 in proteins , to be 0.7 cm/(mv / cm ) . 
 we emphasize that this equation accounts only 
 for the classical electrostatic effects of the environment and does 
 not account for the quantum mechanical aspects of effects such as 
 specific hydrogen - bonding interactions and polarization . 
 while these 
 phenomena have significant classical components , they also have nonclassical 
 aspects that can be incorporated into the calculations by treating 
 interacting residues quantum mechanically , as will be discussed below . 
 recent work explores the role of a local field correction factor that scales the externally applied electric 
 field to obtain the local field experienced by the probe , which is 
 typically larger because of the polarization of the local environment 
 induced by the applied electric field . 
 this local field correction 
 factor has been estimated to be between 1.1 and 1.3 for protein environments , but recent investigations have suggested that it may be as large 
 as 1.8 . 
 we have not considered the role 
 of the local field correction factor in our analysis of the electric 
 fields in dhfr due to the lack of consensus about its value for a 
 protein environment . 
 moreover , applying the local field corrections 
 would result in changes that are within the error bars of our calculations . to calculate the electric field at the nitrile bond , 
 a virtual 
 particle was positioned at the midpoint of the cn axis during the 
 md trajectory . 
 this virtual particle had no electrostatic or van der 
 waals interactions with other particles , and the bond , angle , and 
 dihedral parameters were set to zero . 
 the configurations from the 
 md trajectory were saved , and subsequently the forces acting on all 
 particles were recalculated for these configurations when the charge 
 on the virtual particle was set to + 1 . 
 the electric field , evp , at the virtual particle location 
 was calculated as2where fvp is the force acting on the virtual particle and qvp is the charge of the virtual particle . 
 the total electric 
 field vector , as well as the component along the cn axis , was calculated 
 for each configuration along the md trajectory . 
 calculation of the 
 component of the electric field along the cn axis allows us to calculate 
 the change in the vibrational frequency due to classical electrostatics 
 using eq 1 . 
 the remaining frequency change can 
 be attributed to the quantum mechanical aspects of specific hydrogen - bonding 
 interactions and polarization effects . 
 these effects can be investigated 
 by using a classical polarizable force field or by treating the interacting residues 
 quantum mechanically . 
 note that the experimentally measured and theoretically 
 calculated frequency shifts can be compared directly , but the breakdown 
 into the classical electrostatics and quantum mechanical aspects is 
 available only from the theoretical calculations . 
 we also analyzed 
 the contributions of individual residues and ligands 
 to the electric field at the midpoint of the cn bond . because the 
 classical electric field is the sum of the individual contributions 
 from each atom , the electric field 
 analysis of the contributions 
 from specific residues provides insight into the impact of conformational 
 changes occurring during the closed to occluded transition associated 
 with hydride transfer . 
 we generated 
 the t46c - cn and l54c - cn mutants by site - specifically incorporating 
 a thiocyanate probe at each position ( figure 1 ) . 
 the t46c - cn probe is situated in close proximity to the junction 
 between the two reacting ligands , dhf and nadph , allowing us to monitor 
 the microenvironment close to the reaction center . 
 the l54c - cn probe is located 9  from the 
 center of the hydride donor  acceptor ( d  a ) axis . 
 this 
 probe monitors the folate binding pocket , and its spectral character 
 is expected to respond to the formation of the ternary michaelis 
 menten 
 complex upon folate binding , as well as the release of thf in the 
 enzyme turnover step . 
 ( a ) superposition of the t46c - cn and l54c - cn ecdhfr mutants in the closed conformation with folate and nadp bound , where only the thiocyanate residue is shown for the 
 l54c - cn mutant . 
 ( b ) ecdhfr in the closed ( red ) and 
 the occluded ( blue ) conformations exhibited by the ( c ) five major 
 complexes in its catalytic cycle . 
 the enzyme activity of the two ecdhfr variants 
 was evaluated by their ability to catalyze the nadph - dependent reduction 
 of dhf and the enzyme turnover rates at ph 7.0 and 25 c . 
 the 
 catalytic efficiencies of the t46c - cn ( khyd = 120  10 s ; kcat = 6.5  0.5 s ) and l54c - cn ( khyd = 250  20 s ; kcat = 28  3 s ) variants were 
 comparable to that of wt ecdhfr ( khyd = 220 s ; kcat = 12 s ) . 
 we also found 
 the cys ecdhfr ( c85a / c152s ; khyd = 215  8 s ; kcat = 10.3  0.8 s ) variant to 
 exhibit activity similar to the wt enzyme . 
 furthermore , figure 
 s1 shows that the crystal structures of the ternary complexes 
 of the t46c - cn and l54c - cn mutants in the closed conformation overlap 
 well with the wt crystal structure . 
 these observations suggest that 
 the incorporation of the scn probes into the selected locations of 
 the active site does not significantly alter the structure or activity 
 of the enzyme . 
 it should be noted that we also incorporated 
 the scn probe at the 
 i50 position ( cys - i50c - cn ) . 
 however , such a modification decreased 
 the hydride transfer rate by 10 times at ph 7.0 and 25 c . 
 we felt that the degree of enzyme activity reduction in cys - i50c - cn 
 might result in a less accurate representation of the electrostatic 
 environment in the active site of the wt enzyme . 
 thus , further investigations 
 were not performed for the cys - i50c - cn construct . 
 it is expected 
 that even small modifications to the crowded active site can have 
 large effects on enzyme activity . 
 the ecdhfr 
 catalytic cycle contains the five major species ( figure 1c ) that reflect the significant conformational changes in 
 the active site met20 loop ( figure 1b ) . 
 menten 
 complex ( e : nadph : dhf ) to an occluded conformation in the initial ternary 
 product complex ( e : nadp : thf ) . 
 the enzyme remains in the 
 occluded conformation in the two subsequent product - containing species 
 ( e : thf and e : nadph : thf ) , until the met20 loop reverts back to the 
 closed conformation upon the rate - limiting release of thf to generate e : nadph ( figure 1c ) . 
 nmr relaxation dispersion experiments have found that the rates 
 of the met20 loop conformational change are temporally similar to 
 their corresponding enzymatic processes . 
 in other words , the hydride transfer and enzyme turnover rates are 
 similar to the values determined from the nmr experiments for the 
 conformational transitions that connect the relevant enzyme complexes . 
 while the major change in the met20 loop conformation appears to be 
 induced by the formation of the product thf 
 , theoretical simulations have shown that minor structural rearrangements 
 of the ligands and protein , not restricted to the met20 loop , are 
 necessary to progressively optimize the substrate orientations and 
 the active site environment along the reaction coordinate of the hydride 
 transfer reaction . 
 herein , we utilized the scn probes 
 to experimentally monitor the changes in the local microenvironments 
 in two locations in the active site over the five major catalytic 
 species . 
 the five complexes that constitute the catalytic cycle 
 of ecdhfr ( figure 2 ) were 
 generated using 1.52.5 mm of the enzyme and at least five 
 times excess of the ligands in ph 7.0 phosphate buffer . 
 these concentrations 
 should be more than sufficient to push the equilibria toward full 
 complexation , thus allowing us to obtain vibrational ftir spectra of the five 
 major complexes that constitute the ecdhfr catalytic 
 cycle , as well as for the apoenzyme and the e : nadph : fol complex . 
 note 
 that the e : nadph : dhf state is modeled by e : nadp : fol , as 
 discussed in the literature . 
 for each 
 enzyme complex , the ir spectrum was obtained in duplicate / triplicate 
 using separately prepared samples , and these spectra were found to 
 be very reproducible ( data in supporting information ) . after correcting the ir data for baseline and subsequently normalizing 
 the amplitude of the peak to 1.0 
 , we determined the maximum frequency 
 ( max ) from the first derivative of the corrected 
 ir peak . 
 the peak position is a measure of the transition energy between 
 the vibrational ground state and first excited state of the thiocyanate 
 probe . 
 the transition energy is sensitive to the electric field along 
 the cn bond because the vibrational potential energy function is perturbed 
 by the local electrostatic environment ( surrounding residues , ligands , 
 and solvent molecules ) which modulates the energy difference between 
 the vibrational quantum states . 
 color coding the frequency of maximum 
 absorption detected in each complex illustrates the changes in the 
 local electrostatic interactions experienced by the cn probe over 
 the catalytic cycle ( figure 2 ) . 
 ( b ) 
 nmr ( scn ) measurements along 
 the catalytic cycle of ecdhfr . 
 the color scales for 
 the ftir and nmr data have units of cm and ppm , 
 respectively . 
 the cofactor is orange , and the substrate / product is 
 purple in all complexes ( the coloring of the cofactor and substrate / product 
 does not correspond to the color scales that represent the nmr shift 
 or ir frequency ) . 
 enzyme complex labels in blue are in the occluded 
 conformation , and those in red are in the closed conformation . 
 the 
 measured values for ir frequency and nmr chemical shift are provided 
 in table s4 . 
 figure 2 indicates that the local 
 environment 
 of each cn probe changes significantly over several key steps in the 
 catalytic cycle : formation of the ternary michaelis 
 menten 
 model ground state complex , the hydride transfer step , and the rate - limiting 
 release of thf . 
 the largest frequency shifts observed for the t46c - cn 
 probe occurred across the two most important steps in the catalytic 
 cycle : ( 1 ) upon the conversion between the model michaelis 
 menten 
 e : nadp : fol complex and the initial e : nadp : thf 
 product complex ( 4.1 cm ) , which is analogous 
 to the hydride transfer step ; ( 2 ) across the enzyme turnover step , 
 which is the release of thf ( 5.5 cm ) . for the 
 l54c - cn probe , 
 the most significant change occurred upon the formation 
 of the ternary e : nadp : fol complex ( 2.4 cm ) . 
 crystal structures of the wt enzyme have shown that the t46 and 
 l54 residues retain their positions and orientations in the five catalytic 
 complexes , suggesting that the inserted cn probes likely experience 
 limited freedom of movement in the active site . 
 md simulations confirm that there are no major conformational 
 changes in the probe orientation during 5 ns trajectories . 
 therefore , 
 the observed changes in the microenvironments as the system moves 
 through the various complexes in the catalytic cycle are likely due 
 to protein conformational rearrangements that induce changes in the 
 electrostatic interactions between the probes and their surroundings . 
 note also that the redox state of the cofactor ( nadph / nadp ) has very little effect on both the ir and c nmr data 
 ( table s4 ) . 
 in many cases , we also 
 observed changes in the peak width [ full - width 
 at half - maximum ( fwhm ) ; table s4 ] , which 
 usually reflects the variation of chemical environments sampled by 
 the probe as well as the strength of intermolecular interactions such 
 as hydrogen bonding . 
 for example , the l54c - cn probe , which is located 
 in the folate binding site , reported peak widths that are approximately 
 two times smaller in complexes with bound fol or thf . 
 this observation 
 is consistent with the probe sampling a less fluctuating and more 
 well - defined electrostatic environment resulting from the various 
 interactions that organize the fol / thf to bind in the active site . 
 there is also some evidence indicating that the e : nadph complex and 
 the apoenzyme exhibit a greater degree of hydrogen bonding between 
 a solvent water molecule and the l54c - cn probe . 
 one indication of 
 this phenomenon is obtained from inspection of the c 
 nmr - ir relationship , which shows that the data for the above two complexes 
 deviate from the rest of the data in a manner that implicates greater 
 specific solvent hydrogen - bonding interactions ( figure s4 ) . 
 md simulations support the presence of additional 
 solvent hydrogen - bonding interactions in these two complexes for the 
 l54c - cn probe , with a water molecule hydrogen bonded to the nitrile 
 probe in nearly half of the configurations . 
 we also observed a slightly 
 broader ir peak in the e : nadph : fol complex , which can be attributed 
 to the slow reduction of fol over the course of the ir measurement . 
 this is not unexpected since the l54c - cn probe is located in the folate 
 binding pocket . 
 these trends are less obvious for the t46c - cn probe , 
 which seems to be located in a more fluctuating environment , as indicated 
 by the relative ir peak widths . 
 we also prepared the isotopically 
 enriched cys - cn 
 variants ( t46c - cn and l54c - cn ) to obtain 
 the complementary c nmr data for all of the studied complexes . 
 while the magnitude of the observed changes are different because 
 the c nmr chemical shift is less sensitive to specific 
 solvent hydrogen - bonding interactions than is the cn vibrational stretching 
 frequency , the c nmr data illustrate 
 similar trends to those shown by the corresponding ir data . 
 in particular , 
 we observed a changing microenvironment around the scn 
 probes as the enzyme evolves through the catalytic cycle ( figure 2b ) . 
 similar to the ir data , the microenvironment 
 detected at the t46c - cn probe exhibits larger changes 
 along the catalytic cycle than that at the l54c - cn probe . 
 this trend is expected due to the proximity of the t46c - cn site to the regions of the substrates that undergo the hydride 
 transfer reaction . 
 overall , the experimental data summarized as figure 2 clearly demonstrate a dynamic electrostatic environment 
 in the active site of ecdhfr across its catalytic 
 cycle . 
 the differences in the microenvironments of the two probes 
 and the way they change among the different complexes , especially 
 between the closed and occluded conformations , in the ecdhfr catalytic cycle also provide an indication of the highly heterogeneous 
 nature of the enzyme active site . to identify 
 the various environmental contributions that give rise to the observed 
 cn stretching vibrational frequency shifts along the catalytic cycle , 
 we conducted qm / mm simulations to monitor the electrostatic interactions 
 experienced by the probes for the complexes along the catalytic cycle . 
 these data provide information about how the microenvironments of 
 the probes reorganize across individual steps of the catalytic cycle . 
 we simulated the ir spectra of the t46c - cn and l54c - cn mutants of ecdhfr for two different complexes along the catalytic cycle : 
 ( 1 ) the ternary complex with the reactant analog fol and nadp bound , which mimics the e : nadph : dhf state prior to hydride 
 transfer ; ( 2 ) the ternary complex with the product thf and nadp bound , which is the e : nadp : thf state following 
 hydride transfer . 
 first we examined the local environment around the 
 t46c - cn probe , which is located near the midpoint between the two 
 bound ligands ( figure 1a ) . 
 the qm region includes 
 the t46c - cn residue , the substrate ( either fol or thf ) , and the nearest 
 water molecule to the nitrile nitrogen , as depicted in figure 3a . 
 the qm region was determined by calculating the 
 cn frequency including the entire probe microenvironment in the qm 
 region for a subset of the configurations sampled and comparing these 
 results to those obtained with smaller qm regions . 
 the details of 
 the procedure used to determine the qm region are provided in the supporting information . 
 configurations from md 
 simulations for the ( a ) t46c - cn and ( b ) 
 l54c - cn ecdhfr mutants . 
 the residues with thicker 
 lines are included in the qm region for the qm / mm calculations of 
 vibrational frequencies . 
 figure 4a presents the calculated 
 and experimentally 
 measured ir spectra for the t46c - cn mutant for both the closed ( nadp / fol ) and occluded ( nadp / thf ) states . 
 the calculated 
 ir spectrum for the closed conformation ( solid black curve ) has a 
 maximum at 2165.8 cm , which is within 1.8 cm of the measured peak in the experiments . 
 the agreement 
 between the calculated and experimental absolute frequencies is potentially 
 fortuitous , but the level of agreement is similar to that calculated 
 in our previous studies of ksi . 
 the fwhm 
 of the calculated ir spectrum for the closed conformation is 11 cm , which is in good agreement with the experimentally 
 measured line width of 14 cm . 
 the calculated ir 
 spectrum for the occluded product state ( solid red curve ) has a maximum 
 at 2162.1 cm . 
 the calculated shift between these 
 two states is 3.7 cm , which is in excellent 
 agreement with the experimentally measured shift of 4.1 cm . 
 note that our md simulations and cn frequency calculations 
 are performed on the nanosecond time scale . 
 the probe could experience 
 multiple additional conformations on the longer time scale associated 
 with the ir measurements . during our md simulations of 5 ns , no major 
 structural rearrangements of the probe orientation occurred , indicating 
 that we are only sampling a single conformational state . since the 
 starting configurations for our simulations were based on crystal 
 structures that include the probe , we expect to be sampling the most 
 stable and therefore the dominant configuration of the probe . 
 moreover , 
 the agreement between the calculated and experimental cn frequency 
 shifts provides further validation of the methodology . 
 calculated and experimentally 
 measured ir spectra of the cn vibrational 
 stretching frequency for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr systems with nadp / fol bound ( closed conformation ; 
 black line ) and with nadp / thf bound ( occluded conformation ; 
 red line ) . 
 the solid lines are the simulated spectra from the qm / mm 
 calculations , and the dotted lines are the measured spectra from the 
 ftir experiments . 
 in addition to the ir 
 spectra , we calculated the electric field 
 at the midpoint of the cn bond for configurations sampled by the md 
 trajectory using the methodology described above . from these calculations , 
 we can correlate the measured vibrational frequency shifts to changes 
 in electrostatics from the protein environment . 
 figure 5a depicts the calculated electric field along the cn axis 
 for the two states . 
 the average projected electric field for the closed 
 ( nadp / fol ) and occluded ( nadp / thf ) states 
 are 15.9 and 19.0 mv / cm , respectively . using the experimental 
 stark tuning rate of 0.7 cm/(mv / cm ) in conjunction 
 with eq 1 , the difference of 3.1 mv / cm 
 corresponds to a shift in the vibrational frequency of 2.2 
 cm . 
 the difference between this frequency shift 
 arising from only classical electrostatics and the actual shift of 
 ca . 
 4.0 cm obtained from both calculations 
 and experiments is attributed to the quantum mechanical aspects of 
 hydrogen - bonding interactions and polarization effects that are not 
 included in the linear relation given by eq 1 . 
 calculated electric field along the cn bond at the midpoint of 
 this bond for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr 
 systems with nadp / fol bound ( closed conformation ; black 
 line ) and with nadp / thf bound ( occluded conformation ; 
 red line ) . previously it has been shown experimentally and computationally that significant deviations of 
 10 cm from the vibrational frequency shifts 
 predicted by eq 1 can occur when the nitrile 
 probe participates in specific hydrogen - bonding interactions . according 
 to our md simulations , 
 the t46c - cn thiocyanate probe is solvent accessible 
 in both the closed and occluded conformations and directly hydrogen 
 bonds to a water molecule , as depicted in figure 3a . in both protein conformations , 
 the nitrile probe is also 
 able to act as a hydrogen - bond acceptor from the amine group in the 
 substrate ( fol / thf ) backbone . 
 note that the observation of hydrogen - bonding 
 interactions with the probe for these two states is consistent with 
 the experimental ir - nmr correlation data in figure 
 s4 . 
 thus , the difference between the frequency shift 
 of 2.2 
 cm predicted from eq 1 , 
 which includes only classical electrostatic effects , and the shift 
 of 3.7 cm obtained from the qm / mm calculations 
 of the full system can be attributed to hydrogen - bonding interactions 
 with the water molecule and polarization effects from the water molecule 
 and the substrate ( fol / thf ) . to confirm this hypothesis , we performed 
 the qm / mm frequency analysis with only the probe residue ( t46c - cn ) 
 in the qm region ( i.e. , switching the water molecule and the substrate 
 from the qm region to the mm region ) . in this case , the calculated 
 frequency shift between the two states was 2.6 cm , which agrees well with the 2.2 cm shift 
 obtained from eq 1 . 
 beyond the calculation 
 of the total electric field at the probes , 
 we can decompose the field along the cn axis into contributions from 
 individual residues in the enzymatic complexes . 
 in essence , this methodology 
 allows us to decompose the microenvironment surrounding the cn probe 
 into the constituent parts , which then allows us to identify the major 
 contributing components . for the t46c - cn probe ( figure 3a ) , the sum of the contributions from the main contributors 
 given in table 1 corresponds to a change in 
 electric field along the cn axis of 2.9 mv / cm , which is consistent 
 with the change of 3.1 mv / cm calculated with the entire enzyme . 
 the contributions from the residues in the closed conformation 
 are shown pictorially in figure 6a , where the 
 residues that are colored blue contribute positively and those that 
 are colored red contribute negatively to the total field along the 
 cn bond . 
 for the t46c - cn probe , the nadp cofactor and 
 the nearest water molecule are the dominant contributors to the change 
 in the electric field along the cn axis , although asn18 and ser49 
 also make significant contributions ( table 1 ) . 
 the presence of a positive charge on the nicotinamide ring and 
 the short 4  distance between the nitrile nitrogen and 
 the aromatic nitrogen in the nicotinamide ring lead to a strong contribution 
 to the electric field in the closed conformation . after the transition 
 to the occluded conformation 
 , the cofactor is excluded from the active 
 site , and the magnitude of the contribution to the field from the 
 nicotinamide ring is much smaller due to the larger distance of 14 
  between the two nitrogen atoms . 
 this effect of the movement 
 of nadp on the electric field at the probe demonstrates 
 how atomic motions can significantly alter the local electrostatics 
 in the active site . 
 moreover , the 3.2 mv / cm change in the 
 electric field contributed by asn18 and ser49 is consistent with the 
 structural rearrangement from the closed to the occluded conformation . 
 specifically , the amide nitrogen of asn18 is 1.5  closer 
 to the nitrile probe in the closed conformation than in the occluded 
 conformation , and the hydroxyl group of ser49 is 1.0  
 closer to the nitrile probe in the closed conformation . 
 contributions 
 to the calculated electric field along the cn bond 
 at the midpoint of this bond for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr systems with nadp / fol bound and the met20 
 loop in the closed conformation . 
 the color for each residue corresponds 
 to the calculated values in tables 1 and 2 , respectively , using the color scale provided , 
 although residues contributing a magnitude > 4.0 mv / cm are depicted 
 in the darkest color . 
 the residues that contribute negatively to the 
 field are colored red , the residues that contribute positively to 
 the field are colored blue , and the residues that have no net contribution 
 to the field along the cn bond are colored white . 
 changes 
 in the electric field along 
 the cn bond in going from the closed conformation ( nadp / fol ) to the occluded conformation ( nadp / thf ) . 
 additionally , following the closed 
 to occluded transition , the 
 cn probe becomes more accessible to the solvent . 
 the contribution 
 to the electric field along the cn axis from the closest water molecule 
 changes by 7.46 mv / cm during this transition . 
 moreover , the 
 sign of the average electric field along the cn axis due to this water 
 molecule is different for the closed and occluded conformations , with 
 a value of 2.14 mv / cm for the closed conformation and 5.32 
 mv / cm for the occluded conformation . 
 analysis of the md trajectories 
 indicates that a water molecule is hydrogen bonded to the nitrile 
 probe 26% of the time in the closed ( nadp / fol ) 
 conformation and 52% of the time in the occluded ( nadp / thf ) conformation . 
 the positive contribution 
 to the electric field along the cn axis from the nearest water molecule 
 in the closed conformation is due to configurations in which the nearest 
 water molecule has its negatively charged oxygen atom pointing toward 
 the nitrile . 
 these configurations are stabilized by the hydrogen - bonding 
 interaction of the water molecule with the hydroxyl group of ser49 , 
 as shown in figure s5 . 
 the negative contribution 
 to the field in the occluded conformation arises from configurations 
 in which a hydrogen atom of the nearest water molecule is oriented 
 toward the probe , forming a hydrogen bond between the water molecule 
 and the cn group . 
 thus , this computational approach is also able to 
 probe the detailed active site hydration status , which is not easily 
 accessible with experimental means . 
 we performed the same analysis for the l54c - cn mutant 
 to examine the microenvironment near the folate binding pocket . 
 figure 4b depicts the calculated and experimentally measured 
 vibrational spectra for the l54c - cn mutant in both the closed ( nadp / fol ) and occluded ( nadp / thf ) states . 
 the l54c - cn 
 probe is distal from the active site , and it is located in a relatively 
 hydrophobic pocket near the carboxylate tail of the substrate , as 
 shown in figure 1 . 
 the qm region used for this 
 mutant includes the l54c - cn residue , phe31 , and arg57 , as depicted 
 in figure 3b . 
 the qm region was determined 
 by calculating the cn frequency including the entire probe microenvironment 
 in the qm region for a subset of the configurations sampled and comparing 
 these results to those obtained with smaller qm regions . 
 the details 
 of the procedure used to determine the qm region are provided in the supporting information . 
 the calculated frequency 
 shift between the two states for the l54c - cn probe is 2.5 
 cm , which is in reasonable agreement with the 
 shift of 1.0 cm measured experimentally . 
 figure 5b depicts the calculated electric 
 fields along the cn bond for the l54c - cn mutant in the two states . 
 the calculated average electric fields along the cn probe in the closed 
 and occluded conformations are 8.4 and 13.8 mv / cm , 
 respectively . using the experimental stark tuning rate of 0.7 cm/(mv / cm ) in conjunction with eq 1 
 , this shift of 5.4 mv / cm corresponds to a shift in vibrational 
 frequency of 3.8 cm due to only classical 
 electrostatic interactions . to model the situation with only classical 
 electrostatic interactions , we calculated the shift in the vibrational 
 frequency when only the residue containing the thiocyanate group ( l54c - cn ) 
 is treated quantum mechanically and the remainder of the system , including 
 phe31 and arg57 , 
 the calculated 
 frequency shift when only the probe residue is treated quantum mechanically 
 is 3.5 cm , which is consistent with the 
 value of 3.8 cm obtained from eq 1 . 
 however , this frequency shift is greater than the 
 shift of 2.5 cm calculated with the larger 
 qm region . 
 the greater shift predicted by the purely classical electrostatic 
 model indicates that the polarization effects due to the phe31 and 
 arg57 residues decrease the vibrational frequency shift relative to 
 that predicted by classical electrostatics . 
 table 2 provides the contribution to the 
 electric field along the cn axis for several key residues near the 
 l54c - cn probe . 
 the sum of these contributions is 7.3 mv / cm , 
 which is slightly higher than the change of 5.4 mv / cm calculated 
 with the entire enzyme , indicating that other residues are also contributing 
 to this field . in a similar manner to the analysis for the t46c - cn 
 mutant , 
 the arg57 residue exhibits the largest change 
 between the two states with a net shift of 6.58 mv / cm . in 
 the closed conformation , 
 the line connecting the midpoint of the cn 
 bond to the protonation site of the positively charged arg57 is nearly 
 perpendicular to the cn axis , resulting in almost zero contribution 
 to the electric field along the cn axis . 
 the change in the electric 
 field contribution by arg57 is likely due to a geometrical shift that 
 moved the positive charge on arg57 away from this orthogonal orientation 
 with respect to the probe in the occluded conformation . 
 interestingly , 
 while the substrate exerts a large contribution to the electric field 
 along the cn axis , this contribution remains nearly constant between 
 the two states . 
 the stable orientation of the cn axis relative to 
 the negatively charged carboxylate moiety in the bound substrate results 
 in negligible change in the contribution of the substrate to the electric 
 field at the cn probe . 
 changes 
 in the electric field along 
 the cn bond in going from the closed conformation ( nadp / fol ) to the occluded conformation ( nadp / thf ) . while the calculated electric 
 fields along the cn axes of the probes provide a measure of the electrostatic 
 microenvironments within the active site , the electric field along 
 the hydride transfer d 
 encouraged by the good agreement between the experimental 
 and the simulated data described above , we applied the same methodology 
 to evaluate the total electric field at the midpoint of the d  a 
 axis and the field projected along the d  a axis in the closed 
 ( e : nadp : fol ) state . 
 the negative value of the field indicates 
 that it points from the substrate to the cofactor and that this field 
 will facilitate the transfer of a negatively charged hydride from 
 the cofactor to the substrate . in other words , the projection and 
 the magnitude of the field along the d  a axis in the ternary 
 michaelis complex is highly favorable for the hydride transfer reaction 
 to occur . 
 the substrate and cofactor contribute a total of 32.4 
 mv / cm to the field along the d  a axis . 
 the rest of the protein 
 contributes approximately an additional 31% ( 15.4 
 mv / cm ) of the total field . 
 also , the solvent molecules and ions contribute 
 an additional 1.1 mv / cm to the field along the d  a 
 axis . 
 this analysis suggests that a major function of the enzyme 
 is to bring the substrate and cofactor into a configuration that realizes 
 the highly favorable field for facilitating the hydride transfer reaction 
 ( figure 7 ) . in the absence of the enzyme 
 , it 
 would be less probable for the reacting species , nadph and dhf , to 
 achieve the same relative orientation in solution . 
 moreover , this 
 analysis provides a new perspective on the role of electrostatic catalytic 
 contributions in dhfr . 
 it is currently impossible to experimentally 
 measure the electric field along the d  a axis , but the calculations 
 provide reasonable estimates . a favorable electric field of 15.4 
 mv / cm from the protein and 32.4 mv / cm from the ligands along 
 the d  a axis 
 could potentially reduce the free energy barrier 
 by several kcal / mol . while these values 
 suggest a substantial catalytic contribution generated from the active 
 site electrostatic environment , it is important to acknowledge that 
 the full mechanism of catalysis is far more complex than a simplistic 
 evaluation based solely on the electric field along the d  a 
 axis . 
 depiction of the component of the electric field along the hydride 
 transfer d  a axis calculated from an md simulation of wt ecdhfr with nadp / fol bound and the met20 loop 
 in the closed conformation . 
 the color for each residue corresponds 
 to the calculated electric field values using the color scale provided , 
 although residues contributing a magnitude > 4.0 mv / cm are depicted 
 in the darkest color . 
 the three arrows in the red oval represent the 
 total electric field of 48.9 mv / cm ( purple ) , the field of 
 32.4 mv / cm resulting from the ligands ( yellow ) , and the field 
 of 16.5 mv / cm resulting from the rest of the system ( green ) 
 projected along the d  a axis . 
 we also decomposed this field into the contributions from 
 all of 
 the protein residues , and our analysis identified many residues that 
 contribute to the electric field along the d  a axis . 
 figure 7 depicts the contributions from each of the residues 
 to the field along the d  a axis . 
 the residues that are colored 
 blue contribute positively and those that are colored red contribute 
 negatively to the field pointing from the substrate to the cofactor 
 along the d  a axis . 
 the residues that are shown with side chains 
 contribute to the field with a magnitude > 1 mv / cm . 
 interestingly , there is a strong degree of overlap between the 
 residues that strongly contribute to the field along the d  a 
 axis and the network of coupled motions that has been identified for 
 dhfr catalysis . 
 the residues ile14 , tyr100 , 
 and asp122 contribute 1.2 , 4.2 , and 2.4 mv / cm , 
 respectively , and were all implicated in the network of coupled motions . 
 additional residues that strongly contribute to the electric field 
 along the d  a axis are lys32 ( 3.2 mv / cm ) , arg52 ( 3.9 
 mv / cm ) , and arg57 ( 4.2 mv / cm ) . 
 the residues that contribute 
 strongly against the field along the d  a axis include asp27 
 ( 5.4 mv / cm ) , arg98 ( 3.4 mv / cm ) , and his124 ( 3.0 mv / cm ) . 
 all of these 
 residues represent potential targets to alter the electrostatic contribution 
 to catalysis . 
 we investigated 
 the changing electrostatic environment at two different 
 thiocyanate probes inserted into the active site of ecdhfr for five complexes along the catalytic cycle . 
 our results illustrate 
 a changing electrostatic environment near the hydride transfer site 
 ( t46c - cn ) as well as a smaller change in key regions of the folate 
 binding pocket ( l54c - cn ) . 
 the changes in electrostatic environment 
 are associated with conformational changes occurring along the catalytic 
 cycle of ecdhfr . 
 experimental evidence for the changing 
 electrostatic environment includes both ir frequency and c nmr measurements , which interrogate the electric field along the 
 cn axis and the total electric field at the probe . 
 these experimental 
 data demonstrate fundamental aspects of the electrostatic landscape 
 for catalysis in ecdhfr . 
 molecular dynamics 
 simulations provided an atomic - level picture 
 of the local environment experienced by the thiocyanate probes in 
 complexes that model the states prior to and subsequent to the hydride 
 transfer reaction . 
 the qm / mm vibrational frequency calculations reproduced 
 the experimentally measured frequency shifts for these two states , 
 which span the closed to occluded transition associated with hydride 
 transfer . 
 analysis of the md trajectories provided insight into the 
 conformational changes occurring between these two states . the factors 
 leading to the vibrational frequency shifts were broken down into 
 classical electrostatic effects , specific hydrogen - bonding interactions , 
 and polarization effects . 
 thus , the vibrational frequency shifts are 
 not simply a manifestation of the classical electric fields but rather 
 include additional contributions from the environment . 
 moreover , the 
 electric fields along the cn axis of the probes were decomposed into 
 contributions from specific residues , ligands , and solvent molecules . 
 the electric field along the hydride d  a axis was also calculated 
 with the experimentally validated methodology . 
 the calculations illustrate 
 that the cofactor and substrate , as well as the enzyme , impose a substantial 
 electric field along the d  a axis that facilitates hydride 
 transfer . 
 overall , experimental and theoretical data provide 
 evidence for 
 significant electrostatic changes in the microenvironments that constitute 
 the heterogeneous ecdhfr active site as the enzyme 
 progresses along the catalytic cycle . 
 the electrostatic interactions 
 between the protein and the ligands assist in orienting the reacting 
 species in a geometry that maintains a large electric field favoring 
 hydride transfer from the cofactor to the substrate . 
 the enzyme active 
 site environment also provides a significant portion ( 1/3 ) 
 of the total electric field that facilitates the hydride transfer 
 reaction , suggesting the catalytic importance of enzymatic conformational 
 changes that alter the active site electrostatics . 
 future work will 
 investigate the five trapped states along the catalytic cycle for 
 the two probes studied in this work as well as additional probe sites 
 in the ecdhfr system to provide a comprehensive picture 
 of the electrostatic landscape for the entire catalytic cycle .","electrostatic interactions play an 
 important role in enzyme catalysis 
 by guiding ligand binding and facilitating chemical reactions . 
 these 
 electrostatic interactions are modulated by conformational changes 
 occurring over the catalytic cycle . 
 herein , the changes in active 
 site electrostatic microenvironments are examined for all enzyme complexes 
 along the catalytic cycle of escherichia coli dihydrofolate reductase ( ecdhfr ) by incorporation 
 of thiocyanate probes at two site - specific locations in the active 
 site . the electrostatics and degree of hydration of the microenvironments 
 surrounding the probes 
 are investigated with spectroscopic techniques 
 and mixed quantum mechanical / molecular mechanical ( qm / mm ) calculations . 
 changes in the electrostatic microenvironments along the catalytic 
 environment lead to different nitrile ( cn ) vibrational stretching 
 frequencies and 13c nmr chemical shifts . 
 these environmental 
 changes arise from protein conformational rearrangements during catalysis . 
 the qm / mm calculations reproduce the experimentally measured vibrational 
 frequency shifts of the thiocyanate probes across the catalyzed hydride 
 transfer step , which spans the closed and occluded conformations of 
 the enzyme . 
 analysis of the molecular dynamics trajectories provides 
 insight into the conformational changes occurring between these two 
 states and the resulting changes in classical electrostatics and specific 
 hydrogen - bonding interactions . 
 the electric fields along the cn axes 
 of the probes are decomposed into contributions from specific residues , 
 ligands , and solvent molecules that make up the microenvironments 
 around the probes . 
 moreover , calculation of the electric field along 
 the hydride donor  acceptor axis , along with decomposition of 
 this field into specific contributions , indicates that the cofactor 
 and substrate , as well as the enzyme , impose a substantial electric 
 field that facilitates hydride transfer . 
 overall , experimental and 
 theoretical data provide evidence for significant electrostatic changes 
 in the active site microenvironments due to conformational motion 
 occurring over the catalytic cycle of ecdhfr .",pubmed
"magnetic particle imaging ( mpi ) is a new three - dimensional imaging method using static and dynamic magnetic fields to visualize the distribution of superparamagnetic iron oxide nanoparticles ( spios).1 imaging relies thereby on three characteristics of the spio , ie ; their superparamagnetic nature , their nonlinear magnetization curve , and their ability to be magnetically saturated.2 because of these characteristics , mpi can visualize and quantify the spatial distribution of spio , providing very high temporal and spatial resolution combined with high signal - to - noise ratios . 
 possible application scenarios for mpi might include cardiovascular imaging and even guidance of cardiovascular interventions.3,4 in principle , mpi would allow visualization and quantification of vascular pathologies while also being able to determine additional functional parameters in one examination . 
 further , mpi could be used as an imaging method for percutaneous transluminal coronary angioplasty , if indicated . 
 apart from the need for suitable mpi scanners , this diagnostic and therapeutic pathway relies on appropriate tracer material . ideally , long circulating spio are desirable , and should be able to provide a sufficient signal within the vessels after a single tracer injection at the beginning of the procedure , so a diagnostic scan and intervention can be done consecutively . 
 the circulation time of spio in blood is mainly determined by their overall so - called hydrodynamic diameter . although spio with a greater dimension of the iron oxide core , and consequently a larger hydrodynamic particle size , improve the superparamagnetic behavior of contrast agents in general , the coatings play a major role in the biodistribution and lesional uptake of spio . 
 previous reports demonstrate that spio with a hydrodynamic size greater than 50 nm have in common rapid and specific uptake by the reticuloendothelial system , and thus are cleared rapidly from the blood stream . compared with magnetic resonance imaging ( mri ) 
 , the properties of spio not only influence pharmacokinetics but also greatly influence the quality and sensitivity of the entire mpi system . to achieve high performance in mpi , an ideal tracer should exhibit spio with a homogeneously distributed iron core diameter of 30 nm , resulting in a relatively high hydrodynamic diameter and a shorter half - life in blood.1 although the characteristics of an ideal mpi tracer are well understood in theory , and this issue has been addressed by many working groups , no mpi - specific tracer has reached the marketplace so far . since the advent of mpi , many spio agents have been evaluated for their usefulness in mpi.5 resovist , an approved t2 * contrast agent for mri , is the most important benchmark material , and remains the best performing commercial spio for mpi today and the standard of reference for evaluation of mpi hardware and application , although it has known drawbacks , ie , a hydrodynamic diameter greater than 50 nm and therefore , a limited in vivo distribution.6 however , in the context of cardiovascular mpi , knowledge about the pharmacokinetics of resovist , especially its half - life in blood , is important . 
 naturally , this issue was investigated about 20 years ago , when resovist was evaluated as a contrast agent for mri.7 however , because the imaging principle is fundamentally different in mpi , results from mri can not be easily extrapolated to mpi , as not all resovist particles contribute equally to the mpi signal and larger particles are cleared more rapidly by the reticuloendothelial system ( res ) . 
 thus , the aim of this study was to evaluate the blood half - life of mpi using magnetic particle spectroscopy ( mps ) , given that it may differ significantly from the blood half - life in mri . 
 marketing of resovist by bayer pharma ag ( berlin , germany ) has been discontinued , and it is only available now from the japanese distributor , irom pharmaceutical co ltd ( tokyo , japan ) . 
 however , until now , most mpi experiments were done using resovist from bayer pharma ag , whereas most working groups now use resovist from irom pharmaceutical co ltd . 
 the experimental protocol was approved by the state regulations governing the performance of animal studies . 
 all experiments were conducted under strict adherence to the guidelines for the care and use of laboratory animals as described by the national institutes of health . 
 two different versions of resovist were studied , ie , that distributed by bayer pharma ag in europe and that distributed by irom pharmaceutical co ltd . 
 it was approved for clinical use in 2001 in europe and in 2002 in japan.8,9 marketing of the bayer pharma ag product has now been discontinued in europe . 
 resovist ( 50 mol / kg ) was administered into the ear artery of ten female new zealand white rabbits after drawing of a control blood sample . 
 five animals received resovist by irom pharmaceutical co ltd and five by bayer pharma ag , respectively . 
 each rabbit weighed approximately 4 kg . directly ( about 30 seconds ) after administration of resovist , a second blood sample was collected as a baseline sample ( t0 ) from the contralateral ear artery to avoid contamination with residual resovist . 
 this was repeated after 5 , 10 , 15 , 30 , 45 , 60 , 75 , 90 , 105 , and 120 minutes . during the experiments , 
 the animals were kept under full anesthesia by bodyweight - adapted subcutaneous injection of ketamine hydrochloride ( 0.7 ml / kg bodyweight ; ceva animal health , dsseldorf , germany ) and xylazine hydrochloride ( 0.3 ml / kg bodyweight ; ceva animal health ) . 
 blood samples were collected in accordance with the recommendations of the german society for laboratory animal science.10 the mpi signal was evaluated using mps . 
 mps can be described as a zero - dimensional system that measures the remagnetization spectrum of spio . in mps , only the oscillating magnetic field that excites the spio is used . 
 the magnetic gradient field , which is used for spatial encoding in mpi , is omitted.11 using mps it is possible to evaluate the quality of the mpi signal of particles , so the applicability of the particles for mpi can be characterized . in an ideal superparamagnetic particle , 
 the spectra consist only of odd harmonics due to the symmetry of the particle magnetization curve . in an experimental setup 
 , even harmonics are added by the earth s magnetic field , but can be neglected in evaluation of the particles.12 thus , the signal strength can be determined by evaluating the odd harmonics of spio above noise level . 
 the magnitude of the signal is proportional to the concentration of the spio . as in previous experiments,5 
 the mps excitation frequency was 25 khz ( as is most common in mpi systems ) , the magnetic field amplitude was 20 mt , and the acquisition time was 5 seconds ( corresponding to 125,000 repetitions ) to provide a high signal - to - noise ratio . 
 ten milliliters of each blood sample were transferred into individual eppendorf tubes directly after sampling , and then measured using mps . 
 the signal strength of the odd harmonics from the third to the 49th harmonic was measured at each time point and related to the signal strength of the baseline sample ( t0 ) . 
 thus , the decline of the mpi signal in blood could be described in relation to the signal maximum at baseline directly after administration of resovist . 
 for each group , ie , resovist distributed by bayer pharma ag and irom pharmaceutical co ltd , respectively , the average (  standard deviation ) decline in signal strength was calculated for each time point . 
 ten microliters of pure resovist ( 500 mol fe / ml ) were drawn from each package unit and diluted using ringer s solution to 400 , 300 , 200 , 100 , 80 , 60 , 40 , 20 , 10 , 8 , 6 , 4 , 2 , and 1 mol fe / ml . 
 the hydrodynamic diameter and size distribution of the spio for the two different types of resovist were measured using photon cross correlation spectroscopy ( nanophox , sympatec gmbh , clausthal - zellerfeld , germany ) . in photon 
 cross correlation spectroscopy , the particle size is measured by three - dimensional cross correlation of the scattered light of two intersecting focused laser beams.13 the statistical significance of the difference in the signal decay in blood between the two types of resovist was calculated using the mann 
 further , the statistical significance of the difference between the two package units in the dilution series was calculated in the same way . in both cases , 
 two different versions of resovist were studied , ie , that distributed by bayer pharma ag in europe and that distributed by irom pharmaceutical co ltd . 
 it was approved for clinical use in 2001 in europe and in 2002 in japan.8,9 marketing of the bayer pharma ag product has now been discontinued in europe . 
 resovist ( 50 mol / kg ) was administered into the ear artery of ten female new zealand white rabbits after drawing of a control blood sample . 
 five animals received resovist by irom pharmaceutical co ltd and five by bayer pharma ag , respectively . 
 each rabbit weighed approximately 4 kg . directly ( about 30 seconds ) after administration of resovist , a second blood sample was collected as a baseline sample ( t0 ) from the contralateral ear artery to avoid contamination with residual resovist . 
 this was repeated after 5 , 10 , 15 , 30 , 45 , 60 , 75 , 90 , 105 , and 120 minutes . during the experiments , 
 the animals were kept under full anesthesia by bodyweight - adapted subcutaneous injection of ketamine hydrochloride ( 0.7 ml / kg bodyweight ; ceva animal health , dsseldorf , germany ) and xylazine hydrochloride ( 0.3 ml / kg bodyweight ; ceva animal health ) . 
 blood samples were collected in accordance with the recommendations of the german society for laboratory animal science.10 
 mps can be described as a zero - dimensional system that measures the remagnetization spectrum of spio . in mps , only the oscillating magnetic field that excites the spio is used . 
 the magnetic gradient field , which is used for spatial encoding in mpi , is omitted.11 using mps it is possible to evaluate the quality of the mpi signal of particles , so the applicability of the particles for mpi can be characterized . in an ideal superparamagnetic particle , 
 the spectra consist only of odd harmonics due to the symmetry of the particle magnetization curve . in an experimental setup 
 , even harmonics are added by the earth s magnetic field , but can be neglected in evaluation of the particles.12 thus , the signal strength can be determined by evaluating the odd harmonics of spio above noise level . 
 the magnitude of the signal is proportional to the concentration of the spio . as in previous experiments,5 
 the mps excitation frequency was 25 khz ( as is most common in mpi systems ) , the magnetic field amplitude was 20 mt , and the acquisition time was 5 seconds ( corresponding to 125,000 repetitions ) to provide a high signal - to - noise ratio . 
 ten milliliters of each blood sample were transferred into individual eppendorf tubes directly after sampling , and then measured using mps . 
 the signal strength of the odd harmonics from the third to the 49th harmonic was measured at each time point and related to the signal strength of the baseline sample ( t0 ) . 
 thus , the decline of the mpi signal in blood could be described in relation to the signal maximum at baseline directly after administration of resovist . for each group , 
 ie , resovist distributed by bayer pharma ag and irom pharmaceutical co ltd , respectively , the average (  standard deviation ) decline in signal strength was calculated for each time point . 
 ten microliters of pure resovist ( 500 mol fe / ml ) were drawn from each package unit and diluted using ringer s solution to 400 , 300 , 200 , 100 , 80 , 60 , 40 , 20 , 10 , 8 , 6 , 4 , 2 , and 1 mol fe / ml . 
 the hydrodynamic diameter and size distribution of the spio for the two different types of resovist were measured using photon cross correlation spectroscopy ( nanophox , sympatec gmbh , clausthal - zellerfeld , germany ) . in photon 
 cross correlation spectroscopy , the particle size is measured by three - dimensional cross correlation of the scattered light of two intersecting focused laser beams.13 
 the statistical significance of the difference in the signal decay in blood between the two types of resovist was calculated using the mann 
 further , the statistical significance of the difference between the two package units in the dilution series was calculated in the same way . in both cases , 
 the average mpi signal in blood declined to 30% of the baseline signal 5 minutes after injection . 
 ten and 15 minutes after injection , the average mpi signal was 14.8% and 7% , respectively , of the baseline signal . within 30 minutes 
 , the mpi signal was below the noise level of the mps ( see table 1 ) . 
 regardless of this result , the mpi signal was measured consecutively until 120 minutes after application ; however , it was still not possible to detect any signal above the noise level . 
 for further evaluation , the signal decline for both types of resovist was compared . the signal for resovist from bayer pharma 
 ag decreased to 39.7% of baseline after 5 minutes , to 20.5% after 10 minutes , and to 12.1% after 15 minutes . 
 the signal for resovist from irom pharmaceutical co ltd showed a rapid decrease to 20.4% of the baseline signal after 5 minutes and to 7.8% after 10 minutes ( see table 1 and figure 1 ) ; in contrast with resovist from bayer pharma ag , the signal dropped below noise level even after 15 minutes . 
 this difference was statistically significant ( mann  whitney u - test , p=0.0056 ) . 
 in contrast with the signal decay , the absolute signal strength of the baseline blood mpi signal was not significantly different between the two resovist products ( 1.60e-88 , 09e-9 amhz for resovist from bayer pharma ag and j1.98e-84 , 13e-9 amhz for resovist from irom pharmaceutical co ltd , p=0.3953 , mann 
 in addition , the dilution series showed no statistical significant difference in mpi signal decay between the two compounds ( mann  whitney u - test , p=0.6241 , see figure 3 ) . 
 photon cross correlation spectroscopy showed a median hydrodynamic diameter ( x50 ) of 70.54 nm for resovist from bayer pharma ag and 61.86 nm for resovist from irom pharmaceutical co ltd . 
 the size distribution was x1060.41 nm and x9082.07 nm ( x10/x90=1.36 ) for resovist from bayer pharma ag and x10 and x9051.16 nm and 73.08 nm ( x10/x90=1.4 ) for resovist from irom pharmaceutical co ltd . 
 first , resovist is not suitable as an intravascular tracer in terms of prolonged circulation . given the current lack of alternatives in mpi , it may be used as a bolus tracer either intravenously for a diagnostic scan or be applied during a cardiovascular intervention using an intravascular catheter . 
 the present study underlines once more the need for a dedicated mpi tracer with a prolonged blood half - life of its mpi signal . 
 second , it highlights the fact that the blood half - life of the mpi tracer can not be equalized to the blood half - life time of the mpi signal . 
 the concordance of these two values depends on the fraction of particles contributing to the mpi signal . 
 third , the difference in blood half - life between resovist sourced from bayer pharma ag and that from irom pharmaceutical co ltd has to be taken into account when comparing different studies . 
 further , due to the inhomogeneous size distribution from batch to batch , it is even possible that tracers of the same type show differences in blood half - life . 
 the rapid decay of the mpi signal is in concordance with the initial phase of elimination of resovist in the studies conducted in humans using mri . in their mri studies , 
 hamm et al14 observed two phases of elimination of the contrast agent . in the first phase , 
 about 80% of the resovist concentration was cleared , with a blood half - life of 3.95.8 minutes , whereas that of the second phase was 2.43.6 hours . 
 the blood half - life was therefore independent of the concentration administered.14 the package insert for resovist states that the first phase is 15.611.4 minutes and the second phase is 4.360.75 hours.8 in contrast , no second phase of tracer elimination could be detected using mps in our study . 
 this difference can be explained by the different principles of signal generation in mpi and mri , and the broad size distribution of the spio in resovist . in mri , 
 the effect of the spio is a shortening of the t2 time in the surrounding tissues , leading to a signal loss , especially in t2 * weighted images . 
 thus , in mri , spio work as an indirect contrast agent influencing the signal characteristics of the surrounding tissue . 
 this occurs independently of the size of the spio , as long as they still have superparamagnetic characteristics . in mpi 
 , the spio are visualized directly by their change of magnetization in an oscillating magnetic field . 
 thus , they work like a tracer and not like a contrast agent . as mentioned above 
 , the diameter of the iron core should be around 30 nm for an ideal mpi signal.1 in resovist , theoretically only 3% of particles meet this criterion and contribute substantially to the mpi signal.1 recently , eberbeck et al6 demonstrated by analysis of the m(h ) curve for resovist that there are clusters of smaller spio in resovist that behave like single domain particles and have an effective mean iron core diameter of 241 nm . in their data , this fraction of particle clusters amounted to 30%3% of the nanoparticles in resovist and explain its good mpi performance.6 the other fraction of spio in resovist has an effective mean iron core diameter of 5.53 nm , and does not contribute substantially to the mpi signal.6 it is important to note that the physical mean iron core diameter as measured by , eg , transmission electron microscopy and vibrating sample magnetometry , is between 5 nm and 15 nm , as described by many working groups , and the clusters contributing most of resovist s mpi - signal as described in eberbeck et al6 contain these smaller particles , as can also be shown by transmission electron microscopy.6,1519 these clusters may also account for a fraction with a larger hydrodynamic diameter than the other particle fractions in resovist . 
 these would be cleared more rapidly from the blood by the reticuloendothelial system and the mpi signal would decrease more rapidly than the actual concentration of the tracer measured , eg , on mri . 
 this explanation is supported by a study reported by khandhar et al.19 the authors evaluated resovist and a self - designed tracer for their mpi performance in mice and showed that the decay of the mpi signal from resovist in blood was considerably faster than the decay of the magnetization curve measured by vibrating sample magnetometry . 
 this also indicates that there are resovist particles left in the blood that do not contribute to the mpi signal.19 in contrast with our results , they observed an even faster decrease in the mpi signal to below noise level within 10 minutes . 
 this is probably due to the differing sensitivities of the respective mps systems , as there are no international standardized systems on the market as yet . given that khandhar et al19 did not reveal the dose of resovist related to body weight of the mice , differences in the dose of resovist initially applied might explain the shorter  time above noise level  as well . 
 further , khandhar et al described an initial slow increase of mps signal from 0 to 5 minutes after administration.19 this also seems to be in contrast with the findings of weizenecker et al,20 who were the first group to present in vivo mpi images , which visualized the beating heart of a mouse after application of a bolus of resovist from bayer pharma ag . 
 an approximate equilibrium was achieved in their study about 15 seconds after intravenous injection , and a rapid decay of the tracer concentration determined by the mpi demonstrator in the vena cava from about 3.8 mmol 
 fe / l at first pass to 0.28 mmol fe / l at third pass 10.2 seconds later was described.20 in summary , it seems that the resovist particles contributing most to the mpi signal are cleared most rapidly from the blood . 
 interestingly , we found a significant difference in blood half - life between resovist manufactured by bayer pharma ag and that manufactured by irom pharmaceutical co ltd 
 . however , our measurements do not indicate significant differences in hydrodynamic diameter or size distribution , which would be the most likely explanation for these differences in blood half - life . 
 resovist from bayer pharma ag showed a slightly larger hydrodynamic diameter than that from irom pharmaceutical co ltd . 
 a difference in formulation of the polymeric coating is improbable , given that the japanese company that developed resovist in cooperation with schering ag ( berlin , germany ) still produces the drug substance for resovist , ie , ferucarbotran ( meito sangyo co ltd , nagoya , japan ) . 
 further , there is no discrepancy in the baseline signal strength or signal loss in the dilution series between the two types of resovist , ie , the mpi performance of the tracers . 
 resovist from irom pharmaceutical co ltd performed slightly , albeit not significantly , better than resovist from bayer pharma ag ( figure 3 ) . 
 all mpi - specific research to date regarding particle characterization and dynamics has been conducted using resovist from bayer pharma ag , and there are no such data for resovist from irom pharmaceutical co ltd . 
 tsuchiya et al21 indicate that the iron core diameter of resovist from irom pharmaceutical co ltd is 5 nm , but do not provide a reference for this or a method for how the iron core diameter was measured . 
 given that the mpi performance of resovist sourced from the two distributors is almost the same , it is highly probable that the mechanism of clustering of resovist particles as described by eberbeck et al6 for resovist by bayer pharma ag holds true , otherwise , the mpi performance would be well below that of resovist distributed by bayer pharma ag . 
 thus , for resovist to be used further in the setting of mpi , two issues have to be considered . 
 firstly , the distribution of resovist by bayer pharma ag in europe has been discontinued and it is no longer commercially available . 
 therefore , resovist distributed by irom pharmaceutical co ltd may be the standard of reference until a dedicated spio tracer is marketed , whereas the first in vivo experiments in mpi were conducted using resovist distributed by bayer pharma ag.20 this should be considered when evaluating experimental results . 
 secondly , the difference in blood half - life between the two compounds is significant , but not as high as to indicate a change of application . 
 as we showed , in accordance with other investigators , the mpi signal of resovist in blood lacks the second , slower elimination phase observed in mri and rapidly decays below noise level , so is not ideal for applications requiring long circulating or even blood pool tracers . in this study , 
 the mpi signal was measured using mps , which is known to be the most sensitive method for signal detection ; thus , the time frame in which resovist can be visualized with current and upcoming mpi scanners will probably be even shorter , and resovist will most likely be suitable only as a bolus tracer for vascular applications . 
 in addition to the challenges concerning scanner design , the design of tracers will be a key aspect in future development of mpi . 
 spio formulations originally designed for mri do not exhibit ideal mpi characteristics , so research is now focused on development of a dedicated mpi tracer , ie , an spio tracer with a homogenously distributed iron core diameter of about 30 nm.6,18,19,22 here , resovist serves as an international standard of reference for the performance of mpi using new tracers . 
 mps is the most important tool for evaluation , but magnetorelaxometry , vibrating sample magnetometry , and m(h ) curves are also important tools , particularly for description , and explain the sometimes complex mpi performance of spio.6,18,19 apart from the need for ideal mpi characteristics , mostly determined by the iron core and the homogenous size distribution of the particles , pharmacokinetics have a crucial role in the use of the tracer in vivo . for long circulating tracers , 
 a small hydrodynamic diameter is desirable . because the iron core diameter should be 30 nm , the minimum hydrodynamic diameter is limited , even if the coating is made of , eg , thin monomer layers 
 recently , rahmer et al reported a proof of principle study in which red blood cells in mice were loaded with resovist , enabling mpi of the vascular system for several hours after application.23 another option might be to modify the coating and charge on the spio in order to enhance circulation time.24 outside the mpi community , a lot of research has already been conducted with regard to different spio particle coatings and surface modifications for specific areas of application , eg , for targeted imaging or to prolong the blood circulation time , and mpi can benefit from this knowledge . in order to enable cooperation and dissemination of knowledge , a standardized physicochemical testing protocol as 
 a part of particle development is desirable , as already proposed.25,26 these standards may even simplify the long way from  bench to bedside  . 
 however , resovist remains the only industrially produced ( in japan ) and approved spio formulation applicable for mpi . 
 feraheme ( ferumoxytol , amag pharmaceuticals , waltham , ma , usa ) is a spio formulation approved by the us food and drug administration for the treatment of iron deficiency anemia in adult patients with chronic kidney disease , but is not approved for mpi due to its inadequate mpi signal characteristics . 
 additionally , the diverging performance of the two types of resovist could not be fully elucidated . 
 therefore , studies of different batches of resovist in larger groups of animals may be necessary . 
 further , analysis of resovist distributed by irom pharmaceutical co ltd corresponding to , ie , eberbeck et al , should be executed in the future.6 ideally , the pharmacological formulation of the two distributions should be analyzed in detail , because this may be a reason for the different half - life times . 
 finally , the results need to be confirmed by visualization of vessels in larger animals in real time to determine the effect on mpi in vivo . 
 in conclusion , our study shows that resovist is not a suitable tracer for applications requiring a long circulation time of the mpi tracer in blood . 
 more importantly , our study highlights a difference in a tracer s blood half - life with regard to the mpi signal on the one hand , and the mri signal and iron concentration on the other . 
 interestingly , there are even distinctions between different distributions of the same pharmaceutical agent , in this case resovist by bayer pharma ag and irom pharmaceutical co ltd . 
 consequentially , all spio formulations have to be evaluated for the half - life of the mpi signal in blood before use in mpi .","backgroundmagnetic particle imaging ( mpi ) uses magnetic fields to visualize superparamagnetic iron oxide nanoparticles ( spio ) . today 
 , resovist is still the reference spio for mpi . 
 the objective of this study was to evaluate the in vivo blood half - life of two different types of resovist ( one from bayer pharma ag , and one from irom pharmaceutical co ltd ) in mpi.methodsa resovist concentration of 50 mol / kg was injected into the ear artery of ten new zealand white rabbits . 
 five animals received resovist distributed by irom pharmaceutical co ltd and five received resovist by bayer pharma ag . 
 blood samples were drawn before and directly after injection of resovist , at 5 , 10 , and 15 minutes , and then every 15 minutes until 120 minutes after the injection . 
 the mpi signal of the blood samples was evaluated using magnetic particle spectroscopy.resultsthe average decline of the blood mpi signal from the two distributions differed significantly ( p=0.0056 ) . 
 resovist distributed by bayer pharma ag showed a slower decline of the mpi signal ( 39.7% after 5 minutes , 20.5% after 10 minutes , and 12.1% after 15 minutes ) compared with resovist produced by irom pharmaceutical co ltd ( 20.4% after 5 minutes , 7.8% after 10 minutes , no signal above noise level after 15 minutes).conclusionin mpi , the blood half - life of an spio tracer can not be equalized to the blood half - life of its mpi signal . 
 resovist shows a very rapid decline of blood mpi signal and is thus not suitable as a long circulating tracer . for cardiovascular applications in mpi 
 , it may be used as a bolus tracer .",pubmed
"intracellular pathogens have evolved over many millions of years to coexist in the face of a wide array of host defense mechanisms . in  vitro studies of the host - pathogen interaction 
 exclude the inflammatory milieu , but a major challenge in studying the host response in  vivo is dissecting pathogen - specific responses from the general inflammatory or common host response ( jenner and young , 2005 ) that occurs as a result of tissue insult during the infectious process . 
 this latter response is in part driven by activation of components of the complement cascade ( ricklin et  al . , 2010 ) , the coagulation pathway ( oikonomopoulou et  al . , 2012 ) , and the acute phase response ( suffredini et  al . , 1999 ) , which 
 all come into play within minutes of infection , and is compounded by rapid changes to the inflammatory cytokine and chemokine environment ( oghumu et  al . 
 , 2010 ; sadik and luster , 2012 ; soehnlein and lindbom , 2010 ) . 
 each of these stimuli may have profound effects on phagocyte function ( galvan et  al . 
 , 2012 ; hoffmann et  al . , 2010 ) and reflect , in the broader sense , the normal and/or essential response of phagocytes to inflammatory insult . 
 hence , responses that directly reflect intracellular infection are difficult to distinguish from dominant inflammatory signatures by conventional experimental approaches . 
 we hypothesized that it might be possible to use the transcriptomic response of uninfected cells exposed to inflammation as a measure of the baseline or normal in  vivo response to insult . following this argument 
 , transcriptomic networks specifically observed in infected cells , but not in neighboring uninfected cells , would represent cell - intrinsic pathogen - induced changes . 
 in contrast , transcriptomic networks restricted to uninfected cells would serve to identify responses potentially beneficial to the host , the lack of which ( in infected cells ) might favor pathogen survival . 
 we tested the above hypothesis in a tractable experimental model of leishmania infection , a pathogen associated with a significant global health burden ( alvar et  al . , 2012 ) . 
 leishmania infect a variety of myeloid cells ( including tissue - resident macrophages , inflammatory monocytes , dendritic cells [ dcs ] , and neutrophils ) , but in models of experimental visceral leishmaniasis , leishmania donovani amastigotes have a predilection for resident tissue macrophages , including kupffer cells ( kcs ) , the resident tissue macrophage lining the hepatic sinusoids ( beattie et  al . , 2010 ; murray , 2001 ) . in this study 
 , we discovered that expression of an rxra - centered transcriptomic pathway , which is downregulated in kc in response to inflammation , is aberrantly maintained in infected cells . 
 pharmacological manipulation of rxr activity perturbed the transcriptomic network of infected kcs , leading to enhanced leishmanicidal activity . 
 this study demonstrates that aberrant expression of the rxra - centered transcriptional pathway in leishmania - infected kcs favors parasite survival and validates the use of cells exposed to inflammation as a means to discover pathogen - regulated transcriptomic pathways in  vivo . 
 in the livers of uninfected ( lysm - cre  mt / mg)f1 mice , kcs were obvious from their large size , expression of membrane gfp , and their continued and intimate contact with liver sinusoidal endothelial cells , as has been previously described ( lee et  al . 
 four - dimensional ( 4d ) intravital two - photon imaging further allowed the fine membrane structure and lamellipodia dynamics of steady - state kcs to be observed in exceptional detail ( figure  1a ; movie s1 ) . the continued extension and retraction of kc lamellipodia manifested itself as small fluctuations of cellular volume ( figure  1b ) and surface area over time ( figure  1c ) . 
 in contrast , monocytes and/or neutrophils were identified as moving rapidly through the sinusoidal spaces , whereas liver sinusoidal endothelial cells ( lsecs ) lined the sinusoidal lumen ( figures 1f and 1 g ) . 
 shape factor , a quantitative measure of how close a cell is to a perfect sphere ( shape factor  =  1 ) , showed small fluctuations over time for kcs ( figure  1d ) but was always lower than that for either lsecs or monocytes and/or neutrophils ( figure  1h ) . 
 the effects of kc membrane fluctuation can also be represented by a velocity , which was calculated by measuring the movement around the centroid point of the cell in three dimensions . 
 thus , although kcs do not actively migrate along the sinusoids , velocity as measured here provides an indication of the extent of membrane activity . 
 in contrast , nonmotile lsecs have an even lower velocity ( limited membrane activity ) . 
 many of the monocytes and/or neutrophils that were examined actively migrated along the sinusoids within the time frame of observation , and their velocity was not surprisingly high ( figure  1i ) 
 . as the early response of kcs to infection with l.  donovani amastigotes in  vivo is largely uncharacterized , we examined kc behavior before and after infection in live mice . 
 we injected ( lysm - cre  mt / mg)f1 mice with transgenic td - tomato - expressing  l.  donovani amastigotes ( beattie et  al . , 2010 ) at a dose that resulted in approximately 20% of kcs becoming infected over a 2  hr time period ( a time by which most parasites have been cleared from the blood by kcs and other tissue phagocytes ; figures 2a and 2b ; gorak et  al . , 1998 ) . 
 the binding of amastigotes to the kc membrane ( figure  2a ) was followed by complete phagocytosis within 20  s ( movie s2 ) . 
 neither injection of live , nor of heat - killed , amastigotes resulted in detectable changes in kc volume ( figure  2c ) or surface area ( figure  2d ) , irrespective of whether or not the kcs contained visible amastigotes . 
 in contrast , injection of live , but not dead , amastigotes induced a rapid increase in the average shape factor that was significant for kcs that contained live parasites and showed a trend in this direction for kcs that did not contain amastigotes ( figure  2e ) . when velocity was measured as an indicator of membrane activity ( figure  2f ) 
 , a significant decrease was observed in both infected and uninfected kcs following injection of live amastigotes , suggesting that both populations of cells were responding to the inoculated parasites through rapid activation . 
 such activation was not observed following injection of dead amastigotes ( figure  2f ) . 
 collectively , these data were indicative of a cellular response to active infection , rather than a response merely to nonspecific debris that may be present within the amastigote preparations . 
 the observation that uninfected kcs in the immediate vicinity of infected cells also responded to the infection by loss of membrane activity ( figure  2f ) suggested that the above changes in kc behavior were the result of either signals provided from infected kcs and operating rapidly in trans or from the activation of serum - derived mediators of inflammation , rather than being a direct consequence of intracellular parasitism per se . in either case , the capacity to activate this response was absent from heat - killed amastigote preparations . as we wished to examine the transcriptional events that accompanied the observed change in kc behavior , we developed a method to identify and isolate kcs based on whether they contained parasites ( herein termed infected ) or not ( herein termed inflamed ) . 
 flow cytometry analysis of hepatic mononuclear cell preparations with gating on forward and side scatter ( figure  3a ) , complement receptor of the immunologlobulin superfamily ( crig ) ( helmy et  al . , 2006 ) , and gr-1 ( figure  3b ) demonstrated that both infected and inflamed populations of kcs could be observed and discriminated by the presence of fluorescent amastigotes ( figure  3c ) . both infected and inflamed kcs showed heterogeneous expression of cd11b but relatively uniform expression of f4/80 ( figure  3d ) . 
 backgating on the infected and inflamed kcs showed that both populations contained both crig gr-1 cells and crig gr-1 cells ( figure  3e ) . to confirm that a proportion of kcs express gr-1 in  situ , we labeled liver sections with f4/80 , crig , and gr-1 ( figures 3f3i ) and observed variable expression of gr-1 on kcs , in addition to gr-1 neutrophils that did not express crig or f4/80 and were readily identifiable from their irregular - shaped nuclei ( figures 3h and 3i ) . 
 we then used fluorescence - activated high - speed cell sorting to isolate kcs and separate them into either infected or uninfected cell populations . 
 we identified kcs from infected mice at 2  hr and 12  hr postinfection ( p.i . ) , as described above , based on forward and side scatter ( size and granularity ) ( figure  3j ) and expression of crig , gr-1 ( figure  3k ) , and f4/80 ( figure  3l ) and then further sorted these based on whether or not they contained parasites ( figures 3m3o ) 
 . cytospins of the sorted populations demonstrated that the cells had macrophage - like morphology , that neutrophils were not included in the analysis , and the presence of amastigotes within the infected population ( figure  3q ) , but not in the inflamed population ( figure  3p ) . 
 kcs were also isolated from sham - infected mice to serve as a control population that exhibited steady - state activity . to determine whether there were underlying transcriptional events that differed between kcs infected with amastigotes in an inflammatory setting and those induced in kcs exposed to inflammatory stimuli alone , transcriptomic profiles for each population , in addition to kcs from sham - infected controls , were generated by microarray analysis . instead of performing a direct comparison of the differences in gene expression between the infected and inflamed kcs 
 , we generated a list of genes differentially expressed between infected kcs and sham - infected kcs ( using a cutoff of > 3-fold difference ) . 
 this approach eliminated the possibility that any of the regulated genes were induced or suppressed due to the isolation procedure , as the sham - infected kcs were subjected to the same digestion and sort process . 
 the same analysis was then performed for the inflamed kcs . as a result of this analysis 
 , we therefore generated two gene lists ( infected and inflamed kcs ) for 2  hr p.i . and two gene lists for 12  hr p.i . 
 at 2  hr p.i . , the expression of 1,188 genes ( 909 up and 279 down ) was differentially regulated between sham - treated and infected kcs , whereas only 353 genes ( 327 up and 26 down ) were differentially regulated between sham - treated and inflamed kcs . by 12  hr p.i . 
 ,  the number of genes showing changes in expression was substantially increased ( 2,135 between sham - treated and infected kcs , of which 1,337 had increased , and 798 had decreased , expression ) . string analysis of these genes showed that although there were some interactions between these genes , there were a large number of genes that were outside of the central network ( figure  s1 ) . 
 although substantially fewer genes ( 1,288 ) were differentially expressed at a messenger rna ( mrna ) level between the sham - treated and inflamed kcs overall ( 713 upregulated and 575 downregulated ) , string analysis for these genes showed a higher degree of connectivity between the genes within this list and a substantial network connection that did not appear to be present in the infected cells ( figure  s1 ) . 
 the top 20 genes ( based on fold change compared to sham - infected kcs for each comparison , or according to relative expression ) are provided in table s1 . 
 we interpret these data as indicating that there are genes that are normally regulated as part of the inflammatory response that are not regulated in infected cells . 
 to determine the identity of genes and gene pathways that were common between the infected and inflamed cells , and those that were unique to either population , we performed a subtractive analysis of the gene lists ( figure  4a ) . 
 from this analysis , we could readily identify genes that were either commonly regulated in both infected and inflamed cells or uniquely regulated in either infected or inflamed kcs at both 2  hr and 12  hr p.i . 
 for all genes identified as being selectively or commonly expressed in infected kcs and inflamed kcs , we have provided the top 20 genes ranked by expression level ( table s2 ) . 
 the gene lists were then subjected to pathway and gene ontology ( go ) analysis ( see experimental procedures for detail ) . 
 a total of 1,088 genes were uniquely regulated in their expression at 12  hr p.i . 
 genes that showed differentially regulated expression in both infected and inflamed cells were enriched for go terms suggestive of responsiveness to inflammation , including host defense and inflammatory response and cytokine - cytokine receptor interaction and chemokine activity . 
 significantly , we identified 241 genes that were differentially regulated in their expression in inflamed cells , but not in cells that contained intracellular amastigotes , in spite of exposure of these cells to the same inflammatory insult 
 . ingenuity pathway analysis ( ipa ) was then used to identify genes that fell within ipa - generated pathways at a higher frequency than would normally be expected to occur for a randomly selected set of genes . 
 this analysis identified 8 interacting networks within the inflamed gene list , in which genes involved in cell - to - cell signaling and the inflammatory response ( ipa network score of 38 ) , cell death ( ipa network score of 37 ) , antigen presentation ( ipa network score of 35 ) , cancer ( ipa network score of 26 ) , immune cell trafficking and cell cycle ( ipa network score of 23 ) , and cell morphology and cellular compromise ( ipa network score of 10 ) were all overrepresented . 
 the network that showed the highest level of gene enrichment was that of the lipid metabolism and small - molecule biochemistry pathway ( figure  4c ) , which had an ipa network score of 40 . the fold change and the expression values for this pathway 
 are shown in table 1 . as the above networks were not regulated in infected cells 
 , we hypothesized that they might represent genes and gene pathways for which homeostasis was favorable to parasite survival and , inter alia , that they represented genes potentially associated with antimicrobial defense regulated by inflammation . to test this hypothesis , we selected the most substantially represented network for further proof - of - concept studies ( figure  4c ) . 
 ipa identified that , 11% ( 27/241 genes ) of genes selectively regulated in their expression in inflamed kcs were found within the lipid metabolism and small - molecule biochemistry pathway ( figure  4c ) , which had retinoid x receptor  ( rxra ) as a key hub ( with rxra itself being downregulated 3.5-fold in inflamed cells ) . 
 we then sought to confirm the level of mrna accumulation in infected and inflamed kcs in independent samples by quantitative real - time pcr ( figure  5 ) . 
 although rxra mrna expression was not significantly different in infected and inflamed cells ( figure  5b ) , other genes within this network were validated as being differentially expressed between infected and inflamed cells , including cebpa ( figure  5c ) , vamp4 ( figure  5d ) , lxr/nr1h3 ( figure  5f ) , and lxr/nr1h2 ( figure  5 g ) . as infection with leishmania resulted in maintenance or upregulation of expression of genes within the rxra - associated gene network 
 , we reasoned that if this was associated with parasite survival , then forced down modulation of rxr activity might sufficiently perturb the entire rxra - related gene network within infected cells to generate an enhanced antimicrobial effect . 
 we therefore tested this approach by targeted pharmacological intervention , using a small - molecule antagonist hx531 , specific for rxr ( ebisawa et  al . 
 , 1999 ; yamauchi et  al . , 2001 ) . to first establish that hx531 treatment had a direct effect on the gene network in infected kcs , we treated mice with hx531 or saline , infected the mice with l.donovani , and isolated infected or inflamed kcs to examine gene expression by quantitative real - time pcr ( figure  5a ) . 
 treatment had no significant effect on the expression of rxra at an mrna level ( figure  5b ) , but treatment resulted in a significant modulation of the expression of a number of network - associated genes , including cebpa ( figure  5c ) ,  vamp4 ( figure  5d ) , vdr ( figure  5e ) , lxr ( but not lxr ) ( figures 5f and 5 g ) , and rara ( figure  5h ) . 
 peroxisome proliferator - activated receptor  ( ppar ) was not regulated in infected cells ( figure  5i ) and showed no change in expression following hx531 treatment . 
 we also examined the effect of hx531 treatment on a number of rxr - related genes that were not represented within the identified network ( figure  4c ) , including abca1 ( figure  5j ) , abcg1 ( figure  5k ) , apoe ( figure  5l ) , cyp27a1 ( figure  5 m ) , and cyp27b1 ( figure  5n ) . 
 treatment had no measurable effect on the expression of these genes in kcs , with the exception of abca1 , where treatment appeared to block upregulation in uninfected cells ( figure  5j ) . 
 hx531 treatment blocked the upregulation of il-1 in infected kcs ( figure  5o ) but had no effect on inducible nitric oxide synthase ( inos ) mrna levels in infected or inflamed kcs ( figure  5p ) . to confirm that genes within this network were not subject to regulation merely by phagocytosis , we performed rt - pcr for a subset of genes within this network on kc isolated from mice injected with latex beads . 
 as shown in figure  s2 , uptake of latex did not affect mrna levels for rxra ( figure  s2a ) , lxr ( figure  s2b ) , cebpa ( figure  s2c ) , or vamp4 ( figure  s2d ) , relative to kc isolated from control - injected mice . to determine the effect that the perturbation of the rxr pathway would have on infection , and hence to examine the potential of the expression profiling approach that we have used for the discovery of important host response pathways , we treated mice with hx351 prior to and for 4  days following infection with  td - tomato l.  donovani amastigotes ( figure  6a ) . using a flow cytometric approach to quantify the number of parasites in the tissues , we first assessed splenic and hepatic parasite burden at 2  hr p.i . 
 , when there was no difference in the presence or absence of treatment , and at 96  hr p.i . 
 treatment significantly reduced the number of parasites present in both the liver and the spleen at 4  days postinfection relative to the saline - treated control animals . to examine whether perturbation in this pathway could also be achieved by stimulating , rather than inhibiting , rxr , we treated mice with lg268 ( an rxr agonist ) . 
 lg268 also reduced the number  of parasites present in the spleen but not the liver . to determine whether rxr perturbation could be operating through effects on lxr , we treated mice with gw3965 ( an lxr agonist ) . 
 gw9395 treatment had no effect on parasite burden in either tissue ( figures 6b and 6c ) , suggesting that lxr is not involved in control of parasite load mediated through the rxr network . in order to examine 
 if the therapeutic effect observed reflected leishmanistatic or leishmanicidal activity , we also calculated the fold change in parasite numbers in the 2 organs at 4  days p.i . 
 , parasite numbers increased over a 96  hr period , and this growth was inhibited after drug treatment . 
 in contrast , in liver , parasite numbers did not increase significantly over 96  hr in nontreated mice and were reduced to below the level seen at 2  hr p.i . in drug - treated mice , 
 suggestive of leishmanicidal activity ( figure  6e ) . to confirm this observation , we used a histological approach in the liver and further showed that the percentage of infected kcs was significantly reduced in the lg268-treated animals and showed a trend in this direction in hx531-treated mice but not in gw3965-treated mice ( figures 6f and 6 g ) . 
 although not significant in the single experiment shown in figure  6 , replicate experiments with mice treated with hx531 confirmed that this drug enhanced the leishmanicidal activity of kcs ( p  = 0.0028 ; n  = 11 mice for saline controls and 8 mice for hx531 treatment ) . in conclusion 
 , these data indicate that pharmacological manipulation of rxr function was sufficient to perturb the gene network that we identified as being aberrantly expressed in infected kcs . 
 furthermore , it was unlikely that the effects of hx531 or lg268 were due to direct effects on lxr , as stimulation of lxr had no measurable biological effect . 
 we have examined the early in  vivo response of liver macrophages to infection with leishmania , independently studying the activation of infected and uninfected cells exposed to inflammatory signals within the infected tissue microenvironment . 
 our approach provides a number of unique insights into the transcriptomic response induced by l.  donovani infection in  vivo and provides proof of concept that the use of cells exposed to inflammation is a powerful method for the discovery of pathogen - regulated gene pathways . 
 first , the isolation of kc from infected mice revealed a transcriptional response in infected kcs that is somewhat distinct from that observed by others using different types of mononuclear host cells ( including bone marrow - derived macrophages , monocytes , and dcs ) . 
 it remains to be determined whether this reflects cell specificity in host response or differences due to the analysis being performed on cells infected in  vitro versus in  vivo . in most previous in  vitro experiments , the possible influence of uninfected cells on gene expression profile , 
 e.g. , by dilution of the signal associated with infected cells , was not taken into account ( buates and matlashewski , 2001 ; chaussabel et  al . , 2003 ; 
 , 2009 ; rodriguez et  al . , 2004 ) . in one series of studies , 
 dcs infected in  vitro with l.  amazonensis were sorted to allow microarrays to be conducted only on infected cells ( giraud et  al . , 2012 ; lecoeur et  al . , 
 while these studies revealed unique signatures not evident from bulk population analysis , data were not provided to show whether the uninfected dc within the culture system had also made bystander responses ( e.g. , mediated by cytokine release from infected dcs in culture ) . 
 furthermore , the transcriptomic signatures generated by microarray in this and indeed most other studies ( including those cited above ) have not been validated fully at the protein level . 
 second , our data demonstrate significant quantitative and qualitative differences in transcriptomic responses compared to previous in  vitro gene expression studies . in  vitro experiments have usually been interpreted as indicating that visceralizing species of leishmania result in a relatively silent infection of macrophages ( zhang et  al . , 2010 ) , significant suppression of gene expression ( buates and matlashewski , 2001 ) , or a signature distinct from that associated with interferon ( ifn)- activation ( rodriguez et  al . , 2004 ) . 
 the tendency of l.  donovani to induce a weak inflammatory response in  vitro may be species specific , with l.  donovani metacyclics inducing lower fold changes in inflammatory gene expression compared to l.  major metacyclics in one study ( chaussabel et  al . , 2003 ) . 
 alternatively , there may be life cycle stage - specific determinants of gene expression , as shown for l.  mexicana ( aebischer et  al . , 2005 ) . 
 in an additional study , l.  amazonensis amastigotes were also shown to induce significant gene expression changes in  vitro , including changes to the sterol biosynthesis pathway ( osorio y forta et  al . , 2009 ) . 
 importantly , the impact of l.  chagasi on host macrophage response was significantly modulated by the addition of t  cells to the culture in  vitro ( ettinger and wilson , 2008 ) , suggesting that factors external to the infected macrophage impact the response of the infected cell . 
 our data support and extend these observations by adding the full in  vivo complexity of the microenvironment created in the infected liver . 
 third , we show that uninfected kcs also undergo significant biological and gene transcriptional changes associated with the inoculation into mice of live l.  donovani amastigotes . 
 the activation of uninfected kcs at early time points , with further downstream transcriptional consequences seen at 12  hr , suggests that soluble mediators or signals operating in trans facilitate global activation of macrophages within the infected tissue . 
 although this activation shows both qualitative and quantitative differences between infected and inflamed kcs , many of the genes that have been associated with cell - autonomous defense against intracellular protozoa stimulated by interferons ( macmicking , 2012 ) are induced to similar levels in infected and uninfected kcs at 12  hr postinfection . 
 this would place such genes in the category of commonly - regulated genes within the framework of our analysis . 
 such genes include the guanylate - binding proteins gbp2 and gbp5 , indoleamine 2,3-dioxygenase 1 ( ido1 ) , immunity - related gtpase family m members 1 and 2 ( irgm1 and irgm2 ) , and immunoresponsive gene 1 ( irg1 ) . 
 these data are somewhat intuitive , given that ifn is a soluble mediator , capable of trans - activating neighboring cells . 
 collectively , our comparative analysis of the gene expression profiles of infected and inflamed kcs taken from the livers of infected mice suggests that a degree of caution should be exercised when interpreting whether genes observed to alter their expression in response to infection truly represent autonomously regulated genes associated with the presence of intracellular pathogens . 
 fourth , we have identified and validated the importance of a gene network centered around rxra that is associated with the survival of amastigotes within the liver of l.  donovani - infected  mice . within this rxra network , 
 although lipid metabolism centered around rxr has not previously been implicated in l.  donovani pathogenesis in mice , other studies have observed changes in lipid metabolism genes associated with leishmania infection . 
 for example , changes in components of the sterol biosynthesis pathway were induced by macrophage infection with l.  amazonensis ( osorio y forta et  al . 
 previous studies have characterized a role for two heterodimeric partners of rxr in the control of leishmania major infection , the vitamin d receptor ( vdr ) ( ehrchen et  al . 
 , 2007 ; whitcomb et  al . , 2012 ) and the liver x receptor ( lxr ) ( bruhn et  al . , 
 in addition , upregulation of vdr was previously reported following in  vitro infection of macrophages with l.  chagasi ( rodriguez et  al . , 
 2004 ) as well as l.  donovani and l.  major ( chaussabel et  al . , 2003 ) , suggesting that vdr is an important component of the host response to leishmania infection . 
 we believe that by targeting rxr as the hub of the identified network , we have successfully perturbed an entire gene network that controls l.  donovani survival in kcs . 
 perturbation of rxr in either direction via activation with the agonist lg268 or suppression with the antagonist hx531 had similar effects on the growth of l.  donovani in  vivo . 
 the mechanism by which this network controls parasite survival is currently subject to further investigation . 
 however , we speculate that the gene network may regulate the usage of lipids and cholesterol by the parasite while it is present in the phagolysosome of the host cell ( mcconville and naderer , 2011 ) . alternatively , 
 macrophage sensing of leishmania - derived lipids may be having profound effects on the nature of the innate immune response that is initiated ( nagy et  al . , 2012 ) . finally , and of broader significance 
 , we have described an approach to study host - pathogen interactions , which is capable of revealing important host response pathways that would be invisible in studies of the interaction between macrophages and pathogens in  vitro . 
 importantly , we show that manipulation of such pathways in  vivo can impact parasite survival , reducing parasite load at early stages after liver infection . 
 targeting of nuclear receptors for therapy is a burgeoning field of medicine , with rxr therapeutics showing efficacy in clinical settings of cutaneous t  cell lymphoma ( duvic et  al . , 2001 ; khuri et  al . , 2001 ) and a preclinical model of alzheimer s disease ( cramer et  al . , 2012 ) . 
 rxr antagonists , including hx531 , have also shown efficacy in preclinical models of diet - induced obesity and type 2 diabetes ( yamauchi et  al . , 2001 ) . 
 in this respect , while it is tempting to suggest that our findings may also set the foundation for the use of rxr - mediated therapies for leishmaniasis , detailed investigations into the mechanism of parasite growth arrest and/or killing are now required . 
 mt / mg ( muzumdar et  al . , 2007 ) and lysmcre ( clausen et  al . , 1999 ) 
 mice were bred and housed under specific pathogen - free conditions and used at 612  weeks of age . 
 the ethiopian strain of leishmania donovani ( lv9 ) and tandem tomato fluorescent protein expressing lv9 ( tdtom.lv9 ) ( beattie et  al . , 2010 ) were maintained by serial passage in rag-1 mice . 
 amastigotes were isolated from infected spleens ( smelt et  al . , 1997 ) , and 
 mice were infected with 3   10 
 l.  donovani amastigotes intravenously ( i.v . ) via the tail vein in 200  l of rpmi 1640 ( gibco ) . 
 parasites were killed via heat treatment at 56c for 15  min , followed by labeling with pkh26 red fluorescent cell linker kit ( sigma ) . for latex bead experiments , 1  m fluoresbrite polychromatic red microbeads ( polysciences ) 
 were washed twice in saline , and 3   10 beads per mouse were injected i.v . 
 all experiments were approved by the university of york animal procedures and ethics committee and performed under uk home office license ( immunity and immunopathology of leishmaniasis ref # ppl 60/3708 ) . 
 mice were anesthetized , and surgery was performed similar to that previously described ( beattie et  al . , 2010 ) , except that anesthesia was maintained by inhalation of 4% isofluorane ( abbott laboratories ) . 
 images were acquired on an inverted lsm 780 multiphoton microscope ( zeiss ) maintained at 36c by a blacked - out environmental chamber ( solent scientific ) . 
 images were acquired with a 40 1.1 water immersion objective and fluorescence excitation provided by a chameleon xr ti : sapphire laser ( coherent ) tuned to 870  nm . for 4d analysis , 2035  m 
 z stacks were acquired with a z distance of 2  m approximately every 1530 s. real - time injection was performed via tail vein catheter ( strategic applications incorporated , infusion technologies ) . 
 in addition to collecting baseline data for animals before infection , individual kcs were imaged prior to and for 2  hr directly following infection of mice with l.  donovani . 
 data were rendered and analyzed using volocity software ( perkinelmer ) , and cell analysis was performed automatically with manual checking . 
 confocal microscopy was performed on 20  m frozen sections as previously described ( beattie et  al . , 2011 ) . 
 slides were blinded before imaging on a zeiss lsm 510 axioplan microscope ( zeiss ) . 
 hepatic mononuclear cells were prepared from the livers of tdtom.lv9- or rpmi - injected c57bl/6 mice . 
 we conducted four independent infection experiments to generate samples for analysis . in each experiment , n  = 16 mice , and at each time point studied ( 2  hr and 12  hr postinfection ) , we isolated kcs from the livers of four mice treated with rpmi and four mice infected with l.  donovani . 
 liver mononuclear cells were pooled prior to sorting . for real - time pcr analysis , replicates represent pooled samples from independently sorted samples . 
 livers were perfused with 0.4  mg / ml of warmed liberase tl ( roche ) then digested at 37c for 45  min . 
 digested livers were passed over 100  m cell strainers , washed in pbs containing 2% fetal calf serum ( fcs ) , and resuspended in 33% percoll for centrifugation at 693   g for 12  min . 
 isolated cells were blocked with anti cd16/32 then labeled with f4/80-alexa 488 , gr-1 pecy7 ( ebioscience ) , and crig ( helmy et  al . , 2006 ) that was conjugated to atto 647h via lightning - link conjugation ( innova biosciences ) . 
 a small fraction of cells ( approximately 3,000 sorted cells ) were spun onto glass slides , fixed in methanol , and stained with giemsa for morphological analysis . 
 rna was isolated from purified kc and amplified via agilent low input quick amp labeling kit ( agilent technologies ) . 
 amplified rna was then assayed with agilent sureprint g3 mouse gene expression 860k microarray chips that were scanned with an agilent c scanner with surescan high - resolution technology ( agilent ) . 
 comparison of the gene expression data between the rpmi controls and the infected or inflamed samples was performed using the benjamini and hochberg false discovery rate ( fdr ) correction ( pawitan et  al . , 2005a ) , albeit with the caveat that this approach may have low sensitivity with the sample size used ( pawitan et  al . , 2005b ) . 
 this analysis was performed with genespring software ( version 9 ; agilent ) as a standard 5% fdr , with the variances assessed by the software for each t test performed . 
 all data for long intergenic noncoding rna ( lincrna ) probe sets were removed from the analysis at this point . 
 gene ontology analysis was performed using the david ( huang et  al . , 2009 ) and ingenuity pathway analysis software packages ( ingenuity systems ) , and gene maps generated with string ( www.string-db.org ) . 
 mice were pretreated with saline , 100  mg / kg hx531 ( yamauchi et  al . , 2001 ) , 
 30  mg / kg lg268 ( shen et  al . , 2004 ) , or 20  mg / kg gw3965 ( lei et  al . , 2013 ) 26  hr prior to infection and then every 24  hr until 72  hr for hx531 and lg268 or every 12  hr for gw3965 . 
 at 96  hr postinfection , the livers and spleens were collected , and the parasite burden was determined both by flow cytometry and in fixed liver slices . 
 data were normalized to the baseline 2  hr time point to determine the survival of parasites relative to the initial dose of infection . 
 liver and spleens were collected , homogenized , and resuspended in a specific volume of rpmi 1640 ( gibco ) . 
 a total of 200  l of each sample was aliquoted in duplicate to a 96-well plate , centrifuged at 2,000   g for 10  min , and the pellets were resuspended in 2  mg / ml saponin for 5  min at room temperature ( rt ) . 
 the centrifugation was repeated and followed by resuspension in 1  mg / ml saponin . 
 the remaining pellets were washed a further two times , and the wells were resuspended in 200  l then spiked with 2  l of 6  m polystyrene microspheres ( polysciences , inc . ) at 4.6   10 particles/l . 
 samples were acquired on a cyan adp analyzer ( beckman coulter ) in which both forward scatter detector neutral density filters had been removed to allow for small - particle detection . 
 the number of parasites was quantitated by determining the ratio of beads to parasites and then multiplying to calculate the total number of parasites in the starting volume . 
 rna was reverse transcribed into complementary dna ( cdna ) with a superscript iii first - strand synthesis kit ( invitrogen ) . 
 oligonucleotides used for the specific amplification of target genes are described in the supplemental experimental procedures . 
 real - time quantitative pcr was performed with fast sybr green pcr master mix in a steponeplus real - time pcr system ( applied biosystems ) . 
 accumulation of target genes was normalized to hprt , -actin ( shih et  al . 
 , 2012 ) , or ribosomal protein l32 ( reis et  al . , 2013 ) and expressed as relative expression via the change in cycle threshold ( ct ) analysis method ( relative expression in test sample versus rpmi controls ) .","summaryintracellular pathogens modulate host cell function to promote their survival . however , in  vitro infection studies do not account for the impact of host - derived inflammatory signals . 
 examining the response of liver - resident macrophages ( kupffer cells ) in mice infected with the parasite leishmania donovani , we identified a transcriptomic network operating in uninfected kupffer cells exposed to inflammation but absent from kupffer cells from the same animal that contained intracellular leishmania . to test 
 the hypothesis that regulated expression of genes within this transcriptomic network might impact parasite survival , we pharmacologically perturbed the activity of retinoid x receptor alpha ( rxr ) , a key hub within this network , and showed that this intervention enhanced the innate resistance of kupffer cells to leishmania infection . 
 our results illustrate a broadly applicable strategy for understanding the host response to infection in  vivo and identify rxra as the hub of a gene network controlling antileishmanial resistance .",pubmed
"the control of appetite is complex and involves the coordination of inputs from both physiological and environmental sources . 
 early theoretical approaches were based on the idea that the control mechanism was dedicated exclusively to signals from glucose metabolism , amino acids or proteins , or adipose tissue . respectively , 
 these formed the glucostatic ( mayer , 1955 ) , aminostatic ( mellinkoff et al . , 1956 ) and lipostatic ( kennedy , 1953 ) hypotheses . 
 more recent central nervous system models have emphasised inputs from adipose tissue and the gastrointestinal ( gi ) tract [ along with input from sensory features of food ( e.g. schwartz and morton , 2001 ; keesey and powley , 2008 ) ] . 
 the major signals include leptin from adipose tissue , and hormones such as ghrelin , pyy , glucagon - like peptide 1 ( glp-1 ) , cholecystokinin ( cck ) , amylin and insulin from specialised cells in the gi tract or associated organs . 
 such models are said to account for the regulation of appetite and , in turn , contribute to the regulation of body weight , through the identification of specific molecular signals carrying information from the periphery to a network of pathways centred on the hypothalamus . 
 interest in this type of formulation strengthened during the period when molecular biology began to take over from integrative physiology as an explanatory approach in biological sciences . 
 the major task facing any model of human appetite regulation is to account for the ongoing and recurring drive to eat , together with the intermittent suppression of eating via episodic satiety signalling and tonic inhibition ( halford and blundell , 2000 ) . in this commentary 
 , we outline the basis of a new formulation that explains how aspects of body composition and energy expenditure ( ee ) regulate hunger and appetite . 
 this formulation is based on 15 years of psychobiological research with overweight and obese human subjects , and should help to contribute to understanding the relative impact of biological and environmental cues on body weight . 
 an alternative to the molecular approach to appetite control has arisen from the study of energy balance : the inter - relationships between ee and energy intake ( ei ) . in part , this view stems from the statement that any increase in ee will be met with an equivalent increase in ei ( mayer et al . , 1956 ) . 
 indeed , it was proposed that  the differences between the intakes of food [ of individuals ] must originate in differences in the expenditure of energy 
 edholm s experimental studies , however , revealed no correlation between ee and ei in any one single day , but there was a good correlation over 2 weeks . 
 the lack of any relation in a day is perhaps not surprising , because any episode of ee ( such as work or exercise ) happening towards the end of the day would not leave time for a balancing effect of eating . moreover , daily ee can fluctuate markedly from day to day according to individual behavioural tendencies . 
 by contrast , resting metabolic rate ( rmr ) is the largest component of total daily ee and is quite uniform across the day and between days ( johnstone et al . , 2005 ; johnstone et al . , 2006 ; ravussin et al , 1982 ) , and 
 it is plausible that ee from rmr could provide a tonic signal of energy demand that could act as a driver of daily ei . 
 by contrast , physical activity ee ( being of greater intensity and sporadic ) would be expected to exert a mechanistically different type of control over appetite . 
 exercise can raise resting values of ee several fold depending on the volume , mode and intensity . 
 in addition , the effect of exercise on various body systems means that it will have numerous physiological effects in addition to an increase in ee . 
 such effects include an overall increase in heart rate , and changes in the distribution of blood flow , sympathetic nervous system activity , gut hormone activity and in the absorption of nutrients . 
 therefore , it might be anticipated that there could be a number of separate effects on mechanisms influencing appetite . 
 in addition , it can be presumed that a single acute bout of exercise would have different effects from continued daily sessions of exercise that would exert an increasing cumulative challenge to energy balance . 
 a comprehensive picture therefore requires a comparison between various intensities of exercise over varying periods of time with measurement , wherever possible , of all variables in the energy budget . a central issue underlying the investigation of exercise is to determine [ as mayer proposed ( mayer et al . , 1956 ) ] whether or not exercise inevitably induces a compensatory response characterised by an increase in hunger  and in turn food intake  that would lead to an increase in ei that is sufficient to balance the increase in ee generated by exercise . over the last 15 years , we have developed an experimental platform based on a psychobiological approach and incorporating a multi - level system ( caudwell et al . 
 this has allowed the disclosure of relationships between variables in the domains of body composition [ fat free mass ( ffm ) , fat mass ( fm ) ] , metabolism ( rmr , respiratory quotient , respiratory exchange ratio ) , gi hormones , food composition , psychological sensations , food behaviour , psychometric traits , nutritional composition and allelic variation in a single cohort of overweight and obese subjects . in summary , we find that acute ( single ) bouts of exercise do not lead to a consistent increase in the average food intake in the immediate post - exercise period or in the following 24 hours ( for reviews see blundell and king , 1998 ; martins et al . , 2007 ) 
 . however , there is considerable variability associated with appetite variables ( finlayson et al . , 2009 ) . 
 as exercise sessions are repeated over periods varying from 9 to 16 days , a partial compensatory response begins to appear ( stubbs et al . 
 there seems to be little or no compensation through a decrease in non - exercise - related activity ( turner et al . 
 when exercise was performed daily over a period of 16 days and total ee was measured using the doubly labelled water method , it was calculated that exercise raised average daily ee by 3.5 mj and generated a 30% compensation in ei ( whybrow et al . , 2008 ) . 
 several other studies have measured the impact of different exercise prescriptions over periods of 12 to 32 weeks ( church et al . 
 a typical 12-week study using overweight and obese men and women showed that exercise caused an average weight loss of 3.3 kg , indicating that the exercise ee was not fully compensated ( king et al . , 2008 ) , but individual variability was very large [ also observed in other studies ( church et al . 
 a key feature of this study was that exercise sessions ( five times per week for 12 weeks ) was fully supervised and the ee measured so as to rule out the possibility of the outcome being influenced by lack of compliance . 
 subsequent investigations indicated a dual action of exercise on appetite control that partly explained the large variation in body weight change . 
 the exercise caused a significant increase in fasting levels of hunger ( a compensatory response ) , but this was highly variable between subjects . 
 however , there was an associated increase in the post - prandial satiety signalling from meals , indicated by a change in the satiety quotient ( green et al . , 1997 ; king et al . 
 consequently , the effect of exercise on appetite comprises two components : one generating a tonic stimulation of appetite and the other a counteractive meal - related episodic inhibition . 
 the second response seems to be related to changes in either the release of , or sensitivity to , gi peptides ( broom et al . , 2007 ; martins et al . , 2010 ) . 
 these effects of exercise - related ee on appetite are likely to be quite different from any effect related to the lower intensity and more uniform tonic action of rmr . 
 using the multi - level experimental platform described above ( caudwell et al . , 2011 ) , it has been possible to observe the relationships between variables in separate domains ( biological , nutritional , behavioural , metabolic ) , all measured objectively and to similar degrees of accuracy . 
 importantly , the measurements have been made simultaneously in the same group of overweight and obese people . 
 this has enabled the disclosure of unexpected associations between body composition and appetite . a little more than 10 years ago , the discovery of leptin was decisive in focussing attention on adipose tissue as a major controller of energy balance ( ei and ee ) . 
 several models of appetite regulation have described the role of leptin ( as a signal from adipose tissue ) in influencing hypothalamic neuropeptide pathways that control the stimulation and inhibition of food intake ( morton and schwartz , 2001 ; badman and flier , 2005 ) . with most attention 
 directed to the role of adipose tissue ( or fm ) , it can be considered that ffm has become the  forgotten variable , even though it is clearly important under some conditions ( dulloo et al . , 1997 ) . 
 it is therefore interesting that detailed analysis of body composition and appetite variables using the multi - level platform have demonstrated that ffm , but not fm or body mass index ( bmi ) , is strongly correlated with meal size and daily ei ( blundell et al . 
 the strong implication of this relationship is that some privileged molecules arising from ffm , or some physiological consequences that reflect the activity of ffm , act as a signal to drive food intake . 
 considering total daily ee , it is noticeable that rmr is its largest component ( 5070% ) , of which ffm is the major contributor , accounting for about 6070% , whereas fm accounts for as little as 57% , with gender and age being minor components ( johnstone et al . , 2005 ) . 
 consequently , it is plausible that rmr could act as a mediating variable to reflect the influence of ffm on appetite control . 
 accordingly , in a series of analyses  based on the multilevel platform described earlier  we have demonstrated that rmr is positively associated with meal size and with daily ee ( note that the relationship between rmr and total daily ei mirrors the relationship between ffm and total daily ei , which is shown in fig . 
 in addition , rmr is also a predictor of fasting levels of hunger and influences the profile of hunger across the day ( illustrated by fig . 
 the strong implication of these relationships is that rmr  as a measure of ee  reflects a physiological demand for energy that acts as a  driver of food intake . 
 this is plausible and constitutes a demonstration of edholm s proposal that differences in food intake must originate in the differences in energy expenditure ( edholm et al . , 1955 ) . although edholm did not demonstrate a relationship between ei and the ( highly variable ) total daily ee ( within any single day ) , we have demonstrated a clear relationship with the more stable and uniform factor of rmr . 
 the existence of a relationship ( within a single day ) between rmr and daily ee would depend on the total volume and intensity of the  discretionary physical activity component of ee , and upon the relative strengths of the dual processes of appetite control generated by exercise ( see above ) . 
 most recent models of appetite regulation have drawn attention to the roles for adipose tissue and gi peptides . however , these models are better able to account for the inhibition of eating rather than its initiation . 
 the models embody the view that tonic and episodic inhibitory factors modulate an  intrinsic orexigenic drive to eat . 
 this endogenous drive is not usually well defined . however , in early theorising about appetite control , equal emphasis was given to the inhibitory and excitatory ( drive ) features of appetite . 
 the latter was conceptualised as morgan s  central motive state ( morgan , 1943 ) , and in stellar s embodiment of this in an excitatory centre in the hypothalamus ( stellar , 1954 ) . 
 one major issue was to explain what gave animals ( including humans ) the energy and direction that motivated food seeking . in light of knowledge about the physiology of homeostasis , 
 it is plausible that a drive for food arises from the energy used to maintain physiological and metabolic functioning of the body . 
 this resonates with the proposal of edholm ( edholm et al . , 1955 ) , and represents a merging of parallel thinking from psychobiologists working on animal behaviour and physiologists working on human energy balance . 
 together with modern ( post - leptin ) discoveries , it can be suggested that the physiology of appetite regulation comprises three components : a tonic drive for food arising from the physiological demand for energy ; a tonic inhibition arising from signals of energy storage ( mainly adipose tissue ) ; and episodic signals arising from the mouth and gi tract in response to the periodic consumption of food . 
 this last category is primarily inhibitory  from the classical satiety signals  but also excitatory ( signals registering food palatability ) . 
 ( a ) scatter plot shows the relationship between ffm and daily ei in a group of 41 overweight or obese men and women . 
 the values represent the average values from objective measures taken at specific probe days at weeks 0 , 6 and 12 . 
 ( b ) daily hunger profiles for individuals with the highest ffms ( eight women and five men ) and those with the lowest ffms ( eight women and five men ) . 
 the groups were formed by dividing the whole sample into tertiles of ffm and comparing the upper and lower tertiles ( but keeping similar numbers of men and women in each tertile ) . 
 the difference between the area under the curve for the two profiles is significant ( p<0.01 ) ; asterisks indicate time points at which the profiles are significantly different . 
 the profiles indicate that hunger in the high ffm group is greater before the onset of a meal and in the period leading up to the meal , but not at the end of the meal . 
 this is consistent with the fact that the high ffm individuals eat a larger meal , which would return hunger to a level appropriate for full satiation . 
 together , data displayed in a and b indicate that ffm is positively associated with daily ei and with daily hunger levels . 
 additionally , because rmr is highly and significantly correlated with ffm , the relationships between rmr , ei and hunger very closely match those for ffm shown in the figure . 
 hunger is measured in units on a 100 unit scale on the screen of a hand - held pda . 
 the investigations reviewed here have provided evidence for the physiological drive for food by demonstrating a specific association of ffm , but not fm , with objectively measured food intake variables ( blundell et al . , 2012 ) . 
 in turn , this means that the energy required to maintain the body s lean tissue mass  thus preventing wasting  creates a physiological demand that determines a minimal level of ei at meals and over the day . 
 it is worth noting that the relationship between ffm and ei is consistent with the amino - static hypothesis put forward more than 50 years ago by mellinkoff et al . 
 ( mellinkoff et al . , 1956 ) , and with the more detailed proposal of a role for the protein - stat mechanism described by millward ( millward , 1995 ) . a new formulation for appetite control . a proposed tonic signal for the drive to eat that reflects the body s demand for energy arises ( mainly ) from ffm and rmr 
 in turn , this drive is under tonic inhibition from leptin , whose action reflects the amount of stored energy reserves in the body . as the amount of adipose tissue increases , leptin insensitivity occurs and this tonic inhibition is reduced . 
 the drive to eat is periodically interrupted and suppressed by episodic signals in the form of peptides that are released from the gi tract in response to food consumption . 
 the resulting pattern of eating is a consequence of the interactions between tonic and episodic physiological signals . 
 prolonged exercise displays a dual - process action by stimulating hunger ( an effect that is highly variable between individuals ) but also by increasing post - prandial satiety signalling ( king et al . 
 , 2009 ) through an effect on gi peptides ( martins et al . , 2010 ) . 
 a further step in the formulation comes from the demonstration that rmr is also strongly correlated with objectively measured self - determined ei ( caudwell et al . , 
 this could be expected from the recognition that ffm is the largest contributor to rmr ( accounting for approximately 60% in our studies ) . 
 it is worth noting that ee arising from ffm receives contributions from the liver ( 20% ) , brain ( 20% ) , heart ( 11% ) , gi tract ( 9% ) and skeletal muscle ( 20% ) ( elia , 1992 ) . 
 therefore , the physiological demand is created by the need to provide energy to maintain the functioning of the body s vital systems . moreover 
 , the ee from these organ systems is maintained across the day and is reflected in the relative stability and uniformity of rmr within and between days . 
 consequently , the proposal that rmr acts as a  physiological drive ( or is a marker of such a drive ) underlying appetite is both parsimonious and plausible . 
 this formulation is quite consistent with those models of appetite regulation that assign major roles to adipose tissue and the gi tract . 
 however , it also draws attention to other aspects of body composition as determinants of appetite . combined with other findings , 
 this interpretation provides mechanistic explanations for the link between ffm , rmr and eating behaviour . 
 this formulation can incorporate an extended role for leptin and is in keeping with a widening ergostatic function of additional adipokinetic hormones in energy homeostasis , as envisaged by frhbeck and gmez - ambrosi ( frhbeck and gmez - ambrosi , 2001 ) . 
 the idea of ffm creating an orexigenic or ergostatic drive has also been proposed in other descriptions of the appetite regulatory system ( halford and blundell , 2000 ) . 
 adipokines exert effects not only on adipose tissue but also on skeletal muscle ( sinz et al . , 2009 ) , and that differential expression of genes and pathways that are intrinsic to skeletal muscle might also be involved ( wu et al . , 2011 ) . 
 consequently , it can be envisaged that leptin could act as a major tonic inhibitory signal ( to dampen the physiological drive ) and also as an energostatic factor through its influence on ffm . 
 the proposal that ffm and rmr contribute to a physiological demand for energy that influences appetite is plausible and has implications . 
 first , it is one further reason to be dissatisfied with the use of measures of body weight ( or bmi ) in the research and management of obesity . 
 the recognition that ffm and fm have different functional properties in relation to appetite is a strong reason to use body composition ( rather than the coarse variable of body weight ) in both the research and management of obesity . 
 for example , two individuals with similar bmis ( or body mass ) might have quite different proportions of ffm and fm , and this would confer different properties on their physiological and behavioural responses . those people with a high ffm should have a proportionately higher orexigenic drive to maintain a greater minimal meal intake ( i.e. they should eat more ) than people with less lean tissues and organs . 
 this means that obese people ( with a greater lean mass in support of a large amount of adipose tissue ) and people carrying a large muscle mass ( field athletes , rugby players , swimmers , etc . ) should have a stronger tendency to consume larger meals than smaller people . 
 it follows that such people would have greater difficulty in tolerating dietary restriction ( because the more energetically active lean mass would sustain a drive for a minimal amount of food ) . 
 moreover , in elderly people subject to sarcopenia , the reduced lean mass would result in a diminished appetite . 
 however , it should not be inferred that the influence of ffm and rmr upon food intake is a cause of weight gain or obesity . 
 this mechanism is a physiological way of achieving energy balance ( ensuring that ei does not fall below the energy demand of the body ) . as such , 
 the mechanism influences the strength of the drive to eat ( feeding behaviour ) and determines the level of hunger at the beginning of a meal ( fig . 
 the amount of energy that is actually consumed is strongly modulated by the energy density of the food available ( ello - martin et al . 
 , 2005 ; rolls , 2000 ) . high energy density of food results in passive overconsumption ( blundell and macdiarmid , 1997 ) , which has been identified as a major component of the obesogenic environment ( swinburn et al . , 2011 ) . 
 consequently , a high rmr could influence weight gain by maintaining a high level of hunger , but a positive energy balance would depend on the energy density and palatability of the diet . 
 this is an example of a physiological regulatory process being undermined by the nature of the modern diet in many technologically advanced countries . in turn 
 , the formulation proposed here can help to promote research to clarify the relative strength of biological and environmental variables that contribute to ei and to changes in body weight ( and body composition ) . 
 moreover , we envisage that the system would adapt to progressive changes that occur in body composition over time and could influence energy balance ( e.g. hall et al . , 
 the most likely scenario is that any increase in ffm ( and rmr ) would increase the drive to eat . 
 this amplifying effect on ei would mean that weight gain becomes part of a positive , rather than a negative , feedback system . 
 increasing body weight therefore could facilitate further weight gain and increase the difficulty of weight loss or maintenance .","a long - running issue in appetite research concerns the influence of energy expenditure on energy intake . 
 more than 50 years ago , otto g. edholm proposed that  the differences between the intakes of food [ of individuals ] must originate in differences in the expenditure of energy  . 
 however , a relationship between energy expenditure and energy intake within any one day could not be found , although there was a correlation over 2 weeks . 
 this issue was never resolved before interest in integrative biology was replaced by molecular biochemistry . using a psychobiological approach , 
 we have studied appetite control in an energy balance framework using a multi - level experimental system on a single cohort of overweight and obese human subjects . 
 this has disclosed relationships between variables in the domains of body composition [ fat - free mass ( ffm ) , fat mass ( fm ) ] , metabolism , gastrointestinal hormones , hunger and energy intake . in this commentary , we review our own and other data , and discuss a new formulation whereby appetite control and energy intake are regulated by energy expenditure . specifically , we propose that ffm ( the largest contributor to resting metabolic rate ) , but not body mass index or fm , is closely associated with self - determined meal size and daily energy intake . 
 this formulation has implications for understanding weight regulation and the management of obesity .",pubmed
"one of the most widely available materials in prosthetic dentistry is polymethyl methacrylate ( pmma ) , a material that has been used as a denture base since 1937 . 
 heat - polymerized pmma resin is used as a denture base material because of its excellent esthetics , low water sorption and solubility , relative lack of toxicity , repair ability , and simple processing technique . a conventional method for curing resin 
 advantages of this method include ease of processing , its familiarity for dentists and technicians , and the lack of need for any sophisticated or expensive equipment . 
 disadvantages of this technique are dimensional changes and inaccuracies in the fit of the denture base . 
 therefore , the popularity and relative simplicity of the compression molding technique are usually overshadowed by the high processing stresses induced in the resin during polymerization . 
 chemically activated resins are much less frequently used for denture base fabrication than heat - activated resins . 
 these materials have a higher solubility and inferior color stability due to oxidation of the amine accelerator . 
 chemically activated resins , most often when compression molded , display less polymerization shrinkage than their heat - activated counterparts , which leads to greater dimensional accuracy . this could be attributed to a reduction in residual stresses that have been induced during the processing cycle . 
 the injection molding technique when compared with the compression molding technique has a reduced processing time , lower cost , lower skin sensitivity to the evaporated monomer , and availability of the resin reservoir to compensate for acrylic resin shrinkage . 
 the type of processing could affect the mechanical properties of the resins such as the bond strength between the denture teeth and the denture base resins . 
 debonding of denture teeth from denture bases is a major problem in the prosthodontic practice . 
 previous surveys reported that 26 - 33% of denture repairs are the result of debonded teeth which cause distress and increased cost for patients . 
 this may be related to the basic properties of the materials ( teeth or denture base materials ) , processing factors ( contaminations or curing cycle duration ) , and the available monomer during processing . 
 attempts to increase the bond strength between acrylic resin teeth and denture base resins include grinding the glossy ground surface , painting the ground surface of the teeth with monomer , and cutting mechanical retention features in the ground surface of the teeth . 
 clancy and boyer compared the bond strengths of light- and heat - polymerized , and autopolymerized denture base resins to the denture teeth . in their study , the strongest bonding occurred between heat - polymerized resin and plastic teeth . 
 evaluated the bonding of two resin denture teeth to microwave and heat - processed denture bases . 
 they obtained higher bond strength of denture teeth with the conventional heat - polymerized than the microwave - polymerized acrylic resins . 
 the purpose of this investigation was to evaluate the effect of surface treatments on shear bond strength of denture teeth to heat - polymerized and autopolymerized denture base resins . 
 the null hypotheses were : 1 ) there is no significant difference between the shear bond strength of the denture teeth to the two denture base resins and 2 ) surface treatments of the denture teeth have no effect on their bond strengths to the denture base resins . 
 in this experimental in vitro study , 60 maxillary central incisor acrylic teeth ( ivoclar , vivadent , naturno , italy ) were divided into two groups which were processed with heat - polymerized acrylic resin ( meliodent , heraeus - kulzer gmbh , wehrheim , germany ) ( group m , n = 30 ) and autopolymerized acrylic resin ( futura gen , shutz - dental , rosbach , germany ) ( group f , n = 30 ) , respectively . 
 denture teeth were further subdivided into the following three subgroups with n = 10 per group : 1 ) m1 , f1 : untreated ground surface ; 2 ) m2 , f2 : treated with monomer on ground surface ; and 3 ) m3 , f3 : treated with airborne particle abrasion . 
 the methyl methacrylate monomer was applied to the teeth bonding surfaces using a cotton bud for 180 s. the airborne particle abrasion was done using 50 m aluminum oxide particles for 30 s from a distance of 10 mm under 2 bar pressure . 
 the denture teeth were mounted on two sides of six triangular shaped wax models [ figure 1 ] and covered with silicone putty ( optosil , heraeus - kulzer , leverkusen , germany ) to facilitate deflasking . before processing , all teeth 
 denture teeth in triangular shaped wax model each model was flasked and the wax removed by boiling water . 
 six molds were made in this procedure for groups ( m1 , m2 , m3 , f1 , f2 , f3 ) . 
 three molds were packed with meliodent denture base resin and cured in a curing machine by an automatic curing device ( ewl , type 5518 , kavo , leutkirch , germany ) in accordance with the manufacturer 's instructions . 
 the three other molds were injected with futura gen resin and processed using unipress machine ( shutz - dental ) . 
 after completion of the curing cycle and deflasking , the meliodent and futura gen specimens were stored in distilled water at 37c for 7 days . 
 then , the shear bond strengths of the specimens were evaluated by universal testing machine ( instron corp . , canton , ma , usa ) . 
 force was applied by a stainless steel pin , 1 mm in diameter , at an angle of 130 to the long axis of the tooth at a crosshead speed of 5 mm / min until fracture occurred . in a class 
 i occlusion , this stimulates the average angle of contact between the maxillary and mandibular anterior teeth . 
 the mean shear bond strength values were subjected to two - way analysis of variance ( anova ) followed by tukey 's test . for all statistical analyses , 
 two - way anova demonstrated that the shear bond strength was significantly affected by the type of acrylic resin ( f = 4.71 , p = 0.03 ) and the surface treatment ( f = 21.09 , p < 0.001 ) , but the interaction between these two factors was not statistically significant ( f = 0.004 , p = 1.00 ) [ table 2 ] . 
 mean and standard deviation of the studied groups tukey 's honestly significant difference ( hsd ) multiple comparison test revealed that the control groups had significantly lower bond strength than the treated groups ( p < 0.001 ) , but there was no significant difference between the bond strengths of groups treated by monomer or airborne particle abrasion ( p = 0.29 ) [ table 1 ] . 
 failure mode was predominantly mixed in the meliodent groups , whereas adhesive failure was reported for futura gen specimens [ figure 2 ] . 
 according to the results of the present study , there was significant difference between the shear bond strengths of the denture teeth to the two denture base resins and surface treatment of the denture teeth affected the bond strength significantly . 
 some studies have reported that the bond strength of acrylic teeth to denture base resin is due to the diffusion and polymerization of monomer across the teeth base interface to form an interpenetrating polymer network . 
 the efficiency of the attainment of such bond depends primarily on the rate at which the monomer diffuses from the base resin mixture . 
 higher diffusion rate of the monomer of a denture base polymer mixture into the acrylic resin polymer teeth is achieved with increasing polymerization temperature . 
 injection molding does not have any heat conduction as with the conventional water bath technique , thus the bond strength of the denture teeth in the meliodent group was higher than that in the futura gen group . 
 development of alternative materials such as futura gen ( pmma ) is a breakthrough in denture base materials used for prosthetic dentistry . 
 the advantages of futura gen include less shrinkage , outstanding denture adhesion characteristics , less processing time , and ease of polishing . 
 the manufacturer claims that the quality of this material has been improved because of modification and binding ability which has resulted in higher bond strength . 
 in addition , the lack of a monomer results in higher strength compared to that of heat - cured resin . 
 according to the manufacturer , due to changing of the initiator system and replacement of changed copper and barbituric acid ions , instead of the tertiary amine , the amount of residual monomer in futura gen is similar to that of heat - polymerized resin . in similar studies on the bond strengths of denture teeth to different types of denture base resins , 
 heat - polymerized acrylic resins showed the highest bonding values in comparison to autopolymerized or light - polymerized acrylic resins , which is in agreement with this study . on the contrary 
 , vallitue suggested that heat - polymerized pmma did not adhere to the acrylic resin tooth better than the autopolymerizing pmma . 
 the results of this study showed that the surface treatment with monomer is an effective method for improving the bond strength between denture teeth and denture base resins . 
 acrylic teeth treated with monomer failed predominantly in the cohesive and mixed modes in meliodent groups , which is in agreement with the findings of previous studies on the benefits of chemical locks . 
 the monomer that was applied to the tooth surface before packing provided a solvent effect on the tooth surface which favored a more effective diffusion of the monomer of the denture base polymer across the tooth denture base interface and formed interpenetrating polymer networks . 
 one of the technical factors that contribute to the bond strength of denture tooth to denture base resin is ridge lap surface contamination with wax or tinfoil substitute residues , which can be cleaned with monomer application before packing pmma into the mold . 
 the airborne particle abrasion of the denture teeth ground surface improved significantly the shear bond strength compared to untreated samples . 
 although these results are in agreement with some studies , however , there are other studies which have shown that airborne particle abrasion or grinding decreases the bond strength of the treated teeth to denture bases . 
 other studies have reported that teeth base roughening with cutting or abrasive rotary instruments or aluminum oxide air abrasion provides slightly higher bond strength values than those achieved without surface modification , or with cavity preparation or monomer etching . 
 it has been shown that the use of mechanical retention is a primary means of securing the bond strength between acrylic resin teeth and the denture base resins . 
 the airborne particle abrasion increases the surface area of the ridge lap portion of the denture teeth and , hence , improves their bond strengths to denture base resins . in this study , 
 most of the futura gen specimens ( f1 > f2 > f3 ) showed adhesive failure , but failure mode of the meliodent specimens was predominantly mixed ( adhesive and cohesive ) ( m1 > m2 and m3 ) [ figure 2 ] . hatim and hasan demonstrated that the failure modes are mostly adhesive in all types of selected acrylic teeth with heat - polymerized resins and mixed for microwave acrylic denture bases . fletcher - stark et al . showed adhesive debonding between the acrylic denture teeth and light - polymerized resin without a bonding agent . 
 but when a bonding agent was used with light- and heat - polymerized denture base resins , mixed and cohesive failures were observed . 
 mechanical fatigue , thermal cycling , and chewing simulation were not considered in the present study ; therefore , the results of the present study should be interpreted with caution . 
 within the limitations of this study , these conclusions can be drawn : treatment of the ridge lap surface of denture teeth by monomer or by airborne particle abrasion significantly improved their bond strengths to the denture base resins.there was no significant difference between applying monomer or airborne particle abrasion in improvement of the bond strength.the bond strength of the denture teeth to the studied denture base resins was significantly different.the failure mode was predominantly adhesive with futura gen and mixed in meliodent . 
 treatment of the ridge lap surface of denture teeth by monomer or by airborne particle abrasion significantly improved their bond strengths to the denture base resins . 
 there was no significant difference between applying monomer or airborne particle abrasion in improvement of the bond strength . 
 the bond strength of the denture teeth to the studied denture base resins was significantly different .","background : debonding of denture teeth from denture bases is the most common failure in removable dentures . the purpose of this study was to evaluate the effect of surface treatments on shear bond strength of denture teeth to heat - polymerized and autopolymerized denture base resins.materials and methods : in this experimental in vitro study , 60 maxillary central incisor acrylic teeth were divided into two groups . 
 group m was polymerized with heat - polymerized acrylic resin ( meliodent ) by compression molding technique and group f was processed by autopolymerized acrylic resin ( futura gen ) by injection molding technique . within each group , 
 specimens were divided into three subgroups according to the teeth surface treatments ( n = 10 ) : ( 1 ) ground surface as the control group ( m1 and f1 ) , ( 2 ) ground surface combined with monomer application ( m2 and f2 ) , and ( 3 ) airborne particle abrasion by 50 m al2o3 ( m3 and f3 ) . 
 the shear bond strengths of the specimens were tested by universal testing machine with crosshead speed of 5 mm / min . 
 data were analyzed by two - way analysis of variance ( anova ) and tukey 's honestly significant difference ( hsd ) tests ( p < 0.05).results : the mean shear bond strengths of the studied groups were 96.40  14.01 , 124.70  15.64 , and 118  16.38 n for m1 , m2 , and m3 and 87.90  13.48 , 117  13.88 , and 109.70  13.78 n for f1 , f2 , and f3 , respectively . the surface treatment of the denture teeth significantly affected their shear bond strengths to the both the denture base resins ( p < 0.001 ) . 
 however , there were no significant differences between the groups treated by monomer or airborne particle abrasion ( p = 0.29 ) . 
 the highest percentage of failure mode was mixed in meliodent and adhesive in futura gen.conclusion:monomer application and airborne particle abrasion of the ridge lap area of the denture teeth improved their shear bond strengths to the denture base resins regardless of the type of polymerization .",pubmed
"the light emitted during arc welding contains intense ultraviolet radiation ( uvr ) . in the absence of a barrier , 
 this radiation is emitted into the surrounding environment , and as a result , extremely large number of workers at workplaces where arc welding is performed are exposed to uvr . 
 this includes not only expert arc - welding professionals , whose numbers are estimated at 350,000 in japan , but also welders who perform arc welding occasionally and workers engaged in tasks other than arc welding . 
 however , a precise border between uvr and visible light can not be defined because visual sensations from very bright sources are experienced at wavelengths below 400 nm . 
 therefore , the borders necessarily vary from situation to situation . uvr below approximately 190 nm is known as vacuum uvr because it is strongly absorbed by oxygen molecules and therefore can not propagate through air . 
 uvr is emitted during arc welding over its entire wavelength range excluding vacuum uvr , although the wavelength distribution of uv differs depending on the welding conditions . 
 uvr from the arc welding of steel consists mostly of a large number of spectral lines of iron scattered over this wavelength range . 
 for example , because it is strongly absorbed by proteins and water , uvr incident on a living organism is primarily absorbed at the surface , causing damage confined to surface regions . 
 well - known examples of acute health effects include keratoconjunctivitis and erythema , whereas delayed health effects include cataracts and skin cancer . in practice , 
 such acute health effects because of uvr occur frequently at workplaces where arc welding is performed . 
 the japan welding engineering society surveyed incidences of uv keratoconjunctivitis among 1667 workers at 47 workplaces where arc welding is performed ; the survey included workers who performed arc welding as well as workers who did not . 
 the results indicated that as many as 86% of the workers reported past experience with symptoms of uv keratoconjunctivitis , including foreign body sensation , ophthalmalgia , lacrimation , and photophobia , whereas 45% reported ongoing experience with this ailment with one or more recurrences per month even though the majority of arc welders who experienced uv keratoconjunctivitis wore welding face shields . 
 possible causes for this include ( a ) cases in which workers mistakenly put on their face shields after starting the arc instead of immediately before thus exposing themselves to uvr and ( b ) cases in which workers were exposed to uvr by co - workers performing arc welding nearby . 
 in addition , in a survey by emmett et al . , 92% of welders had suffered one or more flash burns ( keratoconjunctivitis ) , and 40% were afflicted with erythema in the neck . 
 these findings demonstrate the need to introduce protective measures at workplaces that use arc welding to protect all workers from uvr . as the basis for designing such measures , it is desirable to obtain a quantitative understanding of the hazard of uvr emitted during arc welding . in an actual work site 
 , arc welding is performed under a variety of conditions , and the degree of hazard of the emitted uvr is thought to differ for each condition . 
 therefore , it is necessary to examine the hazards of the uvr emitted during arc welding under various conditions . 
 arc welding of metallic materials is conducted primarily on mild steel , aluminum alloys , and stainless steel alloys . 
 gas metal arc welding ( gmaw ) , which uses a continuously fed consumable electrode and a shielding gas , is most often used for mild steel . 
 gmaw is conducted while covering the welding point and the arc by the shielding gas . 
 the shielding gas prevents a decrease in welding quality , such as the formation of an oxide or nitride because of exposure of the weld metal to air . 
 the gmaw electrode is a coiled consumable wire that is automatically supplied throughout the welding . 
 a few previous studies have measured the uvr emitted during arc welding processes , such as the gmaw of mild steel , and assessed their acute health effects . however , in these studies , detailed information on the actual welding was not provided . 
 therefore , the available data about the level of uvr emitted by gmaw is unreliable and insufficient . in particular , the effects of specific conditions have not been examined systematically . 
 one of the authors of this paper performed gmaw using 100% co2 in experiments and examined in detail the uvr emitted under these conditions . 
 it was clearly found that the uvr hazard tends to increase with welding current and is dependent on the type of welding wire . in this study 
 , we conducted a survey of the uvr hazard present during the gmaw of mild steel with a shielding gas of 80% ar + 20% co2 or 100% co2 . 
 the uvr emitted during gmaw performed with the recently popular digital inverter - type pulsed arc welding machine was measured , and its acute health effects were assessed in accordance with the american conference of governmental industrial hygienists ( acgih ) guidelines . in particular , we studied the influence of ( i ) the type of shielding gas and the magnitude of the welding current , and ( ii ) using pulsed vs. non - pulsed welding current . 
 according to the acgih guidelines , the degree of urv hazard as a cause of acute health effects is measured by the effective irradiance . 
 the effective irradiance is defined by where eeff is the effective irradiance ( mw / cm ) , e is the spectral irradiance at wavelength  ( mw/ ( cmnm ) ) , s (  ) is the relative spectral effectiveness at wavelength  , and  is the bandwidth ( nm ) . for the uvr measurements , we used an x13 hazard lightmeter and an xd-45-huv uv - hazard detector head ( both from gigahertz - optik ) , which are designed for measuring effective irradiance . 
 the relative spectral responsivity of the detector head agrees well with the relative spectral effectiveness around 270 nm . 
 some discrepancy between the relative spectral responsivity and the relative spectral effectiveness is visible from 310 to 320 nm . 
 however , because the relative spectral effectiveness in this wavelength regime is small ( 0.015 - 0.0010 ) , we consider the influence of this discrepancy to be too small to cause issues in practice . 
 thus , we conclude that this detector head is well suited to measure the effective irradiance . in actual experiments , 
 the value measured by the devices is the effective radiant exposure ( j / m ) . 
 the measurement device was calibrated by the manufacturer and used within the one - year validity of the calibration . 
 the position of the welding torch was fixed to produce arcs in the same position , and the base metal was fixed on a movable table , which moved the metal for welding . 
 the distance between the arc and the detector head was set to 500 mm to mimic the actual distances to welders . 
 in addition , the detector head was positioned at an angle of 40 from the surface of the base metal and at an angle of 90 from the welding direction . 
 the measurement time was set to 40 s. to exclude the time required for the arc to stabilize immediately after welding begins and the time required for the movable table to accelerate , measurements did not begin until 5 s after the start of welding . in this study , 
 no local exhaust ventilation system was used during the measurement of uvr because local exhaust ventilation is usually not used in welding workplaces ( local exhaust ventilation may disturb the airflow around the arc and cause welding defects ) . experimental setup for measuring effective irradiance ( schematic diagram ) measurements 
 furthermore , following the acgih guidelines , we divided 3 mj / cm by our measured values of effective irradiance to determine the maximum daily exposure time allowable at that irradiance ( equation ( 2 ) ) . in this equation , tmax is the maximum daily exposure time ( s ) and eeff is the effective irradiance . 
 we measured the effective irradiance of the uvr emitted during three types of welding : 100% co2 welding using non - pulsed welding current , non - pulsed 80% ar + 20% co2 welding , and pulsed 80% ar + co2 welding . 
 the welding apparatus was a digital inverter - type pulsed arc welding machine ( dp350 , daihen welding and mechatronics systems co. , ltd . ) , which has recently become popular . 
 the inclination of the welding torch was fixed at 110. the type of welding was bead - on - plate welding in which the base metal is melted while the welding wire is added . 
 the base metal was rolled ss400 , which is a mild structural steel specified by the japanese industrial standards ( jis ) . 
 for the welding wire , we used solid wire ygw12 of diameter 1.2 mm specified by jis . 
 the usable range of welding current for this wire was 80 - 350 a for flat position welding . 
 the shielding gas was 80% ar + 20% co2 or 100% co2 , and the shielding gas flow rate was 15 l / min . 
 the distance between the base metal and the contact tip was 17 mm , and the wire extension length before the start of welding was 12 mm . 
 the welding voltages were preset values corresponding to the welding current determined by the manufacturer of the welding equipment . 
 welding parameters of 100% co2 , non - pulsed 80% ar+20% co2 welding and pulsed 80% ar+20% co2 welding to investigate the influence of the type of shielding gas , we conducted bead - on - plate welding and measured the resulting uvr when using 80% ar + 20% co2 or 100% co2 as the shielding gas and non - pulsed welding current . 
 the range of the welding current was 100 - 350 a as shown in table 1 . because it is less expensive , 100% co2 
 is used more commonly as shielding gas in the gmaw of mild steel in japan , whereas mixed gas is usually used when manufacturing high - quality products efficiently . to investigate the influence of pulsed current 
 , we performed pulsed current gmaw with an 80% ar + 20% co2 shielding gas and measured the resulting uvr . 
 we then compared it with the results for non - pulsed 80% ar + 20% co2 welding . 
 incidentally , the digital inverter - type pulsed arc welding machine provides excellent stability of the arc in the low current range , highly effective reduction of sputter , and is mainly used under 250 a. therefore , the range of welding current used was 100 - 250 a as shown in table 1 . 
 pulsed current is now commonly used in the welding of thin plates of automobile bodies . 
 to investigate the influence of the type of shielding gas , we conducted bead - on - plate welding and measured the resulting uvr when using 80% ar + 20% co2 or 100% co2 as the shielding gas and non - pulsed welding current . 
 the range of the welding current was 100 - 350 a as shown in table 1 . 
 because it is less expensive , 100% co2 is used more commonly as shielding gas in the gmaw of mild steel in japan , whereas mixed gas is usually used when manufacturing high - quality products efficiently . 
 to investigate the influence of pulsed current , we performed pulsed current gmaw with an 80% ar + 20% co2 shielding gas and measured the resulting uvr . 
 we then compared it with the results for non - pulsed 80% ar + 20% co2 welding . 
 incidentally , the digital inverter - type pulsed arc welding machine provides excellent stability of the arc in the low current range , highly effective reduction of sputter , and is mainly used under 250 a. therefore , the range of welding current used was 100 - 250 a as shown in table 1 . 
 pulsed current is now commonly used in the welding of thin plates of automobile bodies . 
 figure 2 and 3 display the effective irradiance for various welding conditions in bead - on - plate welding . 
 the effective irradiance measured in this study at a distance of 500 mm from the arc was in the range of 0.51 - 12.9 mw / cm . 
 the allowable daily exposure times corresponding to these values are 0.23 - 5.9 s. effective irradiance for 100% co2 welding and non - pulsed 80%ar+20%co2 welding . 
 2 shows that the effective irradiance in bead - on - plate welding of mild steel was influenced by the welding current and the shielding gas composition . 
 the effective irradiance increased with increasing welding current for each type of shielding gas . for the case of non - pulsed 80% 
 ar + 20% co2 welding , the effective irradiance measured with welding currents of 100 - 350 a was in the range of 0.51 - 12.9 mw / cm . 
 the increase in effective irradiance between current intervals is very large , starting from 250 a. for 100% co2 welding , the effective irradiance measured with welding currents of 100 - 350 a was in the range of 0.69 - 7.4 mw / cm . 
 the effective irradiances measured with non - pulsed 80% ar + 20% co2 welding and 100% co2 welding at 250 a or less were approximately equal . 
 however , at 300 a and higher , the effective irradiance for non - pulsed 80% ar + 20% co2 welding increased beyond that for 100% co2 welding . 
 3 , the effective irradiance of uvr emitted during mild steel arc welding was strongly influenced by the pulsed current . 
 the effective irradiance measured for pulsed 80% ar + 20% co2 welding with welding currents of 100 - 250 a was in the range of 3.4 - 11.6 mw / cm . 
 in addition , the effective irradiance of uvr occurring during pulsed 80% ar + 20% co2 welding was very high in comparison with that during non - pulsed 80% ar + 20% co2 welding . 
 the effective irradiance observed at a distance of 500 mm from the arc was in the range of 0.51 - 12.9 mw / cm . at these irradiances , 
 the allowable daily exposure times are just 0.23 - 5.9 s , which are extremely short times . 
 these results indicate that exposure to the uvr emitted by the gmaw of mild steel is quite hazardous . 
 it is thought that workers are often exposed to uvr when the arc is started . 
 although the exposure is brief for each start of an arc , this may occur often because workers usually start an arc many times in a day . 
 therefore , the actual total exposure time may easily exceed the allowable daily exposure times determined in this study . 
 thus , we conclude that if workers engage in the gmaw of mild steel without adequate protection , they are exposed to hazardous quantities of uvr for short periods of time . 
 one of the authors previously measured the uvr emitted during the gmaw of mild steel when using 100% co2 as a shielding gas and obtained an effective irradiance of 0.028 - 0.785 mw / cm at a distance of 1 m for welding currents of 120 - 500 a. if we assume that the effective irradiance decreases as the inverse square distance from the arc , the effective irradiance would be 0.106 - 3.14 mw / cm at 0.5 m from the arc , which is roughly half the effective irradiance obtained in the present study for the same type of welding . 
 this difference is considered to be because of differences in welding conditions such as the welding device , welding wire , and ventilation conditions . in this study , 
 no local exhaust ventilation system was used during the measurement of uvr because local exhaust ventilation is usually not used in the welding workplace . 
 local exhaust ventilation removes the welding fume , which strongly mitigates uvr by scatter and absorption . 
 therefore , if local exhaust ventilation had been used during the measurement , the effective irradiance would have been higher . 
 if we assume that the effective irradiance of uvr decreases as the inverse square distance from the arc , the allowable daily exposure times at a distance of 5 m from the arc will be in the range of 23 - 590 s. thus , even at a distance of 5 m from the arc , exposure to uvr is hazardous in cases in which the emitted uvr is intense . 
 moreover , even in cases in which the emitted uvr is weak , we believe that prolonged exposure is hazardous . thus , in cases where the gmaw of mild steel is performed , it is necessary to take precautions to ensure that surrounding workers are not exposed to the uvr emitted by the arc . 
 the effective irradiance measured in this study , regardless of the pulse current and the type of shielding gas , increased with increasing welding current ( figure 2 , 3 ) . 
 this trend was also previously observed for the gmaw of mild steel using 100% co2 and 5% o2 + 95% ar as the shield gas . 
 more recently , we also observed the trend for the gmaw and the gtaw of aluminum and magnesium alloys . 
 thus , the welding current is an important factor influencing the degree of hazard of the uvr emitted during the welding process ; the uvr hazard can be understood to be a rapidly increasing function of the welding current . 
 the effective irradiance of the uvr generated during non - pulsed 80% ar + 20% co2 welding was in the range of 0.51 - 12.9 mw / cm and increased with increasing welding current . in particular , a significant difference in the increase in effective irradiance for welding currents from 100 to 250 a and from 250 a upward was observed . to investigate the reason for this difference , we observed the arc during welding . 
 4 , short - circuit transfer was observed at 100 a. in short - circuit transfer , the tip of the welding wire is short - circuited with the base metal ( molten pool ) , and the molten metal is shifted to the base material . in addition , globular transfer was observed at 250 a. in globular transfer , the welding wire tip melted by the arc is transferred to the base metal in which the grain size grows larger than the wire diameter . as also shown in fig . 
 4 , it was not possible at 300 a to observe the droplets , which were blocked by the arc light 
 . however , the wire tip was pointed , and because no change appeared in the shape of the wire tip with the passage of time , we assumed this was spray transfer . in spray transfer , 
 the welding wire tip melted by the arc is transferred to the base material with a grain smaller than the wire diameter . 
 these results were consistent with the research of takeuchi et al .. the effective irradiance of the uvr emitted by non - pulsed 80% ar + 20% co2 welding increased with increasing welding current from 100 to 250 a , and no change was seen in this increasing trend ( fig . 
 , the effect of changing the metal transfer mode from short - circuiting to globular on the effective irradiance was considered to be small 
 . however , the effective irradiance increased rapidly between 250 a and 300 a. the metal transfer mode changed to spray transfer from globular transfer . 
 the amount of metal vapor in the arc during spray transfer is large compared to that during globular transfer , and the properties of light emitted by the arc welding are affected by the amount of metal vapor blending into the arc . 
 therefore , the cause of the rapid increase in the effective irradiance from 250 to 300 a is considered to be because of the change in the metal transfer mode ( globular transfer to spray transfer ) . 
 the metal transfer for non - pulsed 80% ar+20% co2 welding the effective irradiance of the uvr emitted during 100% co2 welding was in the range of 0.69 - 7.4 mw / cm and increased with increasing welding current . 
 takeuchi et al . reported that for 100% co2 welding , the transition from short - circuit transfer to globular transfer takes place at approximately 250 a. however , in the present study , no clear difference was seen in the effective irradiance vs. current trend between short - circuit transfer and globular transfer . 
 the effective irradiance was approximately the same for non - pulsed 80% ar + 20% co2 welding and 100% co2 welding at 250 a or less . 
 however , a significant difference was observed at 300 a or higher . by observation of the arc during welding , the metal transfer mode of non - pulsed 80% ar + 20% co2 welding was spray transfer and that of 100% co2 welding was globular transfer . 
 therefore , the effective irradiance is considered to depend on the metal transfer mode because of the differences in the shielding gas composition between them , and the effective irradiance increases with the transition to spray transfer . as shown above , the uvr emitted during the gmaw of mild steel was very hazardous during spray transfer . 
 the welding operator must recognize that welding under spray transfer conditions is extremely dangerous , so it is necessary to take adequate protective measures . 
 3 , the effective irradiance of the uvr emitted during pulsed 80% ar + 20% co2 welding increased with increasing current and was 3.0 - 6.7 times larger compared with that emitted during non - pulsed 80% ar + 20% co2 welding at each welding current . the effective irradiance was observed in the vicinity of the arc and the metal transfer mode was spray transfer for the entire current range . 
 5 . the metal transfer for pulsed 80% ar+20% co2 welding ( spray transfer ) . ( the effective irradiance is 3.4 mw / cm . ) for non - pulsed 80% ar + 20% co2 welding , the short - circuit or globular transfer modes were observed at 250 a or less ( fig . 
 the amount of metal vapor in the arc during spray transfer is large compared to that during short - circuit and globular transfer . 
 in addition , the properties of the light emitted during the arc welding are affected by the amount of metal vapor blended into the arc . 
 therefore , we believe that the effective irradiance of the uvr generated by spray transfer for a pulsed current is very large compared with that produced by the short - circuit and the globular transfer modes for a non - pulsed welding current . 
 these results confirm that the effective irradiance is strongly influenced by the pulsed current , and the degree of the effect increases with decreasing pulsed current . in recent years 
 , the demand for mild steel thin plate welding has increased to reduce the weight of equipment that needs to be transported . 
 we used a low welding current below 100 a in the welding of thin plate . 
 in addition , a digital inverter - type pulsed arc welding machine was used to increase the working efficiency . in this study , the effective irradiance of the uvr that occurred during pulsed 80% ar + 20% co2 welding at 100 a was 1.2 times greater than the effective irradiance during non - pulsed 80% ar + 20% co2 welding at 250 a. therefore , welding operators need to recognize the very high hazard of the uvr when they are welding with pulsed current , and supervisors must take adequate protection measures for the peripheral workers who are not welding . 
 the effective irradiance measured in this study , regardless of the pulse current and the type of shielding gas , increased with increasing welding current ( figure 2 , 3 ) . 
 this trend was also previously observed for the gmaw of mild steel using 100% co2 and 5% o2 + 95% ar as the shield gas . 
 more recently , we also observed the trend for the gmaw and the gtaw of aluminum and magnesium alloys . 
 thus , the welding current is an important factor influencing the degree of hazard of the uvr emitted during the welding process ; the uvr hazard can be understood to be a rapidly increasing function of the welding current . 
 the effective irradiance of the uvr generated during non - pulsed 80% ar + 20% co2 welding was in the range of 0.51 - 12.9 mw / cm and increased with increasing welding current . in particular , a significant difference in the increase in effective irradiance for welding currents from 100 to 250 a and from 250 a upward was observed . to investigate the reason for this difference , we observed the arc during welding . 
 4 , short - circuit transfer was observed at 100 a. in short - circuit transfer , the tip of the welding wire is short - circuited with the base metal ( molten pool ) , and the molten metal is shifted to the base material . 
 in addition , globular transfer was observed at 250 a. in globular transfer , the welding wire tip melted by the arc is transferred to the base metal in which the grain size grows larger than the wire diameter . as also shown in fig . 
 4 , it was not possible at 300 a to observe the droplets , which were blocked by the arc light . however , the wire tip was pointed , and because no change appeared in the shape of the wire tip with the passage of time , we assumed this was spray transfer . in spray transfer , 
 the welding wire tip melted by the arc is transferred to the base material with a grain smaller than the wire diameter . 
 these results were consistent with the research of takeuchi et al .. the effective irradiance of the uvr emitted by non - pulsed 80% ar + 20% co2 welding increased with increasing welding current from 100 to 250 a , and no change was seen in this increasing trend ( fig . 
 , the effect of changing the metal transfer mode from short - circuiting to globular on the effective irradiance was considered to be small 
 . however , the effective irradiance increased rapidly between 250 a and 300 a. the metal transfer mode changed to spray transfer from globular transfer . 
 the amount of metal vapor in the arc during spray transfer is large compared to that during globular transfer , and the properties of light emitted by the arc welding are affected by the amount of metal vapor blending into the arc . 
 therefore , the cause of the rapid increase in the effective irradiance from 250 to 300 a is considered to be because of the change in the metal transfer mode ( globular transfer to spray transfer ) . 
 the metal transfer for non - pulsed 80% ar+20% co2 welding the effective irradiance of the uvr emitted during 100% co2 welding was in the range of 0.69 - 7.4 mw / cm and increased with increasing welding current . 
 takeuchi et al . reported that for 100% co2 welding , the transition from short - circuit transfer to globular transfer takes place at approximately 250 a. however , in the present study , no clear difference was seen in the effective irradiance vs. current trend between short - circuit transfer and globular transfer . 
 the effective irradiance was approximately the same for non - pulsed 80% ar + 20% co2 welding and 100% co2 welding at 250 a or less . 
 however , a significant difference was observed at 300 a or higher . by observation of the arc during welding , the metal transfer mode of non - pulsed 80% ar + 20% co2 welding was spray transfer and that of 100% co2 welding was globular transfer . 
 therefore , the effective irradiance is considered to depend on the metal transfer mode because of the differences in the shielding gas composition between them , and the effective irradiance increases with the transition to spray transfer . 
 as shown above , the uvr emitted during the gmaw of mild steel was very hazardous during spray transfer . 
 the welding operator must recognize that welding under spray transfer conditions is extremely dangerous , so it is necessary to take adequate protective measures . 
 3 , the effective irradiance of the uvr emitted during pulsed 80% ar + 20% co2 welding increased with increasing current and was 3.0 - 6.7 times larger compared with that emitted during non - pulsed 80% ar + 20% co2 welding at each welding current . the effective irradiance was observed in the vicinity of the arc and the metal transfer mode was spray transfer for the entire current range . 
 5 . the metal transfer for pulsed 80% ar+20% co2 welding ( spray transfer ) . ( 
 the effective irradiance is 3.4 mw / cm . ) for non - pulsed 80% ar + 20% co2 welding , the short - circuit or globular transfer modes were observed at 250 a or less ( fig . 
 the amount of metal vapor in the arc during spray transfer is large compared to that during short - circuit and globular transfer . 
 in addition , the properties of the light emitted during the arc welding are affected by the amount of metal vapor blended into the arc . 
 therefore , we believe that the effective irradiance of the uvr generated by spray transfer for a pulsed current is very large compared with that produced by the short - circuit and the globular transfer modes for a non - pulsed welding current . 
 these results confirm that the effective irradiance is strongly influenced by the pulsed current , and the degree of the effect increases with decreasing pulsed current . in recent years 
 , the demand for mild steel thin plate welding has increased to reduce the weight of equipment that needs to be transported . 
 we used a low welding current below 100 a in the welding of thin plate . 
 in addition , a digital inverter - type pulsed arc welding machine was used to increase the working efficiency . in this study , the effective irradiance of the uvr that occurred during pulsed 80% ar + 20% co2 welding at 100 a was 1.2 times greater than the effective irradiance during non - pulsed 80% ar + 20% co2 welding at 250 a. therefore , welding operators need to recognize the very high hazard of the uvr when they are welding with pulsed current , and supervisors must take adequate protection measures for the peripheral workers who are not welding . 
 ( 1 ) it is more hazardous at higher welding currents than at lower welding currents . ( 2 ) at higher welding currents , it is more hazardous when 80% ar + 20% co2 is used as a shielding gas than when 100% co2 is used . 
 ( 3 ) it is more hazardous for pulsed welding currents than for non - pulsed welding currents . 
 ( 4 ) it appears to depend on the metal transfer ; the hazard of the uvr emitted during spray transfer is the highest .","objectives : ultraviolet radiation ( uvr ) emitted during arc welding frequently causes keratoconjunctivitis and erythema in the workplace . 
 the degree of hazard from uvr exposure depends on the welding method and conditions . 
 therefore , it is important to identify the uvr levels present under various conditions . 
 methods : we experimentally evaluated the uvr levels emitted in gas metal arc welding ( gmaw ) of mild steel . 
 we used both a pulsed welding current and a non - pulsed welding current . 
 the shielding gases were 80% ar + 20% co2 and 100% co2 . 
 the effective irradiance defined in the american conference of governmental industrial hygienists guidelines was used to quantify the uvr hazard . 
 results : the effective irradiance measured in this study was in the range of 0.51 - 12.9 mw / cm2 at a distance of 500 mm from the arc . 
 the maximum allowable exposure times at these levels are only 0.23 - 5.9 s / day . 
 conclusions : the following conclusions were made regarding the degree of hazard from uvr exposure during the gmaw of mild steel : ( 1 ) it is more hazardous at higher welding currents than at lower welding currents . 
 ( 2 ) at higher welding currents , it is more hazardous when 80% ar + 20% co2 is used as a shielding gas than when 100% co2 is used . 
 ( 3 ) it is more hazardous for pulsed welding currents than for non - pulsed welding currents . 
 ( 4 ) it appears to be very hazardous when metal transfer is the spray type . 
 this study demonstrates that unprotected exposure to uvr emitted by the gmaw of mild steel is quite hazardous .",pubmed
"a wide variety of taxonomically different groups of microorganisms are known to produce intracellular energy and carbon storage compounds , generally described as polyhydroxyalkanoates ( phas ) . 
 it has been shown that bacterial cells with a higher content of phas can survive longer than those with a lower phas content , because they can utilize their reserve material longer and more efficiently . 
 therefore , it was stated that the accumulation of phas might increase the survival capabilities of these bacteria in extreme environments or when nutrient availability is poor . 
 phas are synthesized by many bacteria and archaea   when in environment carbon is available , but other essential nutrients are not . 
 phas are commonly divided into two groups based on the number of constituent carbon atoms in their monomer units : short - chain - length ( scl- ) phas and medium - chain - length ( mcl - phas ) . 
 the former consists of monomers with 35 carbon atoms and the latter consists of monomers with 614 carbon atoms . 
 p(3hb ) is highly crystalline , whereas mcl - phas are elastic with low melting temperature . 
 these variations in the pha polymer family create the potential for a vast array of applications . 
 the surface of pha granules is coated with phospholipids and proteins which play a major role in pha synthesis , degradation , and even in the process of pha synthesis regulation . 
 the type of pha produced and accumulated in the granules depends on the metabolic pathway of the particular microorganism . however , independently of the type of synthesized phas , the enzyme called pha synthase always plays the main role . 
 pha synthases were classified into four classes , according to their substrate specificities and subunits organization . 
 classes i , iii , and iv prefer to synthesize scl - pha ( phac gene ) , whereas the ii class is responsible for mcl - pha synthesis . 
 class i of pha synthase comprises enzyme consisting of single subunit , whereas classes iii and iv comprise enzymes that consist of two different types of subunits . 
 the ii class of pha synthase is also built by single subunit but it is coded by two forms of gene that are called phac1 and phac2 . the nucleotide composition of phac1 and phac2 genes is very similar but their substrate specificity may differ . 
 extremophiles inhabiting polar areas are a source of a novel enzymes , which have a great economic potential in many industrial processes , including agricultural , chemical , and pharmaceutical applications . 
 especially , shallow antarctic reservoirs influenced by the forces of variable trophic conditions are underexploited sources of microorganisms with biotechnological potential . as a result of selection pressures in these reservoirs , 
 there is a potential for the discovery of novel biochemical pathways leading to phas synthesis . 
 psychrotrophic bacteria , which are adapted to variable temperatures and to other fluctuating conditions such as low nutrient availability and low water quality , may offer important advantages for industrial phas production . 
 it is widely accepted that microorganisms from unusual environments provide not only valuable resources for exploiting novel biotechnological processes , but also serve as models for investigating how these biomolecules are stabilized when subjected to changing conditions . 
 the main purpose of this study was to determine whether bacteria inhabiting freshwater reservoirs in glacier forefield areas have the potential to synthesize phas . 
 samples of water and mats were collected in summer of 2011 year from five , shallow freshwater reservoirs and one stream . 
 they were located in the foreland of ecology glacier near to the polish antarctic station  arctowski 
 1map showing sampling location in the foreland of ecology glacier , king georg island , antarctica . 
 ( part of admiralty bay map modified from pudeko   map showing sampling location in the foreland of ecology glacier , king georg island , antarctica . 
 ( part of admiralty bay map modified from pudeko   selected reservoirs were different in age , location , and environmental parameters ( table  1 ) . 
 located near to the front of ecology glacier , pond 7 was two years old . 
 this reservoir was located on the ground moraine and was directly supplied with water from the glacier , with no signs of vegetation . 
 pond 8 was located away from the front of the glacier , with abundant bloom of microalgae . 
 reservoirs 10 and 11 , which have been noticed there since 1977 , were situated on the top of lateral moraine . both were situated close to each other and connected with small stream ( fig .  1 ) . 
 samples were taken with a sampler on a bracket into sterile bottles and delivered to the laboratory within 1  hour . 
 microbial mat samples were collected as cores ( 1    5  cm ) , and one gram of sample was aseptically weighed and homogenized in 9  ml of sterile cold.(4  c ) physiological saline ( 0.86  % nacl ) using a vortex . 
 cultures were obtained by plating 100  l of a sample onto antarctic bacteriological medium ( abm ; ph 7.4 ) containing : peptone ( 0.5  % , w / v ) , yeast extract ( 0.2  % , w / v ) , and agar ( 2  % , w / v ) in three replicates . 
 the well -separated colonies on medium were macroscopically described , photographed , than isolated under binocular , and transferred to slants of abm in the tubes . 
 after transportation to poland the microscopic purity evaluation of gram - stained strains was done.table  1characteristics of sampling sitessampling sitevariables6781011gps position624826,4 
 s 0582853,9 w624825,2 s 0582850,1 w621002,3 s 0582740,9 w620923,2 s 0582820,2 w620937,0 s 0582823,7 welevation . 
 ( m a.s.l.)879227225area ( m)266.91122.30117.60123.80nh4-n ( mg  l)0.024 ( 0.004)0.035 ( 0.011)0.426 ( 0.071)0.161 ( 0.018)0.033 ( 0.002)po4-p ( mg  l)0.148 ( 0.003)0.130 ( 0.00)0.120 ( 0.042)0.079 ( 0.02)0.154 ( 0.044)p - tot ( mg  l)0.03 ( 0.00)0.035 ( 0.007)0.050 ( 0.028)0.020 ( 0.00)0.070 ( 0.00)n - tot ( mg  l)0.650 ( 0.071)1.950 ( 1.768)4.100 ( 0.00)1.400 ( 0.00)1.700 ( 0.00)ph ( mg  l)7.6 ( 0.5)7.7 ( 0.6)8.4 ( 0.7)6.5 ( 1.2)7.1 ( 0.8)temp ( c)2.8 ( 1.8)2.9 ( 1.6)5.0 ( 0.2)6.1 ( 2.1)5.8 ( 0.6)o2 ( mg  l)11.85 ( 0.35)10.40 ( 0.42)10.86 ( 1.22)11.08 ( 0.60)10.92 ( 0.23)conductivity ( us / cm)79.4 ( 26.86)84.7 ( 41.37)166.1 ( 76.23)135.0 ( 32.85)160.3 ( 19.38)chlorophyll a ( g  l)0.4000.4171.6660.9000.900pheophytin ( g  l)2.8002.7912.1253.8001.800total chlorophyll ( g  l)3.2003.2083.7914.7002.700doc ( mg  l)0.5670.6531.1350.7870.826 characteristics of sampling sites in order to obtain dna , cells were scraped from the surface of slants and washed in sterile water . 
 dna extraction was performed using commercial kit genomic mini ( a&a biotechnology ) according to the manufacturers instructions . 
 the purified dna was suspended in 50  l of deionized , dnase free water and stored in 20  c . 
 the partial 16s rrna gene was amplified using the primers 341 : 5-cctacgggaggcagcag -3   and 16sr : 5-taccttgttacgacttcacccca-3 described previously by rossau et al . . 
 in order to detect microorganism producing phas , pcr was performed with two primer pairs . 
 first one , recognizing both scl- and mcl - pha synthase genes was elaborated by romo et al . 
 ( g - d : 5-gtgccgcc(gc)(ct ) ( ag)(gc)atcaacaagt-3 ; g1-r : 5 gttccag(at)acag(gc)a(gt)(ag ) tcg aa-3 ) . 
 the second primers pair ( i-179l : 5-acagatcaacaagttctacatctt cgac-3 ; i-179r : 5-ggtgttgtcgttgttccagtagaggatgtc-3 ) was specific for both genes responsible for mcl - pha synthesis . 
 the mixtures used for pcr amplification contained 50  ng of extracted dna , 0.5  m of each primer , 100  m of deoxynucleoside triphosphate ( promega , winsconsin , usa ) , 1 u of taq dna polymerase ( polgen , poland ) , and 5  l of reaction buffer ( 500  mm kcl , ph 8.5 ; triton x-100 ) . 
 the temperature program for 16s rrna gene amplification was as follows : 94  c for 5  min ; 30 cycles of denaturation at 94  c for 30  s , annealing at 54  c for 30  s , extension at 72  c for 1  min , and single final elongation at 72  c for 5  min . 
 the pcr amplicons of 16s rdna and pha synthase genes were resolved on 1.5  % agarose gels stained with ethidium bromide , and size of pcr products was estimated using molecular weight marker ( 100  bp , promega , winsconsin , usa ) . 
 the sequencing of 16s rdna and scl - pha synthase ( phac ) pcr products was performed using the same pcr primers as for amplification . because pcr primers pair i-179l and i-179r amplifies both genes coding for mcl - pha synthase , we use primers orf2 ( 5-catgacagcggcctgttcacctgg-3   ) and i-179r   using the same conditions as described in the work of ciesielski et al . . using this approach 
 , it was possible to amplify and directly sequence a fragment of phac gene about 720  bp long . 
 dna sequencing was performed using a perkin elmer abi 373 automated dna sequencer ( pe applied biosystems , foster city , ca , usa ) at the institute of biochemistry and biophysics in warsaw , poland . 
 the nucleotide sequences were submitted to the genbank database under accession numbers from kf301568 to kf301599 . 
 the sequences of genes coding for 16s rrna and pha synthases were compared with those from the genbank database using the ncbi blast program . 
 the evolutionary distances were inferred using the neighbor - joining method   with the mega5 program . to determine the degree of statistical support for branches in the phylogeny , 1,000 bootstrap replicates of data were analyzed . 
 the gene sequences that were > 94.0  % identical to sequences of cultured species in the ncbi database were assigned genus names . a canonical correspondence analysis ( cca ) 
 the presence of bacteria possessing specific form of pha synthase genes was analyzed in relation to the environmental background 
 samples of water and mats were collected in summer of 2011 year from five , shallow freshwater reservoirs and one stream . 
 they were located in the foreland of ecology glacier near to the polish antarctic station  arctowski 
 1map showing sampling location in the foreland of ecology glacier , king georg island , antarctica . 
 ( part of admiralty bay map modified from pudeko   map showing sampling location in the foreland of ecology glacier , king georg island , antarctica . 
 ( part of admiralty bay map modified from pudeko   selected reservoirs were different in age , location , and environmental parameters ( table  1 ) . 
 located near to the front of ecology glacier , pond 7 was two years old . 
 this reservoir was located on the ground moraine and was directly supplied with water from the glacier , with no signs of vegetation . 
 pond 8 was located away from the front of the glacier , with abundant bloom of microalgae . 
 reservoirs 10 and 11 , which have been noticed there since 1977 , were situated on the top of lateral moraine . both were situated close to each other and connected with small stream ( fig .  1 ) . 
 samples were taken with a sampler on a bracket into sterile bottles and delivered to the laboratory within 1  hour . 
 microbial mat samples were collected as cores ( 1    5  cm ) , and one gram of sample was aseptically weighed and homogenized in 9  ml of sterile cold.(4  c ) physiological saline ( 0.86  % nacl ) using a vortex . 
 cultures were obtained by plating 100  l of a sample onto antarctic bacteriological medium ( abm ; ph 7.4 ) containing : peptone ( 0.5  % , w / v ) , yeast extract ( 0.2  % , w / v ) , and agar ( 2  % , w / v ) in three replicates . 
 the well -separated colonies on medium were macroscopically described , photographed , than isolated under binocular , and transferred to slants of abm in the tubes . 
 after transportation to poland the microscopic purity evaluation of gram - stained strains was done.table  1characteristics of sampling sitessampling sitevariables6781011gps position624826,4 
 s 0582853,9 w624825,2 s 0582850,1 w621002,3 s 0582740,9 w620923,2 s 0582820,2 w620937,0 s 0582823,7 welevation . 
 ( m a.s.l.)879227225area ( m)266.91122.30117.60123.80nh4-n ( mg  l)0.024 ( 0.004)0.035 ( 0.011)0.426 ( 0.071)0.161 ( 0.018)0.033 ( 0.002)po4-p ( mg  l)0.148 ( 0.003)0.130 ( 0.00)0.120 ( 0.042)0.079 ( 0.02)0.154 ( 0.044)p - tot ( mg  l)0.03 ( 0.00)0.035 ( 0.007)0.050 ( 0.028)0.020 ( 0.00)0.070 ( 0.00)n - tot ( mg  l)0.650 ( 0.071)1.950 ( 1.768)4.100 ( 0.00)1.400 ( 0.00)1.700 ( 0.00)ph ( mg  l)7.6 ( 0.5)7.7 ( 0.6)8.4 ( 0.7)6.5 ( 1.2)7.1 ( 0.8)temp ( c)2.8 ( 1.8)2.9 ( 1.6)5.0 ( 0.2)6.1 ( 2.1)5.8 ( 0.6)o2 ( mg  l)11.85 ( 0.35)10.40 ( 0.42)10.86 ( 1.22)11.08 ( 0.60)10.92 ( 0.23)conductivity ( us / cm)79.4 ( 26.86)84.7 ( 41.37)166.1 ( 76.23)135.0 ( 32.85)160.3 ( 19.38)chlorophyll a ( g  l)0.4000.4171.6660.9000.900pheophytin ( g  l)2.8002.7912.1253.8001.800total chlorophyll ( g  l)3.2003.2083.7914.7002.700doc ( mg  l)0.5670.6531.1350.7870.826 characteristics of sampling sites 
 in order to obtain dna , cells were scraped from the surface of slants and washed in sterile water . 
 dna extraction was performed using commercial kit genomic mini ( a&a biotechnology ) according to the manufacturers instructions . 
 the purified dna was suspended in 50  l of deionized , dnase free water and stored in 20  c . 
 the partial 16s rrna gene was amplified using the primers 341 : 5-cctacgggaggcagcag -3   and 16sr : 5-taccttgttacgacttcacccca-3 described previously by rossau et al . . 
 in order to detect microorganism producing phas , pcr was performed with two primer pairs . 
 first one , recognizing both scl- and mcl - pha synthase genes was elaborated by romo et al . 
 ( g - d : 5-gtgccgcc(gc)(ct ) ( ag)(gc)atcaacaagt-3 ; g1-r : 5 gttccag(at)acag(gc)a(gt)(ag ) tcg aa-3 ) . 
 the second primers pair ( i-179l : 5-acagatcaacaagttctacatctt cgac-3 ; i-179r : 5-ggtgttgtcgttgttccagtagaggatgtc-3 ) was specific for both genes responsible for mcl - pha synthesis . 
 the mixtures used for pcr amplification contained 50  ng of extracted dna , 0.5  m of each primer , 100  m of deoxynucleoside triphosphate ( promega , winsconsin , usa ) , 1 u of taq dna polymerase ( polgen , poland ) , and 5  l of reaction buffer ( 500  mm kcl , ph 8.5 ; triton x-100 ) . 
 the temperature program for 16s rrna gene amplification was as follows : 94  c for 5  min ; 30 cycles of denaturation at 94  c for 30  s , annealing at 54  c for 30  s , extension at 72  c for 1  min , and single final elongation at 72  c for 5  min . 
 the pcr amplicons of 16s rdna and pha synthase genes were resolved on 1.5  % agarose gels stained with ethidium bromide , and size of pcr products was estimated using molecular weight marker ( 100  bp , promega , winsconsin , usa ) . 
 the sequencing of 16s rdna and scl - pha synthase ( phac ) pcr products was performed using the same pcr primers as for amplification . 
 because pcr primers pair i-179l and i-179r amplifies both genes coding for mcl - pha synthase , we use primers orf2 ( 5-catgacagcggcctgttcacctgg-3   ) and i-179r   using the same conditions as described in the work of ciesielski et al . . 
 using this approach , it was possible to amplify and directly sequence a fragment of phac gene about 720  bp long . 
 dna sequencing was performed using a perkin elmer abi 373 automated dna sequencer ( pe applied biosystems , foster city , ca , usa ) at the institute of biochemistry and biophysics in warsaw , poland . 
 the nucleotide sequences were submitted to the genbank database under accession numbers from kf301568 to kf301599 . 
 the sequences of genes coding for 16s rrna and pha synthases were compared with those from the genbank database using the ncbi blast program . 
 the evolutionary distances were inferred using the neighbor - joining method   with the mega5 program . to determine the degree of statistical support for branches in the phylogeny , 1,000 bootstrap replicates of data were analyzed . 
 the gene sequences that were > 94.0  % identical to sequences of cultured species in the ncbi database were assigned genus names . 
 a canonical correspondence analysis ( cca ) was conducted using a statistical package for windows v. canoco 4.5 . 
 the presence of bacteria possessing specific form of pha synthase genes was analyzed in relation to the environmental background 
 a total of 50 bacterial isolates from five ponds and one stream were characterized by sequencing of 1000  bp long fragment of 16s rrna gene . 
 twenty , unique dna sequences were phylogenetically analyzed altogether with their closest relatives derived from genbank ( fig .  2 ) . 
 isolated strains belonged mainly to the phylum of proteobacteria and represent classes betaproteobacteria ( 11 isolates ) , gammaproteobacteria ( 7 isolates ) , alphaproteobacteria ( 2 isolates ) and only two isolates belonged to bacteroidetes phylum ( fig .  2 ) . 
 2phylogenetic tree based on 16s rrna sequences , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . 
 type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . 
 accession numbers of dna sequences are given in parentheses phylogenetic tree based on 16s rrna sequences , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . 
 type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . 
 accession numbers of dna sequences are given in parentheses the most frequently recovered group of isolates was members of the pseudomonas genus ( 7 isolates ) . 
 the second numerous group of isolates belonged to janthinobacterium genus ( 5 isolates ) . in the aim of detection of a microorganism possessing the potential ability of phas synthesis , 
 the first one ( g - d and g-1r ) , is able to recognize both scl- and mcl - pha synthase genes . the second primers pair ( i-179l and i-179r ) was specific only for mcl - pha synthase genes . the amplification using primers g - d and g-1r gave a positive signal in 12 of the isolates corresponding to six pseudomonads strains , four janthinobacterium strains , and two undefined betaproteobacteria ( p21 and p25 ) . 
 the amplification with pcr primers designed by solaiman and co - workers   showed that six pseudomonas strains ( p1 , p4 , p19 , p27 , p32 , and p45 ) and two janthinobacterium strains ( p10 and p24 ) possessed genes responsible for mcl - pha synthesis . 
 pcr products obtained by employing primer pairs g - d : g-1r and orf2 : i-179r were directionally sequenced . 
 the results of dna sequencing of pcr products obtained using g - d and g-1r primers proved that studied dna of two janthinabacterium strains ( p38 and p39 ) , and two undefined betaproteobacteria strains ( p21 and p25 ) is responsible for scl - pha synthesis . 
 nucleotide composition of pcr products amplified with orf2 : i-179r primers pair has provided evidence that all are typical for phac1 gene coding for synthase responsible for mcl - pha synthesis . for deeper analysis , 
 all obtained dna sequences of phac and phac1 genes , after division into two group , were phylogenetically examined altogether with their closest relatives found in genbank . 
 evolutionary tree presenting genetic distance between phac gene dna sequences is shown in the fig . 
 very high similarity ( genetic distance 0.236 ) was revealed between janthinobacterium sp . p39 and antarctic proteobacterium p21 , inhabiting the same pond 7 
 . very close to them dna sequence of janthinobacterium sp . p38 was placed , and all three sequences obtained in this study were accompanied by dna sequence of zooglea ramigera . 
 the fourth sequence , belonging to antarctic proteobacterium p25 was grouped with rhodoferax ferrireducens ( genetic distance 0.237 ) ( fig . 
 the second group , collecting dna sequences of phac1 gene ( mcl - pha ) , was predominated by members of pseudomonas species . the highest similarity ( genetic distance 0.003 ) , was observed between pseudomonas sp . 
 p19 ( genetic distance 0.009 ) and was collected from different , although neighboring ponds . 
 surprisingly , these two sequences were grouped together with janthinobacterium sp . p24 sequence that came also from mat of pond 10 . 
 3phylogenetic tree based on phac gene coding for scl - pha synthase , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . 
 type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . 
 4phylogenetic tree based on phac1 gene coding for mcl - pha synthase , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . 
 type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . 
 accession numbers of dna sequences are given in parenthesestable  2molecular identification of isolated strains , their nearest neighbors based on 16s rrna and phac / phac1 genes and their isolation locationisolateputative phaclosest relatives based on 16s rdna sequence ( accession no / identity  % ) 
 closest cultured based on phac / phac1 gene sequence ( accession no / identity  % ) 
 isolation source 
 pseudomonas sp . 
 p1mcl 
 pseudomonas extremaustralis sy11 ( kc790323/99 ) 
 pseudomonas fluorescens pf0 - 1 ( cp000094/91)mat ( pond 10 ) 
 pseudomonas sp . 
 p4mcl 
 pseudomonas frederiksbergensis ( jf343187/100 ) 
 pseudomonas fluorescens pf0 - 1 ( cp000094/90)mat ( pond 10 ) 
 pseudomonas sp . 
 ( fj472656/91)water ( pond 11 ) 
 pseudomonas sp . p27mcl 
 pseudomonas antarctica ( he586386/99 ) 
 pseudomonas fluorescens sbw25 ( am181176/95)water ( pond 8) 
 pseudomonas sp . p32mcl 
 pseudomonas putida ( he586397/99 ) 
 pseudomonas sp . 
 p45mcl 
 pseudomonas fragi ( ab685634/100 ) 
 pseudomonas stutzeri 1317 ( ay278219/84)stream 6 
 janthinobacterium sp . 
 p10mcl 
 janthinobacterium lividum ( hq824865/100 ) 
 pseudomonas extremaustralis ( fn435843/90)water ( pond 10 ) 
 janthinobacterium sp . 
 p24mcl 
 janthinobacterium lividum ( hq824864/100 ) 
 pseudomonas fluorescens bm07 ( fj472656/92)mat ( pond 10)antarctic betaproteobacterium p21scl 
 burkholderia sp . 
 era35(jq977167/99 ) 
 zooglea ramigera ( u66242/83)water ( pond 7)antarctic betaproteobacterium p25scl 
 rhodoferax ferrireducens ( hg003356/99 ) 
 comamonas testosteroni cnb-2 ( cp001220/81)water ( pond 10 ) 
 janthinobacterium sp . 
 smn 33.6 ( jx624164/99 ) 
 zooglea ramigera ( u66242/83)water ( pond 10 ) 
 janthinobacterium sp . 
 tmt4 - 26 - 6 ( jx949991/99 ) 
 zooglea ramigera ( u66242/83)water ( pond 7 ) 
 pseudomonas sp . 
 p5- 
 pseudomonas fragii ( ab685683/99)-water ( pond 8) 
 iodobacter sp p11- 
 iodobacter fluviatilis ( kc213858/100)-water ( pond 7 ) 
 flavobacterium sp . 
 p14- 
 flavobacterium algicola ( ab455265/99)-water ( pond 10 ) 
 yersinia sp . p16- 
 yersinia intermedia ( nr_027545/99)-water ( pond 7 ) 
 shewanella sp . 
 sphingomonadaceae bacterium n ( dq497241/99)-water ( pond 7)antarctic betaproteobacterium p36- 
 rhodoferax ferrireducens ( nr_074760/99)-water ( pond 8) *  only cultured strains were considered during blast searching phylogenetic tree based on phac gene coding for scl - pha synthase , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . 
 type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . 
 accession numbers of dna sequences are given in parentheses phylogenetic tree based on phac1 gene coding for mcl - pha synthase , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . 
 type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . 
 accession numbers of dna sequences are given in parentheses molecular identification of isolated strains , their nearest neighbors based on 16s rrna and phac / phac1 genes and their isolation location *  only cultured strains were considered during blast searching the statistical analysis of principal components ( cca ) showed correlation between the presence of bacteria possessing particular pha synthase gene and environmental conditions . 
 bacteria having genetic potential to synthesize scl - pha inhabited reservoirs with rather low trophy characterized by low levels of total phosphorus and high amounts of pheophytins ( fig 
 .  5).fig .  5canonical correspondence analysis showing relation between the presence of bacteria possessing specific form of pha synthase genes and environmental data of sampling sites canonical correspondence analysis showing relation between the presence of bacteria possessing specific form of pha synthase genes and environmental data of sampling sites 
 in the ponds that were studied , more than half of the isolated strains possessed genes responsible for phas synthesis . among them were mainly microbes belonging to the pseudomonas genus ( six strains ) and janthinobacterium genus ( four strains ) . 
 they isolated from antarctic soil strains of bacteria having the ability to produce phas , among them twelve were belonging to pseudomonas spp . and three to janthinobacterium genus 
 .   showed that strains of this genus isolated from antarctica could be divided into two groups ; the first is  jantinobacterium lividum group ,  whereas the second was called 
 our analysis also indicates that isolated janthinobacterium strains were divided into two groups on the base of gene coding for 16s rrna ( fig .  2 ) . 
 . are often isolated from cold environments and are known for their metabolic versatility and high genome plasticity . 
 the possibility of phas accumulation and high tolerance to cold and oxidative stress is common features of many pseudomonas species . 
 therefore , it is suggested that pha metabolism in antarctic strains could be an adaptation mechanism that is necessary to withstand hard conditions in polar regions . 
 the high number of isolates possessing genes responsible for phas synthesis suggests that antarctic bacteria evolved , or obtained in another way , this metabolic ability to endure the extreme conditions typical for polar regions . accordingly to our results , isolated strains possessed genes responsible for scl- or mcl - pha synthesis . 
 it is worth pointing out that bacteria having phac1 genes were isolated both from mats and water , and those having phac gene were only planktonic . 
 it has previously been reported that survival in harsh conditions can be increased by belonging to a complex communities like microbial mats . 
 recent studies have evaluated the potential of microbial mats as high - level pha production systems under natural conditions , and as a source of bacterial pha producers [ 3 , 13 ] . accordingly to these studies , microorganisms inhabiting microbial mats from marine coastal zones are able to synthesize and accumulate high quantities of phas . our results also show that microbial mats created in antarctic ponds could be a potential source of pha producing bacteria . 
 our comparative analysis of dna sequences of 16s rdna and phac / phac1 genes suggested that some strains possess pha synthesis genes that might have not been obtained by gradual evolution . 
 the best example are isolates belonging to janthinobacterium spp . among four members of this genus , possessing genes responsible for phas synthesis , two of them had phac genes , whereas two had phac1 genes . 
 p38 and p39 was similar to a variant of this gene previously detected only in zooglea ramigera , whereas the phac1 genes of janthinobacterium sp . p10 and p24 were most similar to genes of pseudomonas fluorescences . 
 the genes responsible for scl - pha synthesis differ so much from those responsible for mcl - pha synthesis that their presence in these closely related bacteria is most likely not the result of gradual evolution , but rather of horizontal gene transfer . 
 it is interesting that isolates that had obtained these particular types of genes were divided into two subgroups on the basis of 16s rdna sequences ( fig 
 it is possible that this evolutionary separation is related to some physiological differences that make a particular pha operon useful for these bacteria . 
 have been reported , and ours is the first report of potential mcl - pha synthesis by members of this genus . 
 16s rdna gene analysis showed that the taxonomic position of each of the six pseudomonas isolates having the phac1 gene was different ( table  2 ) . 
 whereas , variation between phac1 genes was lower , closest dna sequences of phac1 gene of examined strains obtained by blast searching belonged only to three genera . 
 the dna sequences of p1 , p4 , and p19 were closest to the sequence of pseudomonas fluorescens , a well - known mcl - pha producer . the structure of the phac1 gene of pseudomonas sp . 
 in the same study , the same pha operon was found in both cells of pseudomonas sp . 
 p45 , inhabiting a glacial melt water stream , possessed a phac1 gene most similar to that of pseudomonas stutzeri , although the similarity was only 84  % . 
 p45 16s rrna gene was most similar to p. fragi and identical to the dna sequence of the pseudomonas sp . 
 p45 , in cells of strain p5 , genes responsible for pha synthesis were not detected . 
 in general , comparative analysis of 16s rrna and phac / phac1 genes revealed that isolated bacteria gain the genetic ability to synthesize pha rather as a result of horizontal gene transfer than by gradual evolution . 
 furthermore , it seems that some pha operons are favored during this unusual ecological event . 
 bacteria possessing pha synthase coding genes were detected in each of the studied location suggesting that phas accumulation could increase the survival capabilities of microorganisms in this extreme environment . 
 bacteria having genes responsible for both scl- and mcl - phas synthesis were found , but those able of scl - pha synthesis were discovered only in ponds 7 and 10 . 
 the relation between the presence of scl - pha producing bacteria and environmental conditions is supported by cca ( fig . 
 the highest number of different forms of phac / phac1 genes was founded in pond 10 , which was characterized by low ph ( 6.5 ) , highest level of pheophytin ( 3.800  g  l ) , and total chlorophyll ( 4.700  g  l ) , and with the highest temperature measured ( 6.1  c ) . 
 p19 ) and what is interesting similar form of gene was found in cells of janthinobacterium sp . 
 ponds 10 and 11 are connected with the stream and it is likely , that this form of phac1 was channeled from pond 11 to pond 10 . 
 a very distant form of phac1 gene was found in the mat taken from the stream 6 , it might release from the fact that environmental conditions in the streams are different from those in ponds . 
 the spatial distribution of microorganisms possessing particular form of genes in the area of glacier forefield results mainly from glacier melting . 
 the flowing water carries not only nutrients and minerals , but also transfers microorganisms having accelerated ability to colonize new territories . 
 this group can include bacteria having ability to utilize phas produced by themselves or other members of microbial community . 
 it seems that bacteria having genetic ability to store and degrade phas can work as a pioneers during a succession process . 
 thus , glacier forefields provide a unique opportunity as a natural laboratory to study the succession of microorganisms . 
 to sum up , our study shows that many microorganisms inhabiting freshwater lakes charged with glacier meltwater possess the genes responsible for pha synthesis . 
 all isolated pseudomonas species had the phac1 gene responsible for mcl - pha synthesis , whereas janthinobacterium isolates had phac and phac1 genes used for both scl- and mcl - pha synthesis . to our knowledge , this is the first evidence that janthinobacterium spp . 
 another interesting observation is the lack of correspondence between the evolutionary history of the 16s rdna genes and those coding for pha synthases . 
 , suggest that these genes could have been acquired by horizontal gene transfer . the obtained results allow us to speculate that the possession of these genes enabling pha synthesis may improve the fitness and survival of bacteria in harsh conditions 
 additionally , the presented results show that microorganisms inhabiting extreme environments should be considered as potential producers of phas .","the diversity of polyhydroxyalkanoates - producing bacteria in freshwater reservoirs in the ecology glacier foreland , antarctica , was examined by a cultivation - dependent method . 
 isolated strains were analyzed phylogenetically by 16s rrna gene sequencing , and classified as members of alpha- , beta- , or gammaproteobacteria classes . 
 polymerase chain reaction was used to detect pha synthase genes . 
 potential polyhydroxyalkanoates ( phas ) producers belonging mainly to pseudomonas sp . , and janthinobacterium sp . 
 were isolated from all five sampling sites , suggesting that pha synthesis is a common bacterial feature at pioneer sites . 
 all pseudomonas strains had the genetic potential to synthesize medium - chain - length phas , whereas some isolated janthinobacterium strains might produce short - chain - length phas or medium - chain - length phas . 
 it is the first report revealing that janthinobacterium species could have the potential to produce medium - chain - length phas .",pubmed
"the operations required to reclaim arid and semi - arid lands fall into several different categories : modifying topography ( slope , contours , and drainage ) , creating appropriate surface composition ( compaction , particle size , and origin of the final surface ) , and establishing vegetation . of these categories , establishing vegetation entails the most uncertainty . 
 vegetation establishment is important for : reducing erosion from both wind and water ( guiterrez and hernandez 1996 ; de baets et al . 
 2008 ) , improving public perception by reducing the visual impact ( hands and brown 2002 ; svobodova et al . 
 2012 ) , allowing for productive reuse of the land for wildlife habitat or livestock grazing ( e.g. , steinke and majak 2010 ) , and otherwise reintegrating disturbed lands into the existing landscape . 
 uncertainty in vegetation establishment comes primarily from reliance on unpredictable rainfall for germination and establishment . while reliance upon rainfall is not the only option , irrigation is expensive and generally not more successful ( roundy et al . 
 2001 ) possibly because a high intensity / long duration of water application is needed to establish plants , and even when plants are established with irrigation , they can lack water hardiness and die when the irrigation is eventually removed ( josa et al . 
 irrigation can also benefit exotic weeds more than natives ( e.g. , banerjee et al . 
 natural rainfall can allow the establishment of robust native or non - native vegetation albeit at densities similar to nearby undisturbed vegetation ( e.g. , fehmi and kong 2012 ) . 
 while in the past non - natives were preferred for their ability to establish in years with limited rainfall , natives are now more recommended due to their known long - term stability , resource value for wildlife , and better public acceptance ( dantonio and meyerson 2002 ) . 
 non - natives are currently less acceptable due to potential economic losses due to their invasions off - site , increases in the ability of reclaimed lands to carry wildfire ( mcdonald and mcpherson 2011 ) , and other negative impacts ( hobbs and cramer 2008 ) . to maximize the long - term positive impact of re - vegetation 
 , a mixed plant community is needed that contains the same ratio of functional groups as a natural community in the same area . 
 in addition to being self - sustaining , diversity increases the resiliency of the plant community to disturbance ( quijas et al . 
 where they occur , perennial grasses are often the primary contributors to surface stability and erosion prevention ( guiterrez and hernandez 1996 ; de baets et al . 
 2009 ) through their life form which is extensive , low to the ground , and with spreading , fibrous roots near the soil surface . 
 cox and jordan ( 1983 ) expected successful establishment of native perennial warm - season grasses only 10  % of the time ( 1 in 10  years ) in arid to semi - arid southeastern arizona , usa . 
 this is similar to the estimates generated by peters ( 2000 ) for central new mexico . 
 establishment success for a seedling depends on the seminal root staying ahead of the soil drying front . 
 when native warm - season ( c4 ) grasses commonly planted in the southwestern usa germinate , they are supported by the seminal root ( often also called the primary root ) until the adventitious roots begin to grow . 
 because these grasses establish the plant crown near the surface of the soil , adventitious root growth can be suppressed by dry conditions ( hyder et al . 
 other than the predictions mentioned above and research studies that rarely consider more than a few years ( e.g. , woods et al . 2012 ) , little work has occurred to better assess the chance of seeding success for arid areas . 
 other studies have shown that seedling recruitment is not dependent solely on high rainfall years but instead on the amount and distribution of rainfall events ( e.g. , peters 2000 ; zimmerman et al . 
 2012 ) . given the advances in both natural history information and in ease of computer modeling , cox and jordan s ( 1983 ) prediction about southern arizona should be revisited . 
 modeling can resolve important management and ecological questions because it allows comparisons that are not possible in the field ( hardegree et al . 
 the goal of this project is to estimate how frequently native perennial warm - season grasses may be established in southern arizona using 31  years of historical rainfall event data . 
 it is important to research the likelihood of vegetation establishment to better develop mitigation strategies and to allow appropriate budgeting for them . 
 to estimate the frequency of successful establishment , we simulated soil moisture for a 31-year period using a land surface model . 
 the inputs included detailed long - term precipitation event data , data on movement of moisture through the soil , and data on evaporation from the santa rita experimental range ( described below ) . 
 these data allowed analysis of moisture at the surface and at each soil layer as roots grew through them . using real - world data was important because having valid input parameters remains the most difficult aspect of modeling seed germination ( flerchinger and hardegree 2004 ) , and correctly representing rainfall variability is critical for modeling soil moisture ( laio et al . 
 the specific practices selected for analysis included having seeded on 15 may ( 30  days ahead of the normal start of the monsoon season ) onto bare soil in southern arizona , usa . the seeds ( generic native warm - season grasses described below ) were assumed to be in the top 01  cm of the soil when the monsoon rains began . 
 the soil type used in the simulations was a loamy sand because this soil texture was common in the region as well as being the primary soil type on a nearby mineland reclamation site . 
 no existing vegetation was assumed at the time of the seeding ( bare soil ) which had the simplifying effect of having evaporation to be the primary evapotranspiration loss . 
 germination and establishment were evaluated over the first 21  days , so competition among seedlings was assumed not to have an impact on plant growth or soil moisture in this period . rainfall data ( 19802010 , 31  years ) came from gages 5 ( 31.8152  n , 110.8516  w , 1,163  m asl , 398  mm average annual precipitation ) and 6 ( 31.8137  n , 
 110.8544  w , 1,215  m asl , 405  mm average annual precipitation ) from the santa rita experimental range ( srer ) which was roughly 45  km south of tucson , az , usa ( http://www.tucson.ars.ag.gov/dap/ ) . the gages are 311  m apart and record the date , time , amount , and duration of each rainfall event . 
 the monsoon rainfall in this region was generally from convective cells which can result in different amounts of rainfall , even over short distances , so the data from each gage were evaluated separately . 
 seven years ( 6 january 200431 december 2010 ) of eddy flux data were available , including the following measurements : temperature / relative humidity ; photosynthetically active radiation ; ground heat flux ; soil temperature ; precipitation ; volumetric soil water content at 5 , 10 , 20 , 30 , 50 , 70 , 100 , and 130  cm depths ; wind velocity vector ; sonic temperature and concentrations of water vapor ; and carbon dioxide measurements ( scott et al . 2009 ; hereafter , flux tower data ) . 
 the measurements were from the santa rita mesquite savanna site which was also located on the srer ( 31.8214  n , 110.8661  w , 1,120  m  asl ) . 
 to model the expected soil moisture from the 31  years ( 19802010 ) of precipitation data , we ran a land surface model , which was driven by hourly atmospheric forcing data . 
 we calibrated the model s hydraulic parameters to match the observed soil moisture and surface energy and water fluxes from the flux tower data and then applied the optimized parameters to the 31-year precipitation data set . 
 the atmospheric forcing data to drive the model included : hourly downward shortwave and longwave radiation fluxes , precipitation , air temperature , humidity , pressure , and wind speed . 
 these data were computed as distance - weighted averages of those at four surrounding points of the 0.125 degree , gridded north american land data assimilation system ( nldas ) surface atmospheric data ( mitchell et al . 
 2004 ) . to check the accuracy of the nldas data , sensitivity experiments during the calibration period were conducted by replacing an individual variable of the flux tower data with that of the nldas data . through this exercise 
 , we found that nldas data overestimated wintertime precipitation and had the largest effect on the simulation of soil moisture , while other variables had less effect . 
 we then replaced the nldas precipitation with the rainfall from gages 5 and 6 from the santa rita experimental range . 
 thus , for the period from 19802003 , we ran the model using the optimized hydraulic parameters and the nldas atmospheric forcing with its rainfall data being replaced with data from gages 5 and 6 . 
 the noah land surface model was used in this study with multiple options for parameterization schemes of various land surface processes ( niu et al . 
 there were only four soil layers in the standard model that was coupled with the weather research and forecast model . in this study , to match the flux tower measurement depths , we divided the model soil into 16 soil layers ( breaking at 2.5 , 5 , 10 , 15 , 20 , 25 , 30 , 45 , 50 , 60 , 70 , 90 , 100 , 120 , 130 , and 200  cm ) . 
 the model solved the one - dimensional richards equation to account for redistribution of soil moisture among these layers through diffusion and percolation processes . at the 2-m model bottom 
 , we used a groundwater recharge rate , which was provided by a simple groundwater model , as the lower boundary condition to ensure a more precise simulation . 
 the soil hydraulic parameters for loamy sand were set up through a look - up table using the clapp 
 hornberger relationship ( clapp and hornberger 1978 ) . however , the amplitudes of the modeled soil moisture for most of the deep soil layers were too small to match observations , indicating the diffusion processes of soil water are relatively weak . the constant value 1.5    10  m / s was added to the diffusivity which resulted in comparable soil moisture for all measurement depths . the enhanced diffusivity accounted for extra diffusion of vaporized soil water under dry and hot conditions ( niu et al . 
 our analysis assumed that the native , warm - season ( c4 ) grass seeds were in the soil from 01  cm deep as suggested by roundy et al . 
 ( 1997 ) , which corresponds well to visual observations of seed depth after broadcast seeding on this soil type in this area . 
 the seeds were assumed to remain available until they germinate rather than experiencing any other fate such as being eaten , buried , blown , or washed off site , etc . 
 all seeds were assumed to germinate when exposed to suitable monsoon conditions ( per abbott and roundy 2003 ) . 
 wetting events that did not result in germination were assumed to cause no injury to these warm - season perennial grass seeds ( roundy et al . 
 germinating conditions that occurred before 15 may were not considered because this was before we assumed planting to occur . 
 germinating conditions that occurred after 30 september were assumed to result in seeding failure due to the lack of suitable growing conditions ( cool weather ) in october . 
 two scenarios were evaluated for each of the rain gages2  days of suitable conditions resulting in germination and 3  days of suitable conditions resulting in germination . 
 plants were counted as germinated if the water volume in the 02.5  cm layer stayed above 0.09  cm / cm for 2 or 3  days ( analyzed separately ) 
 an intermediate value for water volume ( roughly twice the wilting point , 0.046  cm / cm , laio et al . 
 2001 ) was chosen because the 02.5-cm layer dries quickly , but seeds can germinate if the relative humidity remains high at the soil surface ( wuest 2007 ) . 
 ( 1997 ) that the surface soil becomes too dry within 1  day or less of the cessation of a storm . 
 the 2-day scenario was a more cautious interpretation of the available data on seed germination . 
 most warm - season perennial grasses germinate within 1.5 to 4  days of saturation of the surface soil at summer temperatures ( roundy and biedenbender 1996 ) . 
 the 2-day scenario supported native warm - season grass species like bouteloua curtipendula ( sideoats grama ) which germinated and emerged within 2  days of suitable conditions50  % in 1  day ( simanton and jordan 1986 ) . 
 another study observed emergence of b. curtipendula beginning in 18  h and ending after 3  days ( frasier et al . 
 while different authors have tested germination in different ways , most typically , the reported times were for saturated soil and our standard was dryer than that . 
 our 2-day germination scenario typically corresponded to one large or two medium - sized events . 
 the 3-day scenario was a more lenient estimate of the time needed for germination and corresponds more closely to available data on native warm - season grass germination . 
 ( 2000 ) found digitaria californica ( arizona cottontop ) required a minimum of 140  mm of water in a 3-day period for 50  % emergence . 
 wilson and briske ( 1979 ) found bouteloua gracilis ( blue grama ) needed wet conditions for 24  days to germinate . 
 after 2.3  days in wet field conditions , abbott and roundy ( 2003 ) found b. curtipendula had 90  % germination ; leptochloa dubia ( green sprangletop ) had 75  % ; d. californica had 70  % , and eragrostis intermedia ( plains lovegrass ) had 45  % . 
 germinated plants die when seminal root growth does not keep up with the drying front in the soil which makes soil moisture the primary controlling factor for seedling mortality ( carren et al . 
 1987 ; zimmerman et al . 2008 ; perring and hovenden 2012 ; thapa et al . 
 this process was important for 23  weeks after germination when 24  days of additional moisture was needed at the soil surface for development of the plant crown and adventitious roots ( carren et al . 
 the drying front was defined as the wilting point ( 0.046  cm / cm ) for a loamy sand ( laio et al . 
 the rate of 0.8  cm root elongation per day was chosen as a conservative estimate based on several studies of native warm - season perennial grasses . for b. gracilis , 
 the seminal root grew 0.79  cm per day for the first 2  weeks ( carren et al . 
 b. curtipendula averaged about 0.8  cm per day of seminal root elongation during the first 15  days ( roundy et al . 
 simanton and jordan ( 1986 ) found an elongation rate of 0.97  cm per day for the first 7  days for b. curtipendula . using the modeled soil moisture , 
 if sufficient water were available , plants may have initiated adventitious roots during our 19-day evaluation , but we evaluated soil moisture for the whole soil column and dryness below the wilting point would have affected adventitious roots as well as seminal roots . in our assessment , we checked the simulated water content of the soil column from the surface to the depth of the seminal root , and if the whole column was below the wilting point , the seeding was considered to have failed . 
 rainfall data ( 19802010 , 31  years ) came from gages 5 ( 31.8152  n , 110.8516  w , 1,163  m asl , 398  mm average annual precipitation ) and 6 ( 31.8137  n , 110.8544  w , 1,215  m asl , 405  mm average annual precipitation ) from the santa rita experimental range ( srer ) which was roughly 45  km south of tucson , az , usa ( http://www.tucson.ars.ag.gov/dap/ ) . 
 the gages are 311  m apart and record the date , time , amount , and duration of each rainfall event . 
 the monsoon rainfall in this region was generally from convective cells which can result in different amounts of rainfall , even over short distances , so the data from each gage were evaluated separately . 
 seven years ( 6 january 200431 december 2010 ) of eddy flux data were available , including the following measurements : temperature / relative humidity ; photosynthetically active radiation ; ground heat flux ; soil temperature ; precipitation ; volumetric soil water content at 5 , 10 , 20 , 30 , 50 , 70 , 100 , and 130  cm depths ; wind velocity vector ; sonic temperature and concentrations of water vapor ; and carbon dioxide measurements ( scott et al . 
 the measurements were from the santa rita mesquite savanna site which was also located on the srer ( 31.8214  n , 110.8661  w , 1,120  m  asl ) . 
 the soils at the site are uniform sandy loams . to model the expected soil moisture from the 31  years ( 19802010 ) of precipitation data 
 , we ran a land surface model , which was driven by hourly atmospheric forcing data . 
 we calibrated the model s hydraulic parameters to match the observed soil moisture and surface energy and water fluxes from the flux tower data and then applied the optimized parameters to the 31-year precipitation data set . 
 the atmospheric forcing data to drive the model included : hourly downward shortwave and longwave radiation fluxes , precipitation , air temperature , humidity , pressure , and wind speed . 
 these data were computed as distance - weighted averages of those at four surrounding points of the 0.125 degree , gridded north american land data assimilation system ( nldas ) surface atmospheric data ( mitchell et al . 
 2004 ) . to check the accuracy of the nldas data , sensitivity experiments during the calibration period 
 were conducted by replacing an individual variable of the flux tower data with that of the nldas data . through this exercise 
 , we found that nldas data overestimated wintertime precipitation and had the largest effect on the simulation of soil moisture , while other variables had less effect . 
 we then replaced the nldas precipitation with the rainfall from gages 5 and 6 from the santa rita experimental range . 
 thus , for the period from 19802003 , we ran the model using the optimized hydraulic parameters and the nldas atmospheric forcing with its rainfall data being replaced with data from gages 5 and 6 . 
 the noah land surface model was used in this study with multiple options for parameterization schemes of various land surface processes ( niu et al . 
 there were only four soil layers in the standard model that was coupled with the weather research and forecast model . 
 in this study , to match the flux tower measurement depths , we divided the model soil into 16 soil layers ( breaking at 2.5 , 5 , 10 , 15 , 20 , 25 , 30 , 45 , 50 , 60 , 70 , 90 , 100 , 120 , 130 , and 200  cm ) . 
 the model solved the one - dimensional richards equation to account for redistribution of soil moisture among these layers through diffusion and percolation processes . at the 2-m model bottom 
 , we used a groundwater recharge rate , which was provided by a simple groundwater model , as the lower boundary condition to ensure a more precise simulation . 
 the soil hydraulic parameters for loamy sand were set up through a look - up table using the clapp 
 . however , the amplitudes of the modeled soil moisture for most of the deep soil layers were too small to match observations , indicating the diffusion processes of soil water are relatively weak . the constant value 1.5    10  m / s was added to the diffusivity which resulted in comparable soil moisture for all measurement depths . 
 the enhanced diffusivity accounted for extra diffusion of vaporized soil water under dry and hot conditions ( niu et al . 
 our analysis assumed that the native , warm - season ( c4 ) grass seeds were in the soil from 01  cm deep as suggested by roundy et al . 
 ( 1997 ) , which corresponds well to visual observations of seed depth after broadcast seeding on this soil type in this area . 
 the seeds were assumed to remain available until they germinate rather than experiencing any other fate such as being eaten , buried , blown , or washed off site , etc . 
 all seeds were assumed to germinate when exposed to suitable monsoon conditions ( per abbott and roundy 2003 ) . 
 wetting events that did not result in germination were assumed to cause no injury to these warm - season perennial grass seeds ( roundy et al . 
 germinating conditions that occurred before 15 may were not considered because this was before we assumed planting to occur . 
 germinating conditions that occurred after 30 september were assumed to result in seeding failure due to the lack of suitable growing conditions ( cool weather ) in october . 
 two scenarios were evaluated for each of the rain gages2  days of suitable conditions resulting in germination and 3  days of suitable conditions resulting in germination . 
 plants were counted as germinated if the water volume in the 02.5  cm layer stayed above 0.09  cm / cm for 2 or 3  days ( analyzed separately ) . 
 an intermediate value for water volume ( roughly twice the wilting point , 0.046  cm / cm , laio et al . 
 2001 ) was chosen because the 02.5-cm layer dries quickly , but seeds can germinate if the relative humidity remains high at the soil surface ( wuest 2007 ) . 
 ( 1997 ) that the surface soil becomes too dry within 1  day or less of the cessation of a storm . 
 the 2-day scenario was a more cautious interpretation of the available data on seed germination . 
 most warm - season perennial grasses germinate within 1.5 to 4  days of saturation of the surface soil at summer temperatures ( roundy and biedenbender 1996 ) . 
 the 2-day scenario supported native warm - season grass species like bouteloua curtipendula ( sideoats grama ) which germinated and emerged within 2  days of suitable conditions50  % in 1  day ( simanton and jordan 1986 ) . 
 another study observed emergence of b. curtipendula beginning in 18  h and ending after 3  days ( frasier et al . 
 while different authors have tested germination in different ways , most typically , the reported times were for saturated soil and our standard was dryer than that . 
 our 2-day germination scenario typically corresponded to one large or two medium - sized events . 
 the 3-day scenario was a more lenient estimate of the time needed for germination and corresponds more closely to available data on native warm - season grass germination . 
 ( 2000 ) found digitaria californica ( arizona cottontop ) required a minimum of 140  mm of water in a 3-day period for 50  % emergence . 
 wilson and briske ( 1979 ) found bouteloua gracilis ( blue grama ) needed wet conditions for 24  days to germinate . 
 after 2.3  days in wet field conditions , abbott and roundy ( 2003 ) found b. curtipendula had 90  % germination ; leptochloa dubia ( green sprangletop ) had 75  % ; d. californica had 70  % , and eragrostis intermedia ( plains lovegrass ) had 45  % . 
 die when seminal root growth does not keep up with the drying front in the soil which makes soil moisture the primary controlling factor for seedling mortality ( carren et al . 
 1987 ; zimmerman et al . 2008 ; perring and hovenden 2012 ; thapa et al . 
 this process was important for 23  weeks after germination when 24  days of additional moisture was needed at the soil surface for development of the plant crown and adventitious roots ( carren et al . 
 the drying front was defined as the wilting point ( 0.046  cm / cm ) for a loamy sand ( laio et al . 
 the rate of 0.8  cm root elongation per day was chosen as a conservative estimate based on several studies of native warm - season perennial grasses . for b. gracilis , 
 the seminal root grew 0.79  cm per day for the first 2  weeks ( carren et al . 
 b. curtipendula averaged about 0.8  cm per day of seminal root elongation during the first 15  days ( roundy et al . 
 simanton and jordan ( 1986 ) found an elongation rate of 0.97  cm per day for the first 7  days for b. curtipendula . using the modeled soil moisture , 
 if sufficient water were available , plants may have initiated adventitious roots during our 19-day evaluation , but we evaluated soil moisture for the whole soil column and dryness below the wilting point would have affected adventitious roots as well as seminal roots . in our assessment , we checked the simulated water content of the soil column from the surface to the depth of the seminal root , and if the whole column was below the wilting point , the seeding was considered to have failed . 
 the average germination date for the gage 5 2-day scenario was 20 july ( table  1 ) , and for the 3-day scenario , it was 1 august ( table  2 ) . the average germination dates for gage 6 2-day scenario was 19 july ( table  3 ) , and for the 3-day scenario , it was 27 july ( table  4 ) . in most years , there was little difference in germination date and success among the gages and the scenarios , but in a few years there were substantive differences among the gages and treatment germination dates ( fig .  1 ) . in 2001 , 
 the expected germination in the 3-day evaluation of gage 5 was 111  days later than the 2-day evaluation of gage 6.table 1evaluation of plants taking 2  days to germinate at the gage 5 siteminimum soil moisture cm / cm during root elongation through zonesuccess / failureyeargermination datesurface to 2.5  cm 
 2.5 to 5.0  cm 

 5.0 to 10.0  cm 

 10.0 to 15.0  cm 
 198014 aug0.0510.0490.0530.055198111 jul0.1190.1180.0820.084failed 
 19828 jul0.048 
 0.045 
 0.0580.099198312 jul0.0670.0530.0550.049198430 jun0.0640.0810.0730.078198521 jul0.0730.0600.0750.083failed 
 198617 jul0.0600.057 
 0.041 

 0.043 
 failed 
 198722 may0.0580.052 
 0.039 

 0.037 
 failed 
 198811 jul 
 0.044 

 0.040 
 0.0520.107198928 jul0.0850.0760.0830.08319909 jul0.1290.0950.1330.136failed 
 199131 jul 
 0.035 

 0.033 

 0.041 
 0.067failed 
 19929 jul0.0880.065 
 0.044 
 0.05419934 aug0.1410.1130.0720.08219944 sep0.0600.0500.0890.076failed 
 199522 aug 
 0.046 
 0.049 
 0.039 

 0.040 
 19964 jul0.0720.0780.0940.070199719 aug0.0530.0640.0710.08019986 jul0.1290.0980.0720.085199927 jul0.0900.0670.0650.074200021 jun0.0590.0900.0910.090failed 
 20015 aug 
 0.034 
 0.0640.0680.068failed 
 200218 jul 
 0.045 
 0.0520.1030.087200327 jul0.0990.1110.0820.082failed 
 200415 jul 
 0.036 
 0.0470.0580.08920056 aug0.1160.0910.1010.082200628 jul0.0940.0940.0790.069200721 jul0.0640.0660.0760.089200813 jul0.0510.0470.0670.071failed 
 2009no germ201031 jul0.0750.0560.0540.054numbers at or below the wilting point ( 0.046  cm / cm ) for this soil type are in bold . 
 of the 31  years evaluated , 20  years had successful establishment in the first 19  days after germination 
 too little rain , hits wilting point at 2.5 to 5.0  cm 
 too little rain , hits wilting point at 5.0 to 10.0  cm 
 too little rain , hits wilting point at surface to 2.5  cm 
 no germinationtable 2evaluation of plants taking 3  days to germinate at the gage 5 siteminimum soil moisture cm / cm during root elongation through zonesuccess / failureyeargermination datesurface to 2.5  cm 
 2.5 to 5.0  cm 

 5.0 to 10.0  cm 

 10.0 to 15.0  cm 
 failed 
 198015 aug0.048 
 0.043 
 0.0530.051198112 jul0.1040.1210.0750.084198223 jul0.0680.0630.0610.058failed 
 198313 jul0.064 
 0.044 
 0.055 
 0.046 
 198419 jul0.0910.1030.0980.096198522 jul0.0700.0600.1000.077failed 
 198618 jul0.0470.057 
 0.039 

 0.043 
 19876 aug0.0660.0850.0680.072failed 
 198812 jul 
 0.035 

 0.040 
 0.0850.108198929 jul0.0740.0760.0830.078199010 jul0.1010.0950.1330.130199110 aug0.0550.0710.0660.058failed 
 199213 jul 
 0.046 

 0.042 

 0.044 
 0.06419935 aug0.1410.0970.0720.08619945 sep0.0490.0500.0810.076199530 sep0.0640.0620.0530.051failed 
 199616 jul 
 0.042 

 0.043 
 0.0480.051199716 sep0.0540.0620.0620.05219987 jul0.1050.0980.0720.125199928 jul0.0850.0670.0650.074200025 jun0.0550.0710.0770.075failed 
 200110 oct0.0570.051 
 0.041 

 0.039 
 200225 jul0.1060.0780.0750.085200328 jul0.0960.1230.0780.082failed 
 200417 aug0.049 
 0.044 

 0.044 

 0.039 
 20057 aug0.1030.0910.0920.082200629 jul0.0960.0830.0740.067200722 jul0.0560.0630.0810.083200822 jul0.0990.0710.0490.062failed 
 2009no germ20101 aug0.0570.0510.0490.054numbers at or below the wilting point ( 0.046  cm / cm ) for this soil type are in bold . 
 of the 31  years evaluated , 22  years had successful establishment in the first 19  days after germination 
 too little rain , hits wilting point at 2.5 to 5.0  cm 
 too little rain , hits wilting point at 5.0 to 10.0  cm 
 too little rain , hits wilting point at surface to 2.5  cm 
 germination too late in season 
 no germinationtable 3evaluation of plants taking 2  days to germinate at the gage 6 siteminimum soil moisture cm / cm during root elongation through zonesuccess / failureyeargermination datesurface to 2.5  cm 
 2.5 to 5.0  cm 

 5.0 to 10.0  cm 

 10.0 to 15.0  cm 
 198014 aug0.0550.0510.0550.057198111 jul0.1170.1220.0950.094failed 
 19828 jul0.048 
 0.044 
 0.0570.102198312 jul0.0650.0510.0530.048198430 jun0.0690.0840.0750.095198516 jul0.0630.1180.0810.102failed 
 198617 jul0.0570.054 
 0.042 

 0.043 
 failed 
 198722 may0.0530.048 
 0.037 

 0.036 
 failed 
 198811 jul0.047 
 0.042 
 0.0550.107198928 jul0.0830.0760.0830.067199010 jul0.1320.0980.1350.13719919 aug0.0710.0710.0630.063failed 
 199210 jul0.0900.064 
 0.044 
 0.05319934 aug0.1370.1130.0720.08419944 sep0.0570.0510.0890.079failed 
 199522 aug 
 0.041 

 0.044 

 0.036 

 0.040 
 19964 jul0.0600.0720.0920.067199719 aug0.0510.0620.0710.08019986 jul0.1250.0960.0720.082199927 jul0.0950.0670.0620.068200024 jun0.0660.0710.0840.081failed 
 200122 jun 
 0.031 

 0.035 

 0.030 

 0.034 
 failed 
 200218 jul 
 0.044 
 0.0510.1000.087200327 jul0.0950.1070.0800.077200430 jul0.0650.0700.0480.05620056 aug0.0920.0910.1010.083200628 jul0.0940.0940.0780.069200721 jul0.0640.0670.0760.089200813 jul0.0510.0470.0670.070failed 
 2009 
 no germ 
 201031 jul0.0750.0560.0540.054numbers at or below the wilting point ( 0.046  cm / cm ) for this soil type are in bold . 
 of the 31  years evaluated , 22  years had successful establishment in the first 19  days after germination 
 too little rain , hits wilting point at 2.5 to 5.0  cm 
 too little rain , hits wilting point at 5.0 to 10.0  cm 
 too little rain , hits wilting point at surface to 2.5  cm 
 no germination 
 also failed 5 may which is outside the planting windowtable 4evaluation of plants taking 3  days to germinate at the gage 6 siteminimum soil moisture cm / cm during root elongation through zonesuccess / failureyeargermination datesurface to 2.5  cm 
 2.5 to 5.0  cm 

 5.0 to 10.0  cm 

 10.0 to 15.0  cm 
 failed 
 198015 aug0.051 
 0.045 
 0.0550.054198112 jul0.1100.1220.0860.094198223 jul0.0770.0690.0670.064failed 
 198313 jul0.060 
 0.043 
 0.053 
 0.046 
 19841 jul0.0660.0770.0750.112198520 jul0.0990.0780.0810.085failed 
 198618 jul 
 0.045 
 0.054 
 0.039 

 0.043 
 19876 aug0.0600.0780.0660.069failed 
 198812 jul 
 0.037 

 0.042 
 0.0920.115198929 jul0.0750.0760.0830.063199010 jul0.1050.0980.1350.128199110 aug0.0550.0710.0610.060failed 
 199210 jul0.0750.054 
 0.044 
 0.08019935 aug0.1370.0970.0720.08319945 sep0.0480.0510.0820.079failed 
 199530 sep0.0650.0630.0560.057failed 
 199616 jul 
 0.038 

 0.040 

 0.046 
 0.051failed 
 199716 sep0.0520.0610.0620.05219987 jul0.1020.0960.0710.123199928 jul0.0850.0640.0620.068200025 jun0.0540.0710.0770.077200117 jul0.0660.0620.0610.051200225 jul0.0980.0740.0730.084200328 jul0.0910.1190.0770.077failed 
 200417 aug 
 0.044 

 0.041 

 0.042 

 0.038 
 20057 aug0.1030.0910.0920.083200629 jul0.0960.0830.0740.067200722 jul0.0560.0630.0810.083failed 
 200822 jul0.0990.0700.0490.062failed 
 2009 
 no germ 
 20101 aug0.0570.0510.0490.054numbers at or below the wilting point ( 0.046  cm / cm ) for this soil type are in bold . 
 of the 31  years evaluated , 23  years had successful establishment in the first 19  days after germination 
 too little rain , hits wilting point at 2.5 to 5.0  cm 
 too little rain , hits wilting point at 5.0 to 10.0  cm 
 too little rain , hits wilting point at surface to 2.5  cm 
 germinated too late in season for successful establishment 
 no germinationfig . 
 julian date 150 is 29 may , 200 is 18 july , and 250 is 6 sept evaluation of plants taking 2  days to germinate at the gage 5 site numbers at or below the wilting point ( 0.046  cm / cm ) for this soil type are in bold . of the 31  years 
 evaluated , 20  years had successful establishment in the first 19  days after germination 
 too little rain , hits wilting point at 2.5 to 5.0  cm 
 too little rain , hits wilting point at 5.0 to 10.0  cm 
 too little rain , hits wilting point at surface to 2.5  cm evaluation of plants taking 3  days to germinate at the gage 5 site numbers at or below the wilting point ( 0.046  cm / cm ) for this soil type are in bold . 
 of the 31  years evaluated , 22  years had successful establishment in the first 19  days after germination 
 too little rain , hits wilting point at 2.5 to 5.0  cm 
 too little rain , hits wilting point at 5.0 to 10.0  cm 
 too little rain , hits wilting point at surface to 2.5  cm 
 germination too late in season evaluation of plants taking 2  days to germinate at the gage 6 site numbers at or below the wilting point ( 0.046  cm / cm ) for this soil type are in bold . 
 of the 31  years evaluated , 22  years had successful establishment in the first 19  days after germination 
 too little rain , hits wilting point at 2.5 to 5.0  cm 
 too little rain , hits wilting point at 5.0 to 10.0  cm 
 too little rain , hits wilting point at surface to 2.5  cm 
 also failed 5 may which is outside the planting window evaluation of plants taking 3  days to germinate at the gage 6 site numbers at or below the wilting point ( 0.046  cm / cm ) for this soil type are in bold . of the 31  years evaluated , 23  years had successful establishment in the first 19  days after germination 
 too little rain , hits wilting point at 2.5 to 5.0  cm 
 too little rain , hits wilting point at 5.0 to 10.0  cm 
 too little rain , hits wilting point at surface to 2.5  cm 
 germinated too late in season for successful establishment graph of the germination dates by year from 1980 to 2010 . 
 julian date 150 is 29 may , 200 is 18 july , and 250 is 6 sept for the gage 5 two - day scenario ( table  1 ) , 35  % of the years failed to have successful establishment , and 29  % failed for the 3-day scenario ( table  2 ) . for the gage 6 two - day scenario ( table  3 ) , 29  % of the years failed , and 35  % failed for the 3-day scenario ( table  4 ) . 
 for gage 5 , there were 6  years where the seeding failed for both scenarios ( 19  % failure rate ) , and for gage 6 , there were only 4  years ( 13  % failure rate ) where it failed for both scenarios ( table  5 ) . 
 these same 4  years also failed for gage 5 , giving a failure rate common across both gages and both scenarios of 13  % .table 5the overlap ( consistency ) among establishment failure across the gages and germination scenariosgage 5 sitegage 6 site2-day3-day2-day3-dayfailures119911overlapgage 5 site2-day6963-day658gage 6 site2-day9543-day684average germination date20 
 jul1  aug19  jul27  jul the overlap ( consistency ) among establishment failure across the gages and germination scenarios the worst 10-year span was gage 5 for the 2-day scenario where between 1986 and 1995 , six of ten plantings would have failed ( table  1 ) . 
 the best 10-year span was gage 6 for the 3-day scenario where , between 1998 and 2007 , only one of ten was projected to fail ( table  4 ) . at most , 3  years in a row failed , and 6  years in a row were successful . 
 our modeling results showed six times more potential for seeding success ( 68  % ) than was expected from the 10  % estimate by cox and jordan ( 1983 ) for southern arizona . 
 while this may show substantial potential for reclamation in most years , there are numerous factors , such as germination response , root growth , and other sources of mortality , that bear further consideration . 
 the germination periods of 2 or 3  days which we assumed for analysis do not necessarily reflect the full scope of plant responses in real - world situations . 
 for instance , non - germinating rains ( not long enough in duration ) that cause more than 1  day of adequate soil moisture can reduce seed viability ( emmerich and hardegree 1996 ) . 
 but less than 1  day of soil moisture before the eventual germinating rain can prime seeds and allow them to germinate faster than unexposed seeds once germinating rains occur ( emmerich and hardegree 1996 ) . 
 in addition , a small amount of the native grass seed ( 12  % ) will not germinate until the second or third germinating rain ( abbott and roundy 2003 ) which is also not included in the model . 
 these factors were not included because not enough information was available to determine the expected average outcome , and the magnitude of the effect on reclamation success would likely be small . the period after germination is similarly more complex than our model represents , although we have incorporated plant death due to lack of rain after germination . 
 these deaths are similar to those observed by abbott and roundy ( 2003 ) who found two sowing dates in two separate years where the drying front exceeded seminal root depth for native perennial grasses . 
 the complexity comes in because their field data did not show 100  % mortality but instead had 0.6  % and 7.9  % survival . while this may not result in a stand of vegetation that allows one to claim reclamation success , even with sufficient water , survival rates of native perennial grasses are not near 100  % in arid and semi - arid areas . with 32  days of available 
 water ( the drying front did not exceed the seminal root depth ) , there was 28  % survival of b. curtipendula , 27  % survival of l. dubia , 10  % survival of d. californica , and 11  % survival of e. intermedia ( abbott and roundy 2003 ) . nor are low survival rates unique to arizona . in a study in wyoming 
 , chambers ( 2000 ) found an overall survival rate of 1  % for seeded plants . 
 pyke ( 1990 ) had less than 1  % on a study in utah , and james et al . 
 given the relatively low survival rates of these common native perennial grasses , small differences may be more important than they appear , although poor survival possibly can be managed in the field by increasing the seeding rate or changing seed sources to more drought - tolerant collections . 
 the bottleneck for revegetation appears to be primarily in the establishment phase which occurs in the weeks following germination . 
 this appears applicable across many different systems , including minnesota ( fay and shultz 2009 ) , utah ( pyke 1990 ) , and new mexico ( peters 2000 ) , although other studies from wyoming ( chambers 2000 ) and oregon ( james et al . 
 2011 ) found out that mortality primarily occurred between germination and emergence . in the period after germination , 
 elongation rate of the seminal root and adventitious root initiation are the important factors for survival . 
 b. curtipendula averaged about 0.8  cm per day of seminal root elongation , but the lengths varied 0.51.1  cm per day , depending on the amount and frequency of water with faster elongation associated with more water ( roundy et al . 
 1993 ) . for b. curtipendula , adventitious roots initiated on day 9 when watered every 3  days compared with day 13 when only watered on days 1 and 7 ( roundy et al . 
 this would trigger the need for 24  days of available surface moisture to foster adventitious root elongation . 
 b. gracilis is similar in that it needs wet conditions 28  weeks after germination to allow adventitious root establishment ( frasier et al . 
 the variability of root elongation rates , the lack of studies on the non - genetic causes of rate differences , and inherent real - world variability in soil conditions make it difficult to know how they would change the outcome of our analysis . 
 ( 2008 ) found that the primary driver of seedling survivorship in a climate change scenario was soil moisture and that warming and co2 increases only affected survivorship indirectly through changing soil moisture . 
 ( 2012 ) predicted that southern arizona will have warmer and dryer weather in the future but that much of this will be decreased winter precipitation which may have a limited impact on the dominant warm - season ( c4 ) perennial grasses such as those modeled in this study . 
 consistent with perring and hovenden ( 2012 ) , the total annual rainfall appears only slightly correlated with our predicted seeding success or failure . 
 a logistic regression analysis showed that total seasonal precipitation was a significant predictor of germination success ( p  =  0.027 ) , but the analysis accounted for little of the variability ( nagelkerke r  =  0.059 ) . 
 the 6  years with the lowest rainfall ( below 300  mm compared with a 400  mm average ) had a 50  % failure rate which is more than the average rate of 32  % . 
 the six years with the highest observed average rainfall ( above 465  mm ) had a 33  % failure rate . 
 the distribution of rainfall events is clearly a variable that benefits seeds or seedlings , but larger event sizes during the growing season might not increase establishment . 
 once a storm event saturates the soil , a higher intensity event may not benefit seedling establishment because the seedlings are unable access moisture which has infiltrated deeper in the soil column and the excess water is lost as runoff . 
 plant establishment is sensitive to small changes in rainfall timing which is not well specified in predictions of future rainfall . 
 our observation of the common presence of events conducive to seeding success across the range of annual rainfall values makes it appear that warm - season perennial grasses may not be at risk from the kinds of changes predicted by notaro et al . 
 the overall management implication for this study is that funds for reseeding must be available for at least a second year . 
 this will allow reseeding the following year if there is a reclamation failure the year before . 
 further funds / reseeding effort may be needed because seeding failure 3  years in a row appears possible . a variety of species in a diverse mix , 
 while often recommended for creation of a stable and self - sustaining plant community , may also help reduce the risk of seeding failure in years with marginally poor rainfall distribution because different species have different water and time requirements for germination . 
 field experiments would verify the germination and establishment modeling results and allow better predictions of seeding success .","of the operations required for reclamation in arid and semi - arid regions , establishing vegetation entails the most uncertainty due to reliance on unpredictable rainfall for seed germination and seedling establishment . 
 the frequency of successful vegetation establishment was estimated based on a land surface model driven by hourly atmospheric forcing data , 7  years of eddy - flux data , and 31  years of rainfall data at two adjacent sites in southern arizona , usa . two scenarios differing in the required imbibition time for successful germination were evaluated2 or 3  days availability of sufficient surface moisture . 
 establishment success was assumed to occur if plants could germinate and if the drying front in the soil did not overtake the growth of seminal roots . 
 based on our results , vegetation establishment could be expected to fail in 32  % of years . in the worst 10-year span 
 , six of ten plantings would have failed . in the best 10-year span , 
 only one of ten was projected to fail . across all assessments , at most 3  years in a row failed and 6  years in a row were successful . 
 funding for reclamation seeding must be available to allow reseeding the following year if sufficient amount and timing of rainfall does not occur .",pubmed
"major depression ( md ) is the main cause of disability and the fourth - leading contributor to the global burden of disease . by the year 2020 
 , md is projected to reach second place in the ranking of disability - adjusted life years . 
 trials of available antidepressant medications alone or combined with psychotherapies are effective for 60%80% of those affected with md.1 conversely , up to 40% of patients with md do not show satisfactory improvement attributable to multiple biopsychosocial factors . at its worst 
 , md can lead to suicide , and as a consequence about 850,000 lives are lost every year.2 treatment - resistant depression ( trd ) evades universal definition ; however , a poor response to two adequate ( optimal dosage and 612 weeks duration ) trials of two different classes of antidepressants has been proposed as its operational characterization.3 researchers have categorized trd in accordance to antidepressant trials : stage 0 , has not had a single adequate trial of medication ; stage 1 , failure of an adequate trial of one class of an antidepressant that is monotherapy ; stage 2 , failure of adequate trials of two distinctly different classes  that is , selective serotonin reuptake inhibitors ( ssris ) and tricyclic antidepressants  of antidepressant , involving two monotherapy trials ; stage 3 , stage 2 plus failure to respond to one augmentation strategy of lithium or thyroid augmentation of one of the monotherapies ; stage 4 , stage 3 plus a failure to a second augmentation strategy in terms of monoamine oxidase inhibitors ; and stage 5 , stage 4 plus failure of an adequate course of ect.4 there are other staging methods of trd.5 these staging methods help researchers and clinicians to understand trd patients and accordingly plan interventions for enhancing the response , remission rate , and quality of life 
 . however , trd continues to challenge mental health care providers despite the understanding of psychosocial and biological markers and psychopharmacology of mood disorders and also the availability of multiple therapeutic options including optimization , switching , and combination of antidepressants . 
 notably , currently there is an increasing interest in the utilization of several neuromodulation therapies ( nts ) in the management of patients with trd.6 this is because psychopharmacological therapy exposes the entire body to a potentially therapeutic substance in order to treat a relatively small region of the brain , whereas nts are designed to target specific brain circuits that are important in the pathogenesis of md . 
 additionally , nts are not systemic and , therefore , the side - effect profile is limited and different from medications , and there are minimal , if any , drug interactions.7 furthermore , evidence - based data has been emerging continuously about fda - approved and yet - to - be - approved nts in the trd population over the past decade . 
 multiple computer searches were conducted using pubmed , google scholar , quertle(r ) , and medline databases for the years 20002012 . 
 a number of keywords were used : treatment - resistant depression , treatment - refractory depression , partial - response depression , nonresponse depression , neuromodulation techniques , neurostimulation approaches , and somatic therapies . 
 these words were combined with modified electroconvulsive therapy ( mect ) , repetitive transcranial magnetic stimulation ( rtms ) , vagus nerve stimulation ( vns ) , magnetic seizure therapy ( mst ) , deep brain stimulation ( dbs ) , transcranial direct current stimulation , cranial electric stimulation ( ces ) , epidural cortical stimulation ( ecs ) , focused ultrasound ( fus ) , near - infrared light therapy ( nir ) , low - field magnetic stimulation ( lfms ) , and optogenetic stimulation ( os ) for a second round of computer searches . a third round of searches included words such as mechanisms , 
 brain areas involved , and outcomes combined with aforesaid therapies . as a corollary , relevant articles published in english - language peer - reviewed journals were retrieved . 
 only clinical trials , systematic reviews , and meta - analyses that addressed trd and nts were retained for extensive review and inclusion in this study . 
 some exceptions were made with regard to some unique case reports , open and controlled studies , and small and large case series describing usefulness of nts in patients with trd and md . 
 similarly , studies focusing on neurosurgical ablation approaches in trd populations were not considered for inclusion . 
 references of selected articles were also reviewed for identifying relevant trd trials , which were also included in this review . 
 nts for neuropsychiatric disorders including md are categorized into the following : ( 1 ) seizure therapies , including mect and mst , ( 2 ) noninvasive therapies , including rtms , tdcs , and ces , ( 3 ) neurosurgical approaches , including vns , ecs , and dbs , and ( 4 ) new approaches on the horizon , including fus , nir , lfms , and os.8 another category represents neurosurgical ablation therapies , including cingulotomy and limbic leucotomy used in trd . 
 such technical details as invasiveness , anesthesia needed , seizures induced , target related to deep brain structures , contactness , stimulation being focal or generalized and form of stimulation of each neuromodulation therapy are presented in table 1 . 
 there is an increasing focus on exploring biomarkers underlying the pathogenesis of mood disorders9 that help in the development of new drugs and nts . in several related studies , overactive subcallosal cingulate gyrus ( scg ) 
 glucose metabolism has been reported in md that is reduced with successful antidepressant therapies.10 interestingly , dbs is reported to modulate neural pathways linked with scg in relieving md.8,11,12 according to some studies , antidepressant effects were also found when dbs targeted ventral capsule / ventral striatum ( vc / vs ) in patients with severe obsessive - compulsive disorder ( ocd ) and md.13,14 in a study of single patients with dystonia suffering from depression , dbs of globus pallidus internus ( gpi ) showed improvement in dystonia but also showed antidepressant effects through modulation of mesolimbic dopamine pathways.15 in another study , also of single patients with tardive dyskinesia ( td ) and md , dbs brought about improvement in depressive mood.16 other studies have also reported improvement in both depression and td after dbs of the inferior thalamic peduncle ( itp ) , which modulates orbitofrontal cortex hyperactivity.17,18 bewernick and colleagues reported that dbs of the nucleus accumbens ( nac ) was associated with decreased ratings of depression and anxiety in trd patients.19 rush and colleagues20 noticed antidepressant effects when vns was used for epilepsy . 
 vns modulates neural pathways associated with mood regulation : the nucleus tractus solitaries , raphe nucleus , and locus ceruleus.21 in fact , the vns device stimulates left cervical vagus nerve containing afferent neurons tracking through the brain stem to cortical and subcortical networks.2023 furthermore , some neurobiological studies reported disruptions in right and left dorsolateral prefrontal cortex ( r / ldlpfc ) in mood disorders . 
 also , rtms of r / ldlpfc results in antidepressive effects coupled with increasing cerebral blood supply to this brain areas.2325 certainly , nts target more specific , localized regions in the brain , which are somehow dysfunctional in md . 
 it remains uncertain how the depression is relieved ; this is yet to be understood well , and hence basic neurobiological studies are needed . similarly with regard to ect 
 modified ect has been used extensively in psychotic depression , schizophrenia , mania , and other mental disorders . 
 it requires light anesthesia and is a recognized mode of treatment for trd.31,32 it remains the most effective therapy in trd patients with a response rate of 50%70% , though the strength of recommendation of ect is c.33,34 it targets nonspecific , broad regions of the cortex , and its mechanism of action is elusive . 
 notably , high post - ect relapse rate and safety profile are of great concern for trd patients and health providers as well . in a study of patients with nonpsychotic md that tested whether pre - ect medication resistance is associated with post - ect relapse rates , 
 it was observed that 34.6% of nonmedication - resistant patients who were not exposed to at least one antidepressant medication trial relapsed , while 50.0% of medication - resistant patients relapsed , a difference that was not statistically significant but clinically relevant.35 furthermore , in the first week after acute remission , 9.8% of patients not having at least one antidepressant medication trial met relapse criteria , while 31.4% of medication - resistant patients met relapse criteria , a difference that was statistically significant . it was concluded that md patients who have had at least one adequate antidepressant medication trial or no such trial before ect may be especially prone to early relapse after successful acute remission with mect.35 research is needed to develop strategies in order to prevent relapse following successful ect in md , which may be maintenance ect and a combination of pharmacotherapy and mect . 
 furthermore , it is also important to identify the predictors of nonresponse to mect . in a large sample of patients with trd , mect was effective in 66% of patients . 
 mect nonresponse was associated with bipolar subtype , mixed features , slightly less severe depressive symptoms , and longer duration of the depressive episode.36 in another study that aimed to investigate whether the clinical course of trd patients following a course of mect might be associated with changes of plasma brain - derived neurotrophic factor ( bdnf ) concentrations , it was shown that at baseline , plasma bdnf levels of patients were significantly lower than those of control subjects , and those after ect were significantly increased in parallel with the decrease of the hamilton depression rating scale ( hdrs ) total score . 
 only remitter patients who showed higher baseline bdnf levels than nonremitters reached normalized bdnf levels after mect . 
 these findings suggested the potential usefulness of baseline plasma bdnf levels as predictors of response to mect in trd patients.37 in an earlier study of 18 patients with trd , levels of bdnf and 3-methoxy-4-hydroxyphenylglycol but not homovanillic acid were increased following mect in responders , which suggested that dopamine and bdnf might be involved in the mechanism of action of mect.38 in a recent study of adolescents with trd , both continuation and maintenance of mect were useful and safe for selected adolescents with severe trd , and symptom remission was achieved without experiencing cognitive impairment;39 the latter is a surprising finding and needs replication studies . 
 interestingly , in another development , data support the use of ketamine as anesthetic agent prior to ect for increasing its antidepressant effect as compared to propofol . in a related study , 31 inpatients with 
 the hdrs was used to evaluate these patients before ect and after the completion of the second , fourth , sixth , and eighth ect sessions . 
 the hdrs scores improved earlier in the ketamine group , with decreases in hdrs scores that were significantly greater in the ketamine group . 
 the implication of this finding is that the symptoms of md might be alleviated rapidly if ketamine anesthesia is used in trd patients during ect.40 a retrospective evaluation of 5482 ect treatments in 455 patients with trd found therapeutic advantages in combination therapies versus ect . 
 a total of 18.2% of treatments were ect monotherapy , 8.87% were done with one antidepressant . 
 results revealed that seizure duration was unaffected by most antidepressants , but ssri caused a lengthened seizure activity . 
 postictal suppression was lower in mirtazapine and higher in ssri and snri - treated patients . 
 a significant enhancement of therapeutic effectiveness was seen in the patient group receiving tricyclics , ssri , or mirtazapine , with no serious adverse events . 
 baghai and colleagues suggested that controlled studies are necessary to investigate further the possible advantages of ect and pharmacotherapy combinations , especially the use of modern dual - acting antidepressants , which also have proven their efficacy in trd.41 although mect is effective in trd , it significantly produces transient confusion , anterograde amnesia , and retrograde amnesia . 
 therefore , scientists have focused attention on technological refinements in ect and also developing techniques that do not cause cognitive impairment and at the same time remain effective in md and trd.4246 
 the fda has approved rtms for the treatment of md and trd in adolescents and adults . 
 its other indications include chronic pain , movement disorders , stroke , epilepsy , tinnitus , and other psychiatric disorders . 
 notably , rtms is safer on long - term use and acts more selectively than mect on brain areas implicated in the pathogenesis of md.47,48 the rtms has two forms : high - frequency rapid ( hfr ) ( > 1 hz ) and low - frequency slow ( lfs ) ( 1 hz ) . 
 furthermore , hfr rtms is preferred over lfs sychronized tms , as the former was associated with more antidepressant effects in depressed patients as reflected by significant increases in blood supply to prefrontal cortical and limbic regions.23 a sequential bilateral rtms ( lf right [ lfr ] then hf left [ hfl ] ) is also effective in trd patients but not more effective than unilateral hfl rtms.49,50 in an open - label study , 21 patients who failed two antidepressant trials were given rtms ( hf , 10 hz and intensity of 110% ) for 4 weeks , keeping the dose of preexisting antidepressants unchanged . 
 the majority of patients ( n = 19 ) completed the 4-week study and were assessed . in intention - to - 
 treat analysis , the mean hdrs scores were reduced from 30.80  5.00 to 19.00  6.37 . 
 no patient discontinued rtms due to adverse effects , including headache , which was reported by 16% of patients . 
 the study indicated the potential utility of rtms as an augmenting agent in trd.51 like lfr then hfl sequential bilateral rtms , hfl and lfr unilateral rtms are also efficacious in trd . in a 6-week double - blind , 
 randomized , sham - controlled trial in 50 patients with trd , three trains of lf rtms to the right prefrontal cortex of 140 seconds duration at 1 hz were applied daily , followed immediately by 15 trains of 5 seconds duration of hfl rtms at 10 hz . 
 according to this study , there was a significantly greater response to active than sham stimulation at 2 weeks and across the full duration of the study . 
 a significant proportion of the study group receiving active treatment met response ( 44% ) or remission ( 36% ) criteria by study end compared to the sham stimulation group ( 8% ) , and none remitted ( 0% ) . 
 it was noted that sequentially applying both hfl rtms and lfr rtms to the right prefrontal cortex resulted in substantial improvement in patients with trd . 
 furthermore , the treatment response accumulated to a clinically meaningful level over 46 weeks of active treatment.23 in another controlled investigation , patients with trd were randomized to receive 15 sessions of active or sham rtms delivered to the ldlpfc at 110% the estimated prefrontal cortex threshold . 
 the results showed response rate ( 50% decrease in hdrs score ) for the rtms group was 30.6% , significantly greater than the 6.1% rate in the sham group . 
 the remission rate ( an hdrs score < 8) for the rtms group was 20% , significantly greater than the 3% rate in the sham group . 
 the authors concluded that rtms to ldlpfc can produce statistically and clinically significant antidepressant effects in patients with trd.25 in another study , subjects between the ages of 18 and 85 years were recruited from a tertiary care university hospital . 
 seventy - four subjects with trd and an hdrs score > 21 were randomized to receive unilateral , bilateral , or sham rtms . according to this study , 
 the remission rate was significantly higher in the bilateral group than the sham group , but the remission rate in the unilateral group did not differ from either group . 
 these findings warrant larger controlled studies that compare the efficacy of sequential bilateral rtms and hfl / lfr rtms in md and trd.50,52 from a safety perspective , rtms can rarely induce accidental seizures , especially among patients with brain insult and on medications that reduce seizure threshold . however , this major side effect could be curtailed if expert guidelines are followed.53,54 over the past 10 years , a number of meta - analyses of rtms efficacy studies were conducted and the summary of these studies is as follows : a minimum of five to a maximum of 33 studies included ; almost all included studies except one focused on depression rather than trd ; rtms was more effective than sham rtms ; quality of studies improved successively ; and rtms designs also improved and effect size of rtms was comparable to antidepressant drugs.5560 finally , moreines and colleagues61 have reviewed the neuropsychological effects of somatic therapies including rtms that were associated with reversible mild reductions in sustained attention , spatial planning , and verbal retention . 
 the fda approved the use of vns in patients with md and trd in 2005.62,63 vns principally stimulates the left cervical vagus nerve with a programmable neurostimulator . 
 observations of mood elevation during vns for resistant epilepsy have suggested its potential role in trd.21,22,64 vns targets the nucleus tractus solitarius , frontolimbic network , the locus ceruleus , and dorsal raphe nucleus , which regulate mood . 
 notably , initial studies on vns reported inconsistent findings regarding reduced metabolism and blood flow in targeted brain networks with no putative antidepressant mechanism.21,63,65 similarly , a multicenter study on vns found no significant reductions in depression scores for the experimental group as a whole , but antidepressant responses were observed among 40% of 30 recruited patients with trd.20 however , subsequent studies on vns reported positive results . in a naturalistic , 1-year , follow - up study of 30 trd patients who received vns , 
 the results were as follows : response rate of 40%46% was sustained and the remission rate significantly increased , from 17% to 29% with an additional 9 months of long - term vns . 
 it was concluded that long - term vns was associated with sustained benefit linked with good functional status.66 another naturalistic study with 2 years follow - up of 74 european patients with trd showed a significant reduction at all the three time points , ie , 3 , 12 , and 24 months of vns in the hdrs scores . 
 after 2 years , 53.1% of the patients responded well , and 38.9% fulfilled the remission criteria . 
 the proportion of patients with remission remained constant as the duration of vns increased , with no concomitant antidepressant medication significant impact . 
 this 2-year open - label trial of vns suggested a clinical response and a benign adverse - effect profile among patients with trd.67 in a recent study of 15 consecutive outpatients with trd , vns significantly decreased beck depression inventory ( bdi ) scores compared to baseline at 6 and 12 months , from a mean of 37.8  7.8 before vns activation to a mean of 24.6  11.4 at 12 months . by 1 year , 28.6% of patients responded to vns and 7.1% remitted . 
 hdrs showed similar improvement at 1 year , with a 43% response rate and 14.3% remission rate . reported side effects of vns in decreasing frequency were hoarseness , dyspnea , nausea , pain , and anxiety , and no patient terminated treatment due to side effects . according to this study , a substantial minority of patients with trd benefited from vns.68 vns 
 but it has no adverse neuropsychological effects.61 in a study of single patients , vns produced good results , with cost savings over mect.69 according to a systematic review , vns examined in four clinical trials with 355 patients demonstrated steadily increasing improvement with full benefit after 612 months , sustained up to 2 years . 
 but the primary results of the only controlled trial were negative and attributed to small sample size . 
 further controlled studies with large sample size are warranted to establish its efficacy and tolerability in future.62,70 the issue of predictors of response to vns is addressed sparsely . in an open - label study of trd , 
 the predictors of response to vns were history of resistant depression , mild to moderate resistant depression , not - severe resistant depression , and no history of use of ect.71 trials of vns in combination with pharmacotherapy are also needed in trd populations . 
 transcranial direct current stimulation , a noninvasive technique with no fda approval , has been used in patients with md with mixed results . 
 tdcs of the prefrontal cortex has been proposed as a therapeutic intervention in md.72,73 in a parallel - group , double - blind clinical trial , 40 patients with md who were medication - free were randomized into three groups . 
 they were assessed by a blind rater using hdrs and bdi after ten sessions of tdcs during a 2-week period . according to this investigation , significantly larger reductions in depression scores after dlpfc tdcs 
 moreover , the beneficial effects of tdcs in the dlpfc group persisted for 1 month after the end of treatment . 
 the authors suggested further investigation on the effects of tdcs for the treatment of md.72 another double - blind , randomized study tested tdcs in 40 depressed participants and used the following parameters : 1-ma current strength , five treatment sessions , active or sham , and given on alternate days . 
 anodal stimulation was centered over the left dlpfc , with the cathode placed on the lateral aspect of the contralateral orbit . 
 overall , depression scores improved significantly over ten tdcs treatments , but there was no between - group difference in the five - session , sham - controlled phase . according to this study,73 tdcs was found to be safe , with no adverse effects on a variety of assessed neuropsychological functions.61 it was recommended that the efficacy of tdcs in md be further evaluated over a longer treatment period , using enhanced stimulation parameters.73 in another study , 22 patients with trd were randomly assigned to a crossover protocol comparing tdcs and placebo stimulation add - on to a stable antidepressant medication . 
 the parameters of active tdcs were 1 or 2 ma for 20 minutes / day , anode over the left dlpfc , and cathode over the contralateral supraorbital region . 
 the results showed that there was no significant difference in depression scores after 2 weeks of real compared with 2 weeks of sham tdcs . 
 in contrast , subjective mood ratings showed an increase in positive emotions after real tdcs compared with sham tdcs . 
 anodal tdcs , applied for 2 weeks , was not superior to placebo stimulation in patients with trd . 
 the authors suggested that modified and improved tdcs protocols should be carried out in controlled trials to develop tdcs with better efficacy in trd.74 all aforementioned studies except one74 addressed the usefulness of tdcs in md , and hence more controlled trials are needed in trd patients . 
 deep brain stimulation , yet to be approved by the fda , is a reversible invasive technique that involves stereotactical implantation of electrodes powered by a pulse generator into the specific dysfunctional brain regions implicated in mood disorders , parkinson s disease , alzheimer s disease , movement disorders , and other neuropsychiatric disorders . 
 high frequency dbs of motor , mood , and cognitive neuronal circuits is reported to improve these conditions.75 dbs therapy , dose- and site - dependent , is a less invasive and less extreme alternative to ablative psychosurgeries.76 research data supports dbs that targets cortico - striatal - pallido - thalamocortical loop , the vc / vs , and other neuronal networks in patients with md , trd , ocd , and tourette s syndrome.7781 additionally , nac that contains dopamine , a reward system and involved in the pathogenesis of md , is a promising target for dbs . in a study , 
 twelve months later , five patients reached 50% reduction of the hdrs score , with significantly increased pleasure activities . 
 furthermore , the [ f]-2-fluoro-2-deoxy - d - glucose positron emission tomography data revealed that dbs decreased metabolism in the scg , orbital prefrontal cortex , and amygdala . this study supported antidepressant and antianhedonic effects of dbs in patients with trd . 
 however , the small sample size limits the interpretation of results , and further research recruiting larger samples is needed.82 in a multicenter study of 21 trd patients who received dbs , it was found that patients treated with scg dbs had variable response with time : 57% at 1 month , 48% at 6 months , and 29% at 12 months . 
 the response rate after 12 months of dbs increased to 62% when redefined as a reduction in the baseline hrsd of 40% or more . 
 additionally , reductions in depressive symptoms were associated with amelioration in disease severity in patients who responded to surgery . 
 overall , this study corroborated the results of other research that the outcome of scg dbs may be replicated across multiple centers.83 in two influential review articles , researchers have provided greater details of somatic treatments in terms of target structures , motivation , response rates , mechanism of action , and technical issues.8,9 accordingly , somatic therapies targeted scg , vc / vs , left cervical vagus nerve , r / l dlpfc , gpi , lateral habenula , and itp in md and trd patients , and improvement reported ranged from 30.6% to 66.7%.8,10,12,14 ( table 2 ) . 
 furthermore , an improvement of 100% was reported in two dbs studies that included one patient with dystonia and trd and another patient with md and tardive dyskinesia.16,17 on a long - term basis ( 6 years ) , dbs is safe and effective in patients with trd , as substantiated by recent data.8789 according to these studies,8789 chronic dbs scg was effective in trd and bipolar patients and well tolerated with minor hemorrhagic events,86,90,91 but no neurocognitive impairment was reported61 ( table 3 ) . as a mechanism of action , overactive scg glucose metabolism seen in md is reduced with antidepressant therapies and dbs.10,11 
 magnetic seizure therapy , also known as magnetic convulsion therapy and yet to be approved by the fda , has antidepressant effects . 
 studies conducted in humans and primates suggest that cognitive side effects of mst are more benign than those of mect . 
 notably , postictal orientation recovery time is short and rapid with mst.61,92,93 furthermore , several studies have corroborated improved cognitive outcomes with mst as compared to mect . however , neither therapy causes structural changes , ie , volume , total number , or numerical density in neurons or glia in the frontal cortex , hippocampus , and their subregions in human and nonhuman brain.9496 overall , magnetic seizures with benign side - effect profile are therapeutically better than mect seizures . 
 other than adverse neurocognitive effects , ect is also associated with reversible bradycardia and tachycardia immediate post - ect and ictal and postictal stages , respectively . in nonhuman studies of mst , 
 these effects were minimal , reflecting a more superficial cortical site of action with less impact on deep brain structures , which are implicated in sympathetic and parasympathetic nervous system control , relative to ect.97 both antidepressant activity and cognitive side - effect profile of mst were further addressed in an open - label study , which tested whether it is associated with clinically significant antidepressant effects in trd as an add - on therapy to controlled pharmacotherapy.85 twenty patients with trd were randomly assigned to receive either mst or ect for more than 2 years . 
 the primary outcome measure was antidepressant response assessed by madrs , and secondary outcome measures included hdrs , hamilton anxiety scale , bdi , and 90-item symptom checklist . 
 antidepressant response as defined by 50% improvement in madrs ratings was statistically significant and of similar size in both treatment groups with no cognitive side effects . 
 characteristics in mst- and ect - induced seizures were comparable , especially regarding ictal activity and postictal suppression . 
 kayser and colleagues suggested that mst may be a potential alternative to ect if efficacy and safety are validated in larger clinical trials.85 mst is reported to result in minimal retrograde and anterograde amnesia.61 in summary , more studies are needed to further substantiate the efficacy of mst in mood disorder , including trd patients . 
 notably , there is converging evidence that nts have a lower risk of neurocognitive side effects compared to mect , which are benign.61 ( table 4 ) . by and large , 
 short- and long - term research is needed to establish the efficacy , safety , and cost - effectiveness of neurostimulation therapies.98 in addition , these therapies in general need proper selection of patients in line with tailored treatment guidelines.99 also , treatment teams should strictly follow ethical guidelines , especially those concerning autonomy , voluntary consent , beneficence , and nonmaleficence prior to using nts in individual patients.48,80,100 there are other nts , including ces and ecs , used uncommonly for a variety of disorders , such as anxiety , headaches , pain , stroke recovery , movement disorders , insomnia , and depression , but the data are largely limited in trd patients.101,102 in a systematic review , rosa and lisanby have described the technical details of all nts , including indications , safety , and effectiveness of ecs and ces.8 at the neurophysiological level , ces is quite different from tdcs.103 in one study , with ecs that used prefrontal cortical modulation , an average 55% improvement in depression scores was demonstrated.104 ces is associated with headache and nausea followed by skin irritation.105 unlike dbs , epidural cortical stimulation has fewer side effects.8 
 there are other neuromodulation therapies on the horizon , which include fus , lfms , and nir.106109 the data about these approaches are limited and need further research , especially concerning their role in mood disorders , including trd populations . with regard to os 
 , microbial light - sensitive proteins called opsins are introduced into neurons and function as ion channels that open or close according to light exposure . 
 channelrhodopsin-2 is one that allows na+ ions to enter the cell following exposure to ~470 nm blue light.110 according to rosa and lisanby,8 the advent of this technique has multiple implications : targeting specific fiber tracts that overlap in space ; selectively activating or inactivating specific projection neurons to the same target ; being a contactless form of stimulation relying on photoactivation ; and its potential use in treating mood disorders . like dbs 
 , os will also require surgical implantation of the light - emitting electrode ; however , os certainly has other advantages over dbs.8 in one nonhuman study , antidepressant effects of os of medial prefrontal cortex have already been reported in a chronic social defeat stress model in rodents.111 more studies on newer nts are needed in human subjects with md and trd . 
 this is a qualitative review of literature on somatic therapies used in the management of md and refractory depression . about 30% of patients with trd not responding to several intervention approaches , including optimization , augmentation and a combination of antidepressant drugs , are the principle candidates for nts.59 among these therapies , mect is most extensively and effectively used in severe depression and trd but associated with serious neurocognitive adverse effects because of nonspecific , broad excitation of cortical and deeper structures of the brain , and its mechanism of action is continuingly debatable.2633 other noninvasive somatic treatments such as rtms , tdcs , mst , and ces target more specific neuronal networks in the brain that are dysfunctional in md , trd , and other neuropsychiatric disorders , and reported to have fairly good safety and clinical profiles with more benign neuropsychological side effects.8,9,2325,4861,7274,85 invasive nts , ie , vns , dbs , and ecs with nonserious adverse effect profile , 
 are also reported to be effective in patients with md and trd.810,1222,61,104 new nts on the horizon are also promising in patients with md and trd . 
 although short- and long - term evidence - based comparative - effectiveness data on the role of nts in adult patients trd is emerging at a rapid pace,112 further research on their technical optimization , mechanisms of action , efficacy , side effect profile , and cost - effectiveness in larger populations of trd patients are warranted in future . 
 there is converging evidence that up to 40% patients with md fail to respond to an initial antidepressant therapy . 
 modified ect has a definite place in the management of patients with trd ; however , it carries well - known potential for neurocognitive impairment . like ect , mst also has neuropsychological adverse effects but of a milder nature . 
 the role of other neuromodulation methods , including vns , rtms , dbs , and tdcs , in trd patients is expanding with greater efficacy and fewer side effects . 
 these treatment modalities could be used alone or in combination with antidepressant therapy and/or psychotherapy . besides their therapeutic utility 
 , neuromodulation techniques can further open windows into the biological basis of disordered neurocircuits related to md and trd . 
 most studies on somatic therapies are of small sample size and hence reflect less reliable and valid results . therefore , collaborative 
 , multisite and/or multicountry studies that use the same protocols and also recruit larger samples with trd are urgently needed . 
 this methodological dilemma could be circumvented by determining a hypothesis a priori and others as exploratory . 
 most importantly , trd evades a universally accepted definition , and hence tools to measure refractoriness of depression and strict eligibility criteria need to be developed . 
 evidently , poor results of recent md and trd trials indicate the heterogeneous nature of depression and trd as well . therefore , treatment trials of somatic therapies should target more specific subpopulations together with the detection of endophenotypes to predict their response another challenge is blinding , which is vulnerable , and both the use of external raters and avoiding contact between subjects will solve this problem . additionally , open - label studies , especially of vns and dbs , tend to produce weak results , and therefore alternative designs including partial crossover and comparison against waiting list are needed . 
 there is a relative lack of follow - up studies on somatic therapies , and hence more naturalistic studies are required in future . 
 it is observed that the optimal parameters of somatic therapies are not defined , which could be managed by the use of adaptive designs and collaborative networks . 
 finally , unlike nonpharmacologic research in adults with trd,112 there is a relative lack of direct comparison with antidepressant drugs , and hence comparative research is needed . 
 most of these recommendations were constructed closely matching the challenges reported in the literature on nts , md , and trd populations.8,72,113","backgroundpatients with treatment - resistant depression ( trd ) who showed partial response to pharmacological and psychotherapeutic interventions need a trial of neuromodulation therapies ( nts).objectivethis paper aims to review evidence - based data on the use of nts in trd.methodusing keywords and combined - word strategy , multiple computer searches of pubmed , google scholar , quertle(r ) , and medline were conducted for retrieving relevant articles published in english - language peer - reviewed journals ( 20002012 ) . 
 those papers that addressed nts in trd were retained for extensive review.resultsdespite methodological challenges , a range of 30%93% of trd patients showed substantial improvement to one of the nts . 
 one hundred 
 percent improvement was reported in two single - case studies on deep brain stimulation . 
 some studies reported no benefits from transcranial direct current stimulation . 
 nts were reported to have good clinical efficacy , better safety margin , and benign side - effect profile . 
 data are limited regarding randomized clinical trials , long - term efficacy , and cost - effectiveness of these approaches . 
 both modified electroconvulsive therapy and magnetic seizure therapy were associated with reversible but disturbing neurocognitive adverse effects . besides clinical utility , nts including approaches on the horizon may unlock the biological basis underlying mood disorders including trd.conclusionnts are promising in patients with trd , as the majority of them show good clinical response measured by standardized depression scales . 
 nts need further technological refinements and optimization together with continuing well - designed studies that recruit larger numbers of participants with trd .",pubmed
"small rna molecules are important in the regulation of various molecular and biological activities in the cell , and it is now well known that short rna sequences play a critical role in regulating the expression levels of specific genes in a targeted manner . 
 rna interference ( rnai ) has become widely recognized to be an important gene regulatory mechanism that causes sequence - specific downregulation of mrnas . 
 acting through this mechanism , double - stranded short interfering rnas ( sirnas ) can knock down expression levels via the rna - induced silencing complex , which mediates degradation or translational inhibition of the targeted mrnas . 
 moreover , in addition to rna - induced silencing complex - mediated regulation at the post - transcriptional level , rnai can also modulate gene transcription itself . 
 in fission yeast , homologs of the rna - induced silencing complex can regulate chromatin through recruitment of histone - modifying proteins to loci transcribing small noncoding rna , a mechanism also seen in plants , ciliates , nematodes , and flies . 
 small promoter - targeted rnas have also been shown to repress transcription and induce epigenetic changes in eukaryotic cells through a mechanism called transcriptional gene silencing . in human cells 
 , it has recently been reported that short rnas targeted to the promoter regions of certain genes can activate expression at the transcriptional level . 
 this phenomenon has been called rna activation ( rnaa ) , and has been shown to be conserved in other mammalian species , including mouse , rat , and nonhuman primates . 
 promoter - targeted small hairpin rnas have also been shown to efficiently upregulate genes in vivo . while the mechanism is not completely understood 
 , it appears that naturally occurring antisense transcripts arising from or near the same genetic locus are able to direct recruitment of argonaute proteins and histone methyltransferases . 
 short - activating rnas ( sarna ) may regulate transcription by targeting these antisense transcripts for degradation , resulting in a reversal of this epigenetic silencing and upregulation of sense mrna . 
 based on this approach , we have developed a method to design sarnas for upregulation of specific cellular genes . in the present study , we focused on the feasibility and genetic consequences of upregulating important target genes involved in stem cell regulation and reprogramming . 
 combined expression of the transcription factors kruppel - like factor 4 ( klf4 ) , pou5f1 ( also called oct3/4 ) , sox2 , and c - myc has been shown to reprogram mouse and human fibroblasts into induced pluripotent stem ( ips ) cells . 
 in particular , klf4 is important for maintenance of embryonic stem cells , and has been reported to be a master regulator in embryonic stem cells that controls the expression of other pluripotency factors including pou5f1 , sox2 , c - myc , and nanog . 
 it has previously been reported that direct reprogramming can be achieved in murine fibroblasts with only klf4 , oct4 , and sox2 . 
 however , it has recently been shown that c - myc is critical for efficient induction of pluripotency in the early phases of reprogramming , by altering the metabolic state of the cell . accordingly , in the present study , we employed a genomic  bioinformatic approach to design sarnas that specifically target two of these key reprogramming genes , klf4 and myc . 
 these sarnas were tested for their ability to upregulate the targeted reprogramming factors , as well as their respective downstream genes , in human mesenchymal stem cells ( mscs ) , adult bone marrow - derived tissue - specific stem cells that already have multilineage differentiation potential . 
 the effects of sarna transfection on endogenous gene expression profiles were compared with those resulting from lentiviral vector - mediated overexpression of the exogenous klf4 and c - myc transgenes . to date , there have been no studies comprehensively examining the cellular gene expression profiles after sarna - mediated gene activation of endogenous genes , particularly pluripotency - related genes , and few studies comparing how different reprogramming methods might differentially affect various cellular pathways . 
 these results indicate that the use of sarna shows significant potential , both as a tool for studying stem cell biology , as well as a safe method to manipulate stem cell gene expression without altering the genome . 
 to design sarna candidates for activation of stem cell factors , we developed a novel bioinformatic approach . 
 the klf4 gene , located on chromosome 9 ( 9q31.2 ) , and c - myc gene , located on chromosome 8 ( 8q24.21 ) , were our initial targets for activation ( figure 1a , b ) . to identify potential antisense transcripts from the klf4 and c - myc loci , we searched the genomic region surrounding each locus for spliced expressed sequence tags ( ests ) which mapped to the positive strand . although it is usually difficult to determine the transcriptional orientation of ests , orientation can be determined by using splice site signatures of spliced ests . 
 we found no spliced ests that overlapped klf4 , but the scan identified one antisense est ( db461753 ) ~15  kb upstream of klf4 's annotated transcription start site ( tss ) . 
 we were also unable to find a spliced ests that overlapped c - myc , but identified one antisense est ( bc042052 ) ~2  kb upstream of c - myc 's tss . 
 recent deep sequencing experiments have revealed that antisense rnas often are found in the region surrounding tsss . 
 therefore , this region was chosen to design sarna sequences that targeted potential antisense transcripts from the promoter region . 
 we used the antisense sequence 500 nts upstream and downstream from the tss ( abbreviated klf4_as_tss+/500 and myc_as_tss+/500 ) as a second target candidate . 
 our goal was to design short sense rnas that could potentially bind and degrade antisense rnas generated from the two candidate sequences ( ests and the regions surrounding tsss ) with the hypothesis that this binding and degradation of the antisense rnas would activate gene expression . 
 candidate sarnas targeting the 500 nt upstream of the tss were designated pr1 , whereas those targeting the 500 nt downstream of the tss were designated pr2 . to give effective antisense targeting and degradation and to minimize off - target effects 
 , we used the gpboost sirna design algorithm to identify potential short rnas for downregulating the two candidate sequences . from the lists of predicted sirna candidates 
 , we selected the two most promising non - overlapping sirna target sites on the antisense est db461753 and bc042052 , and the most promising sirna target site on each side of the klf4 and c - myc tss within the antisense promoter sequence ( klf4_as_tss+/500 and myc_as_tss+/500 ) . 
 reasoning that activation of pluripotency factor gene expression might be more readily achieved in adult tissue - derived stem cells that retain restricted multilineage potential , we then tested whether these sarna candidates could upregulate klf4 or c - myc expression , respectively , in primary human mscs derived from adult bone marrow . 
 target gene expression levels were examined by quantitative pcr of reverse - transcribed mrna from msc cultures after transfection of each individual sarna candidate oligonucleotide , as compared with transfection with an alexa fluor 555- or fam - labeled negative control oligo . 
 a transfection efficiency of > 95% was determined by flow cytometry and by knockdown with control sirnas using the same transfection conditions ( see supplementary materials and methods and supplementary figures s1 and s2 ) . 
 initially , none of the sarna oligos appeared to show any effect on target gene expression after 48 hours . 
 however , upon continued transfection with klf4-pr1 sarna every other day , significant upregulation of klf4 mrna ( figure 2a ) was observed , on the order of 2.5-fold over controls by day 4 , and reaching approximately fourfold by day 6 of treatment ( p < 0.01 ) . 
 target gene mrna levels after treatment with klf4-pr1 were significantly higher when mscs were exposed to sarna at concentrations of 25 or 50  nmol / l , as compared with 5  nmol / l ( figure 2b ) . 
 increased klf4 protein was confirmed in mscs treated with klf4-pr1 sarna ( figure 2c , left panel ) by western blot analysis , and densitometric quantitation of these blots showed over threefold upregulation of klf4 protein relative to -actin internal control ( figure 2c , right panel ) , correlating closely with the level of mrna upregulation . 
 this time - dependent upregulation of klf4 gene expression by the klf4 promoter - targeted pr1 sarna was observed consistently over multiple experiments . 
 none of the other three klf4-targeted sarna candidate sequences was able to upregulate target gene expression , although conversely , klf4-db1 appeared to reduce klf4 mrna levels in mscs to about 60% that of scrambled oligo - treated controls by day 6 . 
 similarly , we found that among the four sarna candidate sequences targeted to the c - myc promoter and antisense ests , both myc - pr1 and myc - pr2 were able to induce consistent upregulation of myc mrna ( figure 2d ) by up to 1.8-fold ( p < 0.01 ) and 1.6-fold ( p < 0.01 ) , respectively , during the 6-day interval of treatment , as compared with scrambled sequence controls . 
 while myc - bc1 and myc - bc2 also appeared to affect myc mrna levels to some extent , these effects were more modest , and were not consistent between days 4 and 6 . 
 again , target gene mrna levels after treatment with myc - pr1 and myc - pr2 were significantly higher when mscs were exposed to sarna at concentrations of 25 or 50 
 nmol / l , as compared with 5  nmol / l ( figure 2e ) . 
 image densitometry analysis of western blots from mscs treated with myc - pr1 or myc - pr2 again confirmed upregulation of c - myc protein by over 2.5-fold relative to -actin internal control ( figure 2f ) . 
 all western blots were performed in triplicate with replicates shown in supplementary figure s3 . to determine whether this upregulation of klf4 and myc is due to true transcriptional activation , expression levels of nascent rna were assessed . 
 mscs were pulsed with ethynyl uridine ( eu ) during sarna treatment and total rna was isolated . 
 newly transcribed eu - rna was separated from total rna by biotinylation of eu in a copper - catalyzed  click  reaction , followed by purification on streptavidin magnetic beads . 
 quantitative pcr of reverse - transcribed nascent rna showed that the level of nascent klf4 mrna was significantly upregulated with klf4-pr1 treatment across a 6-day timecourse ( figure 3a ) . 
 the level of nascent myc mrna was significantly upregulated with myc - pr2 treatment , whereas no significant change in nascent myc mrna expression was seen with myc - pr1 treatment ( figure 3b ) . to verify that these sarnas do not act through interferon response pathways , 
 no significant upregulation of interferon response genes was detected 24 hours after sarna treatment ( figure 3c ) . 
 in contrast , overexpression of klf4 and c - myc by lentiviral transduction showed dose - dependent induction of interferon response genes ( supplementary figure s4 ) . to further elucidate the mechanism of activation by sarna , 
 the presence of promoter - associated antisense rnas was investigated using 5 rapid amplification of cdna ends ( race ) . 
 since any antisense rnas involved in regulating gene expression may not be polyadenylated , random hexamers were used to prime cdna synthesis from total msc rna . 
 antisense strand - specific primers matching the klf4-pr1 , myc - pr1 , and myc - pr2 sarna sequences were used for 5 race to amplify potential antisense rnas that are targeted by each sarna and may be involved in regulation of expression . 
 race reactions were run on an agarose gel , followed by purification , cloning , and sequencing of any products ( see supplementary materials and methods ) . despite the presence of bands from each sarna primer , 
 each was identified to be a known mrna with homology to the 3 end of the sarna sequence , likely a result of mispriming at permissive annealing temperatures ( supplementary figure s5 ) . 
 repeats of this assay with more restrictive annealing temperatures yielded no products ( data not shown ) . to further rule out involvement of possible antisense rnas arising downstream of each sarna target sequence , cdna was synthesized from total msc rna using antisense strand - specific primers for klf4-pr1 , myc - pr1 , and myc - pr2 . 
 this cdna was used as template for pcrs using primers downstream of the sarna target sequence , which would be in the direction of the 5 end of any putative antisense transcript . 
 no antisense transcripts were amplified up to 525  bp from the klf4-pr1 target site ( supplementary figure s6a ) . of the priming sites up to 667 
 bp from myc - pr1 , including and surpassing the myc - pr2 target site , two products were amplified , cloned , and sequenced corresponding to the 63 and 136  bp downstream of myc - pr1 ( supplementary figure s6b ) . 
 the expression level of this antisense transcript was significantly upregulated with myc - pr2 treatment , whereas its upregulation with myc - pr1 was not statistically significant ( p = 0.0682 ) ( supplementary figure s6c ) . to assess the biological significance of sarna activation of klf4 and myc in mscs 
 , cells were monitored for morphological changes during oligo treatment with klf4-pr1 and myc - pr2 , which had been confirmed to be associated with increased nascent mrna transcribed from their respective target genes . 
 after 6 days of treatment , klf4-pr1treated mscs showed marked differences in cell morphology compared with control , whereas myc - pr2 had modest changes ( figure 4 ) . 
 in contrast to scrambled sequence oligo - treated controls , which appeared as progressively more densely packed fibroblastic cells over time , klf4-pr1treated mscs were less confluent and predominantly arranged in clusters with epitheloid cell - like morphology . 
 myc - pr2treated cells were less confluent than controls but appeared more heterogeneous with numerous epitheloid cells containing enlarged nuclei . 
 thus , target gene mrna levels after treatment with klf - pr1 and myc - pr2 , were significantly higher when mscs were exposed to these sarna oligos at concentrations of 25 or 50  nmol / l over a 6-day treatment period . hence , these promoter - targeted sarna oligos consistently induced increases in both mrna and protein expression of klf4 and c - myc , respectively , in a time- and dose - dependent manner . 
 neither of these sarnas activated the interferon response , and both appear to act through specific activation of transcription . 
 we therefore focused on these two positive sarnas , using the 6-day treatment protocol , in further experiments examining how sarna - mediated upregulation might affect downstream targets . 
 microarray analysis was performed to determine the global gene expression profile and to investigate possible off - target effects with sarna treatment . 
 differential gene expression profiles after upregulation of endogenous klf4 and c - myc by treatment with their respective sarnas versus control oligo were examined , and compared with that after overexpression of exogenous klf4 and c - myc delivered by viral gene transfer using commercially available second - generation lentivirus vectors . 
 mscs were transduced with 0.1 pg p24 protein per cell as determined by p24 elisa , a viral dose that does not result in significant induction of interferon response genes ( supplementary figure s4 ) . 
 only those cellular genes showing upward or downward changes in their expression levels at a significance level of at least p < 0.1 , as compared with their levels in scrambled sequence oligo - treated controls , were included in these analyses . 
 interestingly , analysis of the overall gene expression profile in human mscs after treatment with klf4 pr-1 ( figure 5a ) and myc - pr2 ( figure 5b ) showed that the majority of cellular genes exhibited concordant changes in expression , but with some notable differences , between upregulation by sarna and overexpression by lentiviral transduction . for mscs treated with klf4-pr1 sarna , 
 68% of the cellular genes showing significant changes in their expression levels ( 971 out of 1,429 genes ) showed the same pattern of regulation as klf4 lentivirus - transduced mscs . for mscs treated with myc - pr2 , 64% ( 273 out of 429 genes ) exhibited the same pattern of regulation . 
 however , this indicates that roughly a one - third of the cellular genes with significant changes in expression after sarna transfection or lentiviral gene transfer showed discordant regulation between these two groups . to determine whether these differences were due to off - target effects or resulted from the different methods used to activate expression , we used metacore pathway analysis from genego ( carlsbad , ca ) to determine what pathways were significantly enriched in those genes that were differentially regulated between sarna and virus samples . 
 notably , the pathways that were most significantly differentially regulated between klf4-pr1 sarna - treated mscs and klf4 virus - treated mscs were those involved in cytoskeletal remodeling , macropinocytosis , and regulation of epithelial - to - mesenchymal transition ( emt ) , including transforming growth factor ( tgf)- induction of emt ( table 1 ) . the pathways that were most significantly differentially regulated between myc - pr2 sarna - treated mscs and the c - myc virus - treated mscs included cell survival and proliferation pathways such as granzyme a signaling , tgf- regulation , and telomere length , as well as macropinocytosis and emt pathways ( table 2 ) . 
 that is , for klf4-targeted sarna versus klf4-virus , as well as myc - targeted sarna versus myc - virus , in both cases genes involved in the same pathways were found to be discordantly regulated in the same manner . 
 the high degree of similarity in differentially regulated pathways observed with two different sets of sarnas versus two different lentivirus vectors suggests that the discordant gene expression patterns primarily arise due to characteristic cellular changes in response to oligo transfection versus viral transduction , rather than off - target effects that are shared by each set of sarnas . to determine whether narrowing the focus of the differential expression analysis to those types of genes we expect to be regulated by klf4 and c - myc would yield greater similarity , we generated lists of genes involved in stem cell maintenance , development , and proliferation , as well as cell cycle - related genes from the amigo gene ontology database . 
 lists of genes used in this analysis are included in supplementary tables s2 and s3 . 
 heatmaps visualizing the differential expression profiles of klf4 sarna - treated and klf4 virus - treated msc compared with scrambled oligo - treated control msc for stem cell - related genes ( figure 5c ) and cell cycle - related genes ( figure 5d ) showed much greater similarity , with 74% ( 26 out of 35 ) exhibiting concordant regulation for stem cell - related genes , and 80% ( 132 out of 165 ) exhibiting concordant regulation for cell cycle - related genes . 
 heatmaps focused on cell cycle - related genes in myc - pr2 ( figure 5e ) sarna - treated msc compared to myc virus - treated msc also showed a higher degree of similarity , with 67% of cell cycle - related genes ( 8 out of 12 ) for myc - pr2 exhibiting concordant regulation . 
 further david gene ontology ( go ) analysis of all genes revealed that the most significantly enriched go terms in the klf4-pr1 sarna - treated samples were the same as those in the klf4 virus - treated samples ( table 3 ) . 
 similarly , the majority of the most significantly enriched go terms in the myc - pr2 sarna - treated samples were also significantly enriched in the c - myc virus - treated samples ( table 4 ) . 
 to further validate the gene expression profile , we chose several well - known transcriptional gene targets of klf4 and c - myc to verify the results seen in the microarray data by real - time pcr . 
 we found that klf4-pr1 sarna transfection resulted in significantly increased expression of the klf4 target genes cyclin d1 , ornithine decarboxylase ( odc1 ) , p21 , and p53 ( figure 6a ) ; klf4 virus - transduced cells also showed significantly increased expression of cyclin d1 and p21 to a similar degree , although not of odc1 or p53 . similarly , myc - pr2 sarna transfection resulted in significantly increased expression of c - myc target genes odc1 , p21 , and p53 , as did transduction with c - myc virus ( figure 6b ) . 
 finally , to assess the ability of the klf4-pr1 sarna to activate the expression of other reprogramming factors , we analyzed mrna expression of oct4 , sox2 , and myc , as well as the stem cell marker nanog , by real - time pcr . 
 we found that klf4 activation by sarna was also able to activate expression of oct4 , sox2 , myc , and nanog ( figure 6c ) . 
 further analysis of oct4 mrna isoforms showed that oct4a was being significantly upregulated , whereas oct4b showed no difference ( figure 6d ) . 
 conversely , the ability of myc - pr2 sarna to activate endogenous klf4 gene expression was also analyzed . in response to myc - pr2 transfection 
 , there was an approximately twofold increase in klf4 mrna levels ( figure 6e ) . 
 notably , this effect of myc - pr2 sarna was corroborated by the microarray analysis ( supplementary table s1 ) . 
 to design sarna candidates for activation of stem cell factors , we developed a novel bioinformatic approach . 
 the klf4 gene , located on chromosome 9 ( 9q31.2 ) , and c - myc gene , located on chromosome 8 ( 8q24.21 ) , were our initial targets for activation ( figure 1a , b ) . to identify potential antisense transcripts from the klf4 and c - myc loci , we searched the genomic region surrounding each locus for spliced expressed sequence tags ( ests ) which mapped to the positive strand . although it is usually difficult to determine the transcriptional orientation of ests , orientation can be determined by using splice site signatures of spliced ests . 
 we found no spliced ests that overlapped klf4 , but the scan identified one antisense est ( db461753 ) ~15  kb upstream of klf4 's annotated transcription start site ( tss ) . 
 we were also unable to find a spliced ests that overlapped c - myc , but identified one antisense est ( bc042052 ) ~2  kb upstream of c - myc 's tss . 
 recent deep sequencing experiments have revealed that antisense rnas often are found in the region surrounding tsss . 
 therefore , this region was chosen to design sarna sequences that targeted potential antisense transcripts from the promoter region . 
 we used the antisense sequence 500 nts upstream and downstream from the tss ( abbreviated klf4_as_tss+/500 and myc_as_tss+/500 ) as a second target candidate . 
 our goal was to design short sense rnas that could potentially bind and degrade antisense rnas generated from the two candidate sequences ( ests and the regions surrounding tsss ) with the hypothesis that this binding and degradation of the antisense rnas would activate gene expression . 
 candidate sarnas targeting the 500 nt upstream of the tss were designated pr1 , whereas those targeting the 500 nt downstream of the tss were designated pr2 . to give effective antisense targeting and degradation and to minimize off - target effects 
 , we used the gpboost sirna design algorithm to identify potential short rnas for downregulating the two candidate sequences . from the lists of predicted sirna candidates 
 , we selected the two most promising non - overlapping sirna target sites on the antisense est db461753 and bc042052 , and the most promising sirna target site on each side of the klf4 and c - myc tss within the antisense promoter sequence ( klf4_as_tss+/500 and myc_as_tss+/500 ) . 
 reasoning that activation of pluripotency factor gene expression might be more readily achieved in adult tissue - derived stem cells that retain restricted multilineage potential , we then tested whether these sarna candidates could upregulate klf4 or c - myc expression , respectively , in primary human mscs derived from adult bone marrow . 
 target gene expression levels were examined by quantitative pcr of reverse - transcribed mrna from msc cultures after transfection of each individual sarna candidate oligonucleotide , as compared with transfection with an alexa fluor 555- or fam - labeled negative control oligo . 
 a transfection efficiency of > 95% was determined by flow cytometry and by knockdown with control sirnas using the same transfection conditions ( see supplementary materials and methods and supplementary figures s1 and s2 ) . initially , none of the sarna oligos appeared to show any effect on target gene expression after 48 hours . 
 however , upon continued transfection with klf4-pr1 sarna every other day , significant upregulation of klf4 mrna ( figure 2a ) was observed , on the order of 2.5-fold over controls by day 4 , and reaching approximately fourfold by day 6 of treatment ( p < 0.01 ) . 
 target gene mrna levels after treatment with klf4-pr1 were significantly higher when mscs were exposed to sarna at concentrations of 25 or 50  nmol / l , as compared with 5  nmol / l ( figure 2b ) . increased klf4 protein 
 was confirmed in mscs treated with klf4-pr1 sarna ( figure 2c , left panel ) by western blot analysis , and densitometric quantitation of these blots showed over threefold upregulation of klf4 protein relative to -actin internal control ( figure 2c , right panel ) , correlating closely with the level of mrna upregulation . 
 this time - dependent upregulation of klf4 gene expression by the klf4 promoter - targeted pr1 sarna was observed consistently over multiple experiments . 
 none of the other three klf4-targeted sarna candidate sequences was able to upregulate target gene expression , although conversely , klf4-db1 appeared to reduce klf4 mrna levels in mscs to about 60% that of scrambled oligo - treated controls by day 6 . 
 similarly , we found that among the four sarna candidate sequences targeted to the c - myc promoter and antisense ests , both myc - pr1 and myc - pr2 were able to induce consistent upregulation of myc mrna ( figure 2d ) by up to 1.8-fold ( p < 0.01 ) and 1.6-fold ( p < 0.01 ) , respectively , during the 6-day interval of treatment , as compared with scrambled sequence controls . while myc - bc1 and myc - bc2 also appeared to affect myc mrna levels to some extent , these effects were more modest , and were not consistent between days 4 and 6 . 
 again , target gene mrna levels after treatment with myc - pr1 and myc - pr2 were significantly higher when mscs were exposed to sarna at concentrations of 25 or 50  nmol / l , as compared with 5  nmol / l ( figure 2e ) . 
 image densitometry analysis of western blots from mscs treated with myc - pr1 or myc - pr2 again confirmed upregulation of c - myc protein by over 2.5-fold relative to -actin internal control ( figure 2f ) . 
 all western blots were performed in triplicate with replicates shown in supplementary figure s3 . to determine whether this upregulation of klf4 and myc is due to true transcriptional activation , expression levels of nascent rna were assessed . 
 mscs were pulsed with ethynyl uridine ( eu ) during sarna treatment and total rna was isolated . 
 newly transcribed eu - rna was separated from total rna by biotinylation of eu in a copper - catalyzed  click  reaction , followed by purification on streptavidin magnetic beads . 
 quantitative pcr of reverse - transcribed nascent rna showed that the level of nascent klf4 mrna was significantly upregulated with klf4-pr1 treatment across a 6-day timecourse ( figure 3a ) . 
 the level of nascent myc mrna was significantly upregulated with myc - pr2 treatment , whereas no significant change in nascent myc mrna expression was seen with myc - pr1 treatment ( figure 3b ) . to verify that these sarnas do not act through interferon response pathways , 
 no significant upregulation of interferon response genes was detected 24 hours after sarna treatment ( figure 3c ) . 
 in contrast , overexpression of klf4 and c - myc by lentiviral transduction showed dose - dependent induction of interferon response genes ( supplementary figure s4 ) . to further elucidate the mechanism of activation by sarna , 
 the presence of promoter - associated antisense rnas was investigated using 5 rapid amplification of cdna ends ( race ) . 
 since any antisense rnas involved in regulating gene expression may not be polyadenylated , random hexamers were used to prime cdna synthesis from total msc rna . 
 antisense strand - specific primers matching the klf4-pr1 , myc - pr1 , and myc - pr2 sarna sequences were used for 5 race to amplify potential antisense rnas that are targeted by each sarna and may be involved in regulation of expression . 
 race reactions were run on an agarose gel , followed by purification , cloning , and sequencing of any products ( see supplementary materials and methods ) . despite the presence of bands from each sarna primer , 
 each was identified to be a known mrna with homology to the 3 end of the sarna sequence , likely a result of mispriming at permissive annealing temperatures ( supplementary figure s5 ) . 
 repeats of this assay with more restrictive annealing temperatures yielded no products ( data not shown ) . to further rule out involvement of possible antisense rnas arising downstream of each sarna target sequence 
 , cdna was synthesized from total msc rna using antisense strand - specific primers for klf4-pr1 , myc - pr1 , and myc - pr2 . 
 this cdna was used as template for pcrs using primers downstream of the sarna target sequence , which would be in the direction of the 5 end of any putative antisense transcript . 
 no antisense transcripts were amplified up to 525  bp from the klf4-pr1 target site ( supplementary figure s6a ) . of the priming sites up to 667  bp from myc - pr1 , including and surpassing the myc - pr2 target site , 
 two products were amplified , cloned , and sequenced corresponding to the 63 and 136  bp downstream of myc - pr1 ( supplementary figure s6b ) . 
 the expression level of this antisense transcript was significantly upregulated with myc - pr2 treatment , whereas its upregulation with myc - pr1 was not statistically significant ( p = 0.0682 ) ( supplementary figure s6c ) . to assess the biological significance of sarna activation of klf4 and myc in mscs 
 , cells were monitored for morphological changes during oligo treatment with klf4-pr1 and myc - pr2 , which had been confirmed to be associated with increased nascent mrna transcribed from their respective target genes . 
 after 6 days of treatment , klf4-pr1treated mscs showed marked differences in cell morphology compared with control , whereas myc - pr2 had modest changes ( figure 4 ) . 
 in contrast to scrambled sequence oligo - treated controls , which appeared as progressively more densely packed fibroblastic cells over time , klf4-pr1treated mscs were less confluent and predominantly arranged in clusters with epitheloid cell - like morphology . 
 myc - pr2treated cells were less confluent than controls but appeared more heterogeneous with numerous epitheloid cells containing enlarged nuclei . thus , target gene mrna levels after treatment with klf - pr1 and myc - pr2 , were significantly higher when mscs were exposed to these sarna oligos at concentrations of 25 or 50  nmol / l over a 6-day treatment period . 
 hence , these promoter - targeted sarna oligos consistently induced increases in both mrna and protein expression of klf4 and c - myc , respectively , in a time- and dose - dependent manner . 
 neither of these sarnas activated the interferon response , and both appear to act through specific activation of transcription . 
 we therefore focused on these two positive sarnas , using the 6-day treatment protocol , in further experiments examining how sarna - mediated upregulation might affect downstream targets . 
 microarray analysis was performed to determine the global gene expression profile and to investigate possible off - target effects with sarna treatment . 
 differential gene expression profiles after upregulation of endogenous klf4 and c - myc by treatment with their respective sarnas versus control oligo were examined , and compared with that after overexpression of exogenous klf4 and c - myc delivered by viral gene transfer using commercially available second - generation lentivirus vectors . 
 mscs were transduced with 0.1 pg p24 protein per cell as determined by p24 elisa , a viral dose that does not result in significant induction of interferon response genes ( supplementary figure s4 ) . 
 only those cellular genes showing upward or downward changes in their expression levels at a significance level of at least p < 0.1 , as compared with their levels in scrambled sequence oligo - treated controls , were included in these analyses . 
 interestingly , analysis of the overall gene expression profile in human mscs after treatment with klf4 pr-1 ( figure 5a ) and myc - pr2 ( figure 5b ) showed that the majority of cellular genes exhibited concordant changes in expression , but with some notable differences , between upregulation by sarna and overexpression by lentiviral transduction . for mscs treated with klf4-pr1 sarna , 68% of the cellular genes showing significant changes in their expression levels ( 971 out of 1,429 genes ) showed the same pattern of regulation as klf4 lentivirus - transduced mscs . for mscs treated with myc - pr2 
 , 64% ( 273 out of 429 genes ) exhibited the same pattern of regulation . 
 however , this indicates that roughly a one - third of the cellular genes with significant changes in expression after sarna transfection or lentiviral gene transfer showed discordant regulation between these two groups . to determine whether these differences were due to off - target effects or resulted from the different methods used to activate expression , we used metacore pathway analysis from genego ( carlsbad , ca ) to determine what pathways were significantly enriched in those genes that were differentially regulated between sarna and virus samples . 
 notably , the pathways that were most significantly differentially regulated between klf4-pr1 sarna - treated mscs and klf4 virus - treated mscs were those involved in cytoskeletal remodeling , macropinocytosis , and regulation of epithelial - to - mesenchymal transition ( emt ) , including transforming growth factor ( tgf)- induction of emt ( table 1 ) . 
 the pathways that were most significantly differentially regulated between myc - pr2 sarna - treated mscs and the c - myc virus - treated mscs included cell survival and proliferation pathways such as granzyme a signaling , tgf- regulation , and telomere length , as well as macropinocytosis and emt pathways ( table 2 ) . 
 that is , for klf4-targeted sarna versus klf4-virus , as well as myc - targeted sarna versus myc - virus , in both cases genes involved in the same pathways were found to be discordantly regulated in the same manner . 
 the high degree of similarity in differentially regulated pathways observed with two different sets of sarnas versus two different lentivirus vectors suggests that the discordant gene expression patterns primarily arise due to characteristic cellular changes in response to oligo transfection versus viral transduction , rather than off - target effects that are shared by each set of sarnas . to determine whether narrowing the focus of the differential expression analysis to those types of genes we expect to be regulated by klf4 and c - myc would yield greater similarity , we generated lists of genes involved in stem cell maintenance , development , and proliferation , as well as cell cycle - related genes from the amigo gene ontology database . 
 lists of genes used in this analysis are included in supplementary tables s2 and s3 . 
 heatmaps visualizing the differential expression profiles of klf4 sarna - treated and klf4 virus - treated msc compared with scrambled oligo - treated control msc for stem cell - related genes ( figure 5c ) and cell cycle - related genes ( figure 5d ) showed much greater similarity , with 74% ( 26 out of 35 ) exhibiting concordant regulation for stem cell - related genes , and 80% ( 132 out of 165 ) exhibiting concordant regulation for cell cycle - related genes . 
 heatmaps focused on cell cycle - related genes in myc - pr2 ( figure 5e ) sarna - treated msc compared to myc virus - treated msc also showed a higher degree of similarity , with 67% of cell cycle - related genes ( 8 out of 12 ) for myc - pr2 exhibiting concordant regulation . 
 further david gene ontology ( go ) analysis of all genes revealed that the most significantly enriched go terms in the klf4-pr1 sarna - treated samples were the same as those in the klf4 virus - treated samples ( table 3 ) . 
 similarly , the majority of the most significantly enriched go terms in the myc - pr2 sarna - treated samples were also significantly enriched in the c - myc virus - treated samples ( table 4 ) . 
 to further validate the gene expression profile , we chose several well - known transcriptional gene targets of klf4 and c - myc to verify the results seen in the microarray data by real - time pcr . 
 we found that klf4-pr1 sarna transfection resulted in significantly increased expression of the klf4 target genes cyclin d1 , ornithine decarboxylase ( odc1 ) , p21 , and p53 ( figure 6a ) ; klf4 virus - transduced cells also showed significantly increased expression of cyclin d1 and p21 to a similar degree , although not of odc1 or p53 . 
 similarly , myc - pr2 sarna transfection resulted in significantly increased expression of c - myc target genes odc1 , p21 , and p53 , as did transduction with c - myc virus ( figure 6b ) . 
 finally , to assess the ability of the klf4-pr1 sarna to activate the expression of other reprogramming factors , we analyzed mrna expression of oct4 , sox2 , and myc , as well as the stem cell marker nanog , by real - time pcr . 
 we found that klf4 activation by sarna was also able to activate expression of oct4 , sox2 , myc , and nanog ( figure 6c ) . 
 further analysis of oct4 mrna isoforms showed that oct4a was being significantly upregulated , whereas oct4b showed no difference ( figure 6d ) . 
 conversely , the ability of myc - pr2 sarna to activate endogenous klf4 gene expression was also analyzed . in response to myc - pr2 transfection 
 , there was an approximately twofold increase in klf4 mrna levels ( figure 6e ) . 
 notably , this effect of myc - pr2 sarna was corroborated by the microarray analysis ( supplementary table s1 ) . 
 we have been able to identify and characterize two sarnas , klf4-pr1 and myc - pr2 , that specifically activate transcription of endogenous klf4 and myc genes , respectively , in primary human mscs . 
 interestingly , in both cases , it was a promoter - targeted sarna that gave the desired effect . given that studies implicate antisense rnas that overlap the gene of interest as the targets for degradation in rnaa , it is conceivable that these promoter - targeted sarnas are targeting an antisense rna that has not yet been discovered . 
 however , we were unable to identify any antisense transcripts arising from the region of the klf4-pr1 target site . 
 antisense transcripts have been reported to arise from the vicinity of the c - myc promoter in human cells such as prostate cancer cell lines , and we were able to identify one such antisense rna in primary human mscs . however , this antisense rna does not overlap the myc - pr2 target site and is therefore unlikely to be the rna target of myc - pr2 . 
 interestingly , this antisense rna does overlap the myc - pr1 target site , but cells transfected with myc - pr1 sarna did not show increased transcription of nascent myc mrna . 
 hence it is difficult to ascertain whether myc - pr1 does target this antisense rna , perhaps resulting in increased accumulation of myc sense mrna at a post - transcriptional level , or if the observed upregulation of myc mrna levels by myc - pr1 is an off - target effect . due to the relatively small activation of myc 
 , it would be difficult to completely rule out off - target effects even with the use of more sophisticated techniques such as chromatin immunoprecipitation analysis . 
 furthermore , specific sarna - targeted effects on myc expression by transcriptional or post - transcriptional mechanisms , and coexisting off - target effects , are not necessarily mutually exclusive . 
 although it may not be possible for every gene to be activated by rnaa , our results suggest that this method could be used to generate sarna candidates for activation of other endogenous genes for which a promoter - associated antisense rna has not yet been defined . focusing on stem cell- and cell cycle - related genes , which were expected to be altered by klf4 and c - myc expression 
 , we found the similarities between sarna - transfected and virus - transduced samples to be quite striking . 
 this was confirmed by go analysis , as the most significantly enriched terms were nearly identical in klf4-pr1 oligo and klf4 virus samples , and most of the top myc - pr2 terms were also significantly enriched in the c - myc virus samples . 
 in addition , several well - known klf4 and c - myc target genes were validated by real - time pcr , and showed a similar pattern of expression in the sarna - treated samples as in the virus - transduced samples . 
 furthermore , we have shown that klf4 sarna is able to activate expression of endogenous pluripotency factors including oct4 , sox2 , nanog , and myc . 
 activation of oct4 was specific to oct4a , which has been reported to be essential for stemness in human embryonic stem cells . 
 the highly specific nature of oct4a upregulation in response to transfection of sarna targeting klf4 makes this likely to be a true downstream event caused by specific activation of klf4 . 
 furthermore , the sarna - treated mscs showed marked differences in cell morphology , supporting our conclusion that this activation is biologically relevant . 
 interestingly , for some downstream gene targets , our results were different from what was expected , as klf4 is expected to downregulate expression of cyclin d1 , ornithine decarboxylase , and p53 , whereas c - myc is expected to downregulate p21 . 
 however , these previously observed results are often cell- and tissue type - dependent , as evidenced by more recent reports showing activation of cyclin d1 and p53 by klf4 , and activation of p21 through p53 by c - myc in different cell types . 
 in addition , our data are consistent across both sarna- and virus - treated samples in independent microarray and real - time pcr experiments . as with rnai , 
 a primary concern for using rnaa to study biological processes is the minimization of potential off - target effects . to evaluate possible off - target effects , we compared the gene expression profile of sarna activation of klf4 and myc to lentiviral - mediated expression . 
 some significant differences were observed in the total gene expression profile , but this is perhaps not surprising , as one method employed transfection of sarna and the other lentiviral transduction . 
 indeed , the use of any transduction method involving introduction of long stretches of exogenous nucleic acids , including both viral vectors as well as plasmids , will likely activate a variety of innate signaling mechanisms . 
 this may result in unwanted upregulation of interferons and related genes that may not only cause protein synthesis shutdown and effects on cell proliferation , but may also impair normal stem cell function , as has been described in hematopoietic stem cells . 
 notably , our interferon response gene expression analysis as well as our microarray analysis of gene functional annotation and differentially regulated pathways showed no evidence of interferon response upregulation after sarna oligo transfection . 
 in contrast , we observed significant induction of interferon responses upon viral transduction at a dose of 1 pg p24 per cell . 
 this corresponds roughly to an multiplicity of infection of 10 , which is within the range of multiplicity of infections typically used in reprogramming protocols . 
 hence , it may be highly advantageous that rna duplexes < 23  bp in length will not cause induction of interferon , while effecting transcriptional gene activation of endogenous pluripotency factors . 
 in fact , when we focused on the genes that were differentially expressed between the sarna and virus samples , the most significantly enriched pathways implicated the use of lentiviral vector - mediated gene transfer as the cause of many of the expression profile changes that were discrepant . notably , cytoskeletal remodeling , including tgf - mediated remodeling , were upregulated in both klf4 virus- and c - myc virus - treated samples . 
 it is well known that retroviruses manipulate the host cytoskeleton to facilitate virus entry and integration . 
 furthermore , the presence of tgf signaling pathways is also not surprising , considering that carryover of hiv tat can occur after packaging of second - generation lentivirus vectors , and that tat protein has been associated with the induction of tgf- , which likely serves multiple functions for the virus , including immunosuppression and facilitation of cytoskeletal remodeling . 
 the differential regulation of macropinocytosis in both klf4 virus- and c - myc virus - treated samples is also likely due to viral entry , as both native hiv- and vesicular stomatitis virus - g ( vsv - g)pseudotyped hiv vectors have been reported to use macropinocytosis for entry . 
 notably , several emt regulation pathways were observed to be differentially regulated between sarna- and virus - treated samples , irrespective of whether it was klf4 or c - myc being targeted or transduced . 
 this was quite striking , as it has been reported that suppression of emt signals is required for reprogramming mouse fibroblasts . 
 klf4 serves to activate the mesenchymal - to - epithelial transition ( met ) that is required for reprogramming , whereas oct4 , sox2 , and c - myc suppress tgf-induced emt . 
 the differential regulation of these pathways in our experiments fits well into this model , as in both cases klf4 and c - myc are acting in competition with the tgf- induced by lentiviral infection . 
 this suggests that using lentiviral vectors to activate reprogramming factors may actually hinder the reprogramming process , as these vectors have been shown to activate tgf- signaling . 
 this underscores the need for alternative methods of gene activation in reprogramming . in recent years 
 , invaluable information has been obtained from the use of rnai to study stem cell biology by inhibiting expression of specific genes involved in the regulation of pluripotency within embryonic as well as somatic stem cells . here , we have shown the potential of using sarnas that , conversely , upregulate expression of endogenous genes in stem cells . as each gene can be selectively targeted for activation , the use of sarnas may also provide a highly useful tool in studying the contribution of individual factors in ips cell reprogramming . 
 several studies have used inducible systems to study the reprogramming process , but these have been limited by the inability to activate or repress the activity of each factor individually . since rnaa is a transient process , it may be possible to develop an optimized protocol for ips cell production wherein each factor can be activated when needed by the transfection of its specific sarna , and similarly removed when it is no longer necessary . in this context , 
 current methods for upregulating klf4 and c - myc require transfection or viral transduction of klf4 or c - myc expression vectors into cells . 
 as noted above , this study suggests that the tgf- induced by lentiviral vectors may actually be detrimental to reprogramming . 
 further , oncogenic reactivation of stably integrated c - myc transgenes poses a serious safety issue to the use of ips cells . 
 in addition , evidence that latent viral expression of reprogramming factors impairs normal differentiation of ips cells , and intolerance to genomic damage caused by exogenous dna or transposon integration further emphasizes the need for a method of ips cell generation that uses endogenous cellular processes and requires no foreign dna . in this regard , while reprogramming to full pluripotency has not , to date , been demonstrated with this method , other groups have recently shown sarna - mediated upregulation of endogenous oct4 in a breast cancer cell line , and endogenous klf4 in prostate cancer cell lines . notably , 
 downstream gene expression and phenotypic changes induced by sarna - mediated upregulation of klf4 in prostate cancer cell lines were reported to be comparable to those obtained by retroviral vector transduction . 
 this is consistent with our results obtained in primary human mscs , and suggests that the use of synthetic sarna oligos may prove highly advantageous as a safe and efficient alternative for upregulation of endogenous reprogramming genes . 
 four parameters were used : ( i ) download target gene annotations ; ( ii ) identify antisense rna target sequences ; ( iii ) select promoter antisense sequences ; and ( iv ) identify candidate sarnas . 
 first , the method downloads information about the target 's genomic location , orientation , and transcriptional structure from available databases such as the refseq database at ucsc ( university of california , santa cruz ) . 
 second , given a database of rna transcripts with known read direction , such as the ucsc spliced est track , our method searches the database for transcripts that are antisense to and in the vicinity of the target gene . 
 more specifically , the method identifies antisense transcripts that ( i ) overlap the target 's promoter and the target mrna 's 5 end ; ( ii ) overlap the target mrna ; ( iii ) are at most 20100  kb upstream of the target 's tss ; or ( iv ) are at most 20100  kb downstream of the target 's polyadenylation site . 
 the method uses these four criteria as hierarchical filters such that if it finds antisense transcripts that for example satisfy criterion ( i ) , the method does not consider the three other criteria . 
 third , based on the target 's tss , the method downloads the antisense genomic sequence from a fixed size region upstream and downstream of the tss . 
 the typical region size used by the method is 500 nts upstream and downstream of tss , but larger or smaller sizes can also be used . 
 fourth , the method designs sirnas that give effective and specific downregulation of the antisense target sequence . 
 the method ( i ) uses a sirna design algorithm , such as gpboost , to identify candidate effective sirnas ; ( ii ) removes all candidate sirnas with aaaa , cccc , gggg , or uuuu motifs and gc content < 20% or > 55% ; ( iii ) removes all candidates that have hamming distance < 2 to all potential off - target transcripts ; and ( iv ) returns a given number of remaining non - overlapping sirnas sorted by their predicted sirna knockdown efficacy . 
 bone marrow - derived adult human mesenchymal stem cells ( lonza , basel , switzerland ) were cultured in the manufacturer 's media as instructed . the klf4 , myc , and control duplex rna oligonucleotides were transfected into mscs using lipofectamine rnaimax reagent ( invitrogen , carlsbad , ca ) following the manufacturer 's protocol with 30  pmol oligo to 1 l reagent in a 24-well plate to a final oligo concentration of 50  nmol / l . 
 the block - it alexa fluor red fluorescent control ( invitrogen ) and silencer fam labeled negative control # 1 sirna ( applied biosystems , carlsbad , ca ) , which have no homology to any known gene , were used as negative controls and to assess transfection efficiency by fluorescence microscopy and flow cytometry . 
 images were taken at 100 magnification on a nikon ts100 microscope ( nikon instruments , melville , ny ) . 
 the plasmids psin - ef2-klf4-pur and psin - ef2-c - myc - pur were generated by cloning human klf4 and c - myc transgenes from plasmids pmxs - hklf4 or pmxs - hcmyc , respectively , into the psin - ef2-pur lentiviral vector backbone . 
 vsv - g  pseudotyped second - generation lentivirus preparations were produced using standard protocols ; briefly , packaging plasmids pmd2.g , pspax2 , and transfer vector were cotransfected into 293 t cells with jetprime reagent ( polyplus - transfection , new york , ny ) , and 48 hours later virus - containing supernatant was collected , filtered , and concentrated by ultracentrifugation . 
 vector titers were determined by p24 elisa , performed by the ucla virology core . quantitative reverse transcription - pcr . 
 total rna was isolated from mscs using the rneasy micro plus kit to remove gdna ( qiagen , valencia , ca ) . 
 rna was reverse - transcribed to cdna using the high capacity cdna kit ( applied biosystems ) . for nascent rna analysis 
 , experiments were performed using the click - it nascent rna capture kit ( invitrogen ) according to the manufacturer 's protocol with a 1 hour eu pulse before sample collection on each day of the experiment . 
 quantitative real - time pcr was performed using taqman gene expression master mix ( applied biosystems ) on a myiq2 thermal cycler ( bio - rad , hercules , ca ) according to the manufacturer 's standard protocols . 
 the taqman primer sets used were as follows : klf4 , hs00358836_m1 ; pou5f1 ( oct4a and oct4b isoform ) , hs00999632_g1 ; pou5f1 ( oct4a isoform ) , hs01895061_u1 ; pou5f1 ( oct4b isoform ) , hs00742896_s1 ; sox2 , hs00602736_s1 ; nanog , hs02387400_g1 ; myc , hs00153408_m1 ; ccnd1 , hs00277039_m1 ; cdkn1a , hs00355782_m1 ; odc1 , hs00159739_m1 ; tp53 , hs99999147_m1 ; actb , hs00357333_g1 ( applied biosystems ) . for interferon response gene expression , primers from the interferon response detection kit ( 
 system biosciences , mountain view , ca ) were used for sybr green real - time pcr with ssofast evagreen supermix ( bio - rad ) . as suggested by the manufacturer 's protocol 
 , samples were collected for expression analysis 24 hours after sarna transfection or viral transduction . 
 expression of -actin mrna was used as an internal control and samples were normalized to the scrambled sequence control oligonucleotide or untreated samples . 
 statistical significance was determined by student 's t - test , with p values < 0.05 considered significant . 
 cells were lysed and protein concentration was determined using coomassie plus assay reagent ( thermo scientific , waltham , ma ) . 
 each sample was loaded onto a nupage bis - tris gel ( invitrogen ) at 30 g / well and electrophoresed and transferred according to the manufacturer 's specifications . 
 primary antibodies used were gklf ( sc-20691 ; santa cruz biotechnology , santa cruz , ca ) and c - myc ( sc-764 ; santa cruz biotechnology ) . 
 -actin primary antibody ( ab8227 ; abcam , cambridge , ma ) was used as an internal control and for quantitation . 
 protein was detected using anti - rabbit hrp conjugated secondary antibody ( haf008 ; r&d systems , minneapolis , mn ) , developed using immun - star westernc reagent ( bio - rad ) , and visualized on a chemidoc xrs+ ( bio - rad ) . 
 statistical significance was determined by student 's t - test , with p values < 0.05 considered significant . 
 rna was processed and hybridized to a genechip human gene 1.0 st array ( affymetrix , santa clara , ca ) in triplicate by the city of hope microarray core facility ( duarte , ca ) . 
 samples were normalized using the exonrma16 summarization algorithm and filtered on expression percentile in the raw data ( 20100% ) . 
 differential expression analysis compared to control samples was performed using an unpaired t - test with asymptotic p value computation and benjamini - hochberg multiple testing correction . 
 heatmaps were generated using hierarchical clustering using centroid linkage and euclidean similarity measure . for pathway analysis , 
 lists of genes differentially regulated between sarna and virus samples were used to generate significantly enriched pathways in metacore ( version 6.3 build 25177 by genego ) . for 
 go analysis , lists of differentially expressed genes with the indicated adjusted p values and absolute fold changes were generated for the sarna- and virus - treated samples versus control . 
 these lists were used to generate functional annotation charts using david bioinformatic analysis with the goterm_bp_fat category on a hugene-1_0-st - v1 background . 
 transfection efficiency of positive control sirnas as evaluated by rt - qpcr for expression levels of targeted genes . 
 rt - qpcr of klf4 and c - myc virus transduction in mscs for interferon response genes at the indicated p24 amount per cell . 
 list of genes with stem cell - related gene ontology , used to generate figure 3c . 
 table s3 . list of genes with cell cycle - related gene ontology , used to generate figure 3d  f . 
 transfection efficiency of positive control sirnas as evaluated by rt - qpcr for expression levels of targeted genes . 
 rt - qpcr of klf4 and c - myc virus transduction in mscs for interferon response genes at the indicated p24 amount per cell . 
 list of genes with stem cell - related gene ontology , used to generate figure 3c . 
 list of genes with cell cycle - related gene ontology , used to generate figure 3d  f .","it is now recognized that small noncoding rna sequences have the ability to mediate transcriptional activation of specific target genes in human cells . using bioinformatics analysis and functional screening , we screened short - activating rna ( sarna ) oligonucleotides designed to target the promoter regions of the pluripotency reprogramming factors , kruppel - like factor 4 ( klf4 ) and c - myc . 
 we identified klf4 and c - myc promoter - targeted sarna sequences that consistently induced increases in their respective levels of nascent mrna and protein expression in a time- and dose - dependent manner , as compared with scrambled sequence control oligonucleotides . 
 the functional consequences of sarna - induced activation of each targeted reprogramming factor were then characterized by comprehensively profiling changes in gene expression by microarray analysis , which revealed significant increases in mrna levels of their respective downstream pathway genes . 
 notably , the microarray profile after sarna - mediated induction of endogenous klf4 and c - myc showed similar gene expression patterns for stem cell- and cell cycle - related genes as compared with lentiviral vector - mediated overexpression of exogenous klf4 and c - myc transgenes , while divergent gene expression patterns common to viral vector - mediated transgene delivery were also noted . 
 the use of promoter - targeted sarnas for the activation of pluripotency reprogramming factors could have broad implications for stem cell research .",pubmed
"chronic kidney diseases , which lead to end - stage kidney failure , are associated with changes in kidney structure and fibrosis regardless of the underlying cause . 
 urinary tract obstruction is characterized by tubular atrophy or dilation , tubular cell death by apoptosis and necrosis , interstitial leukocyte infiltration , and increased interstitial matrix deposition . . 
 such an obstruction might be observed after benign prostatic hyperplasia ; renal , ureteral , or bladder calculi ; urethral stricture ; and neoplasm of the bladder , prostate , or urethra . 
 the hydrostatic pressure , which is the result of the blockage , initiates renal injuries . 
 the injuries are characterized by tubular dilatation or atrophy , inflammatory infiltration of leucocytes , fibroblast activation , proliferation , increase in matrix proteins , and progressive interstitial fibrosis with the loss of renal parenchyma . 
 unilateral ureteral obstruction ( uuo ) is an experimental rat model of renal injury that imitates the process of obstructive nephropathy in an accelerated manner . 
 evidence supports a key role of the inflammatory process in the renal structural changes that occur after uropathy . 
 taurine ( 2-aminoethanesulfonic acid ) is an organic acid and a major component of the bile . 
 taurine has been shown to have beneficial effects on the renal fibrosis that occurs after cisplatin - induced injuries , diabetic nephropathy , and age - related renal changes . 
 it also plays an important role in some biochemical and physiological processes in the kidney . in a review , 
 chesney et al .   described the renal - taurine interactions related to ion reabsorption , renal blood flow , renal vascular endothelial function , and antioxidant properties ( especially in the glomerulus ) . 
 in addition , it has been reported that taurine plays a major role in the renal cell cycle as well as in apoptosis and also functions as an osmolyte during the stress response . 
 taurine has been shown to play an important role in four different forms of kidney diseases : glomerulonephritis , diabetic nephropathy , chronic renal failure , and acute kidney injury . 
 paclitaxel stabilizes microtubules and as a result interferes with the normal breakdown of the microtubules during cell division . 
 sun et al .   and zhang et al .   have reported that paclitaxel ameliorates fibrosis in the uuo rat kidney model . 
 in addition , sommardahl et al .   showed the efficacy of taxol in the mouse model of polycystic kidney disease . 
 in addition , zhou et al .   reported the ameliorative effects of paclitaxel on liver fibrosis . in this study 
 , we tried to determine whether taurine and taxol can attenuate the tubulointerstitial fibrosis and preserve the normal renal histology in a rat model of uuo . 
 moreover , stereological survey was applied to investigate structural changes in the uropathic kidney , including the volume of the cortex , medulla , glomeruli , proximal convoluted tubules ( pct ) , distal convoluted tubules ( dct ) , henle tubules , collecting ducts , vessels , and fibrous tissue . 
 the animals were treated according to the standard directive as recommended and approved by the research authorities of shiraz university of medical sciences . 
 the rats were randomly divided into four groups each including six animals , which is considered acceptable for stereological studies . in group one ( sham - operated animals ) , laparotomy only was done without any procedures and the rats did not receive any treatments . on the other hand , groups 
 the third group received taxol ( 0.3 mg / kg twice per week intraperitoneally ) for 28 days . 
 finally , the fourth group was treated with taurine ( 7.5 mg / kg / d ) dissolved in a distilled water vehicle given by gavage for 28 days . 
 after the rats were anesthetized with ketamine / xylosine ( 25 mg / kg i.p . and 2.5 mg / kg i.p . , 
 respectively ) , the abdominal cavity was opened by an incision to reach the left ureter . 
 a 3 - 0 silk ligature was tied around the ureter , after which the incision was closed . after 28 days , the rats ' kidneys were removed . 
 the primary volume of the kidney , vprimary , was measured by using sherle 's immersion method . 
 then , the tissue shrinkage produced by fixation , processing , and staining was estimated . 
 estimation of the shrinkage and the length of the renal tubules as well as the vessels requires isotropic , uniform random sections [ 15 - 17 ] . 
 2 . the estimated shrinkage was also used to estimate the final volume of the kidney to avoid the consecutive sectioning that is required for the cavalieri method . 
 the final volume of the kidney ( the reference space ) was corrected by using the following formula : vfinal = vprimary(1-volume shrinkage ) . 
 each sampled section was analyzed by using a video microscopy system that was made up of a microscope ( e-200 , nikon , tokyo , japan ) linked to a video camera , a computer , and a monitor to determine the parameters . between 8 and 12 microscopic fields 
 then , the stereological grids were superimposed on the images by means of the stereology software designed at histomorphometry & stereological research center , shiraz university of medical sciences , shiraz , iran . 
 the volume density of each structure ( the fraction of the unit volume of the kidney that is occupied by the structure ) was estimated by using the point - counting method . 
 the volume fraction of the cortex and the medulla were estimated at a final magnification of 375 and other parameters at 1,500 . the length density ( the length of each tubular structure in the unit volume of the kidney ) of the proximal and the dct , 
 collecting ducts , henle 's loop , and vessels was estimated by randomly overlaying an unbiased counting frame   with an area of 2,700 m on the live monitor images . 
 finally , the total volume of the parameters and the total length of the tubules as well as the vessels were estimated by multiplying the fractional volume or the length density by the final volume of the kidney to prevent the "" reference trap "" [ 15 - 17 ] . 
 statistical comparisons between the group means were done by kruskall - wallis and mann - whitney u - test . moreover , p0.01 was considered as statistically significant . 
 the animals were treated according to the standard directive as recommended and approved by the research authorities of shiraz university of medical sciences . 
 the rats were randomly divided into four groups each including six animals , which is considered acceptable for stereological studies . in group one ( sham - operated animals ) , laparotomy only was done without any procedures and the rats did not receive any treatments . on the other hand , groups 
 the third group received taxol ( 0.3 mg / kg twice per week intraperitoneally ) for 28 days . 
 finally , the fourth group was treated with taurine ( 7.5 mg / kg / d ) dissolved in a distilled water vehicle given by gavage for 28 days . 
 after the rats were anesthetized with ketamine / xylosine ( 25 mg / kg i.p . and 2.5 mg / kg i.p . , 
 respectively ) , the abdominal cavity was opened by an incision to reach the left ureter . 
 a 3 - 0 silk ligature was tied around the ureter , after which the incision was closed . after 28 days , the rats ' kidneys were removed . 
 the primary volume of the kidney , vprimary , was measured by using sherle 's immersion method . 
 then , the tissue shrinkage produced by fixation , processing , and staining was estimated . 
 estimation of the shrinkage and the length of the renal tubules as well as the vessels requires isotropic , uniform random sections [ 15 - 17 ] . 
 2 . the estimated shrinkage was also used to estimate the final volume of the kidney to avoid the consecutive sectioning that is required for the cavalieri method . 
 3 . after estimating the volume shrinkage [ 15 - 17 ] , the final volume of the kidney ( the reference space ) was corrected by using the following formula : vfinal = vprimary(1-volume shrinkage ) . 
 each sampled section was analyzed by using a video microscopy system that was made up of a microscope ( e-200 , nikon , tokyo , japan ) linked to a video camera , a computer , and a monitor to determine the parameters . between 8 and 12 microscopic fields were selected in a systematic random manner . 
 then , the stereological grids were superimposed on the images by means of the stereology software designed at histomorphometry & stereological research center , shiraz university of medical sciences , shiraz , iran . 
 the volume density of each structure ( the fraction of the unit volume of the kidney that is occupied by the structure ) was estimated by using the point - counting method . 
 the volume fraction of the cortex and the medulla were estimated at a final magnification of 375 and other parameters at 1,500 . the length density ( the length of each tubular structure in the unit volume of the kidney ) of the proximal and the dct , collecting ducts , henle 's loop , and vessels was estimated by randomly overlaying an unbiased counting frame   with an area of 2,700 m on the live monitor images . 
 finally , the total volume of the parameters and the total length of the tubules as well as the vessels were estimated by multiplying the fractional volume or the length density by the final volume of the kidney to prevent the "" reference trap "" [ 15 - 17 ] . 
 statistical comparisons between the group means were done by kruskall - wallis and mann - whitney u - test . moreover , p0.01 was considered as statistically significant . 
 the histological changes of the kidney after the uuo procedure as well as after the uuo procedure plus taxol or taurine treatment can be observed in fig . 
 renal disruption , tubular atrophy , degeneration , necrosis , and fibrosis were observed in the uuo animals . after treatment with taxol or taurine , 
 the mean and the standard deviation of the kidney volume , the volume of the cortex , and the volume of the medulla are presented in table 1 . 
 the total volume of the glomeruli , pct , dct , henle 's loop , collecting duct , degenerative tubules , and necrotic and fibrotic tissue in the different groups is shown in table 2 . 
 the volume of the glomeruli , pct , dct , henle 's loop , and collecting ducts was reduced in the rats after uuo . less than -8% of these structures remained intact on average . after treatment of the uuo rats with taxol , -87% , -32% , -51% , -72% , and -88% of the glomeruli , pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . 
 after treatment of the uuo rats with taurine , -25% , -16% , -16% , -40% , and -46% of the glomeruli , pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . 
 table 2 depicts the increase in degenerative tubules and necrotic and fibrotic tissues in the uuo groups . 
 in addition , after treating the uuo animals with taxol , the volume of the degenerative tubules and necrotic and fibrotic tissues was decreased -42% and -53% , respectively , compared with that in the uuo rats . after treating the uuo rats with taurine , 
 the volume of the degenerative tubules increased 1.8-fold , whereas necrotic and fibrotic tissues decreased -63% compared with that in the uuo rats without any treatments ( p<0.01 ) . on average , less than -3% of the lengths of these structures remained intact in the uuo rats . 
 after treatment of the uuo rats with taxol , -65% , 70% , 56% , and 61% of the pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . on the other hand , after treatment of the uuo rats with taurine , -49% , -53% , -43% , and -45% , of the length of the pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) ( table 3 ) . 
 the histological changes of the kidney after the uuo procedure as well as after the uuo procedure plus taxol or taurine treatment can be observed in fig . 
 renal disruption , tubular atrophy , degeneration , necrosis , and fibrosis were observed in the uuo animals . after treatment with taxol or taurine , 
 the mean and the standard deviation of the kidney volume , the volume of the cortex , and the volume of the medulla are presented in table 1 . 
 the total volume of the glomeruli , pct , dct , henle 's loop , collecting duct , degenerative tubules , and necrotic and fibrotic tissue in the different groups is shown in table 2 . 
 the volume of the glomeruli , pct , dct , henle 's loop , and collecting ducts was reduced in the rats after uuo . 
 less than -8% of these structures remained intact on average . after treatment of the uuo rats with taxol , -87% , -32% , -51% , -72% , and -88% of the glomeruli , pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . 
 after treatment of the uuo rats with taurine , -25% , -16% , -16% , -40% , and -46% of the glomeruli , pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . 
 table 2 depicts the increase in degenerative tubules and necrotic and fibrotic tissues in the uuo groups . 
 in addition , after treating the uuo animals with taxol , the volume of the degenerative tubules and necrotic and fibrotic tissues was decreased -42% and -53% , respectively , compared with that in the uuo rats . after treating the uuo rats with taurine , 
 the volume of the degenerative tubules increased 1.8-fold , whereas necrotic and fibrotic tissues decreased -63% compared with that in the uuo rats without any treatments ( p<0.01 ) . 
 on average , less than -3% of the lengths of these structures remained intact in the uuo rats . 
 after treatment of the uuo rats with taxol , -65% , 70% , 56% , and 61% of the pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . on the other hand , after treatment of the uuo rats with taurine , -49% , -53% , -43% , and -45% , of the length of the pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) ( table 3 ) . 
 the present study confirmed through a quantitative survey the protective role of taxol and taurine on renal tissue damage after the induction of uuo in rats . 
 furthermore , the findings of the present study revealed that taxol was more protective than taurine . 
 paclitaxel is an alkaloid plant that is derived from the bark of the pacific yew tree . 
 paclitaxel works by disrupting the microtubular network essential for cell division and other normal cellular functions , which eventually leads to cell death . 
 the positive effects of taxol have been reported in cisplatin - induced and ischemia - reperfusion injuries . moreover , a study demonstrated that taxol can attenuate oxidative / nitrosative stress , glutathione depletion , enhanced urinary hydrogen peroxide excretion , and the decrease in antioxidant enzymes ( catalase , glutathione peroxidase , and glutathione - s - transferase ) . 
 in addition , yoon et al .   reported that taxol can induce cytoprotective enzymes and thereby improve the ischemia - reperfusion - induced changes in lipid hydroperoxides , glutathione , creatinine clearance , kidney weight , and histological abnormalities . 
 the protective effect of taurine on nephrotoxicity after cyclosporine or cisplatin treatment has been reported . 
 it is believed that taurine , owing to its antioxidant properties , avoids cyclosporine - induced reactive oxygen species and , consequently , nephrotoxicity . in a recent article by yousef et al . 
 , it was shown that taurine induces a protective property after paracetamol - induced oxidative injury of the kidney . 
 articles have discussed various mechanisms by which taxol and taurine may protect renal structure changes after uuo . for example , the results of a study by sun et al .   revealed that a low dose of paclitaxel ameliorates renal fibrosis via the modulation of microrna-192 pathobiology and transforming growth factor- signaling . transforming growth factor- has been shown to play a central role in the development of the tubulointerstitial fibrosis . 
 guerrero - beltran et al .   reported that taxol can prevent cell death and inflammation in nephropathy . 
 the findings of the study by zhou et al .   indicate that 200 nmol / l paclitaxel ameliorates hepatic fibrosis by modulating transforming growth factor - beta signaling . 
 in addition , koz et al .   showed that paclitaxel provided antifibrotic effects during conjunctival wound healing . 
 these major roles include regulating the blood flow in the vasculature of the kidney , scavenging the reactive oxygen species in glomerulus , na transport in the proximal tubules , and osmoregulation as well as cell volume regulation in the medulla . 
 in addition to the preventive roles of taurine in renal fibrosis , other tissues can also be protected . 
 reported that taurine reduces iron - potentiated alcoholic liver fibrosis by reducing oxidative stress , inflammatory and fibrogenic mediators , and stellate cell activation . 
 in addition , the results of the study by devi and anuradha   showed that the restorative effect of taurine in an experimental model of liver fibrosis involved amelioration of protein and lipid damage by decreasing oxidative and nitrosative stresses . 
 therefore , a variety of mechanisms can be considered responsible for the protective effects of taxol and taurine in the present study . 
 it can be concluded that taxol and taurine are both effective in preventing some structural renal damage in the direct obstruction model in rats . 
 the volume of the necrotic and fibrotic tissue was found to be less in the taxol- and taurine - treated groups .","purposeblockage of the urinary tract induces changes in renal structure including tubular dilatation or atrophy , tubular cell death , inflammatory processes , and progressive interstitial fibrosis with the loss of renal parenchyma . 
 the present study was conducted to survey the protective effects of taxol and taurine on the renal structure after unilateral ureteral obstruction ( uuo).materials and methodsuuo was induced in three groups of rats ( n=6 ) who then received distilled water , taxol ( 0.3 mg / kg / d ) , or taurine ( 7.5 mg / kg / d ) . 
 stereological methods were used to gather quantitative as well as comparative data.resultsless than -8% of the volume of the glomeruli , proximal convoluted tubules ( pct ) , distal convoluted tubules ( dct ) , henle 's loop , and collecting ducts were preserved after uuo . 
 after treatment of the uuo rats with taxol , between -32% and 88% of the parameters mentioned above remained intact , and after treatment of the uuo rats with taurine , between -16% and 46% of the parameters remained intact ( p<0.01 ) . compared with the untreated uuo animals , the volume of necrotic and fibrotic tissues decreased -53% and -63% in the uuo rats treated with taxol and taurine , respectively ( p<0.01 ) . 
 less than -3% of the lengths of the renal tubules ( pct , dct , henle 's loop , and collecting ) were preserved in the uuo rats . 
 after treatment with taxol and taurine , -61% to 70% and -43% to 53% of the length of the renal tubules were preserved , respectively ( p<0.01).conclusionstaurine and taxol are effective in preventing some structural renal damage in a direct ureteral obstruction model . 
 taxol was more effective in renal protection .",pubmed
"pathogen recognition receptors ( prrs ) trigger innate immune responses after sensing microbial components such as lipopolysaccharide ( lps ) , lipoprotein , flagellin , and nucleic acids and are necessary for the protection of a host suffering from microbial infection ( beutler , 2009 ; kawai and akira , 2009 ) . after detecting microbial components , 
 prrs drive the coordinated activation of transcription factors , leading to the expression of inflammatory cytokines , chemokines , type i interferons ( ifns ) , and antimicrobial genes . although innate immunity is essential for host defense , aberrant activation of innate immune responses results in the development of inflammatory diseases ; e.g. , autoimmune disease and septic shock . 
 thus , the signaling pathways of the innate immune system are tightly regulated to avoid either deficient or excessive responses . 
 protein degradation is critically involved in the control of innate immune responses ( beutler , 2009 ; kawai and akira , 2009 ) . 
 although the regulation of immune responses by the ubiquitin  proteasome ( a selective protein degradation system ) has long been studied ( liu et al . 
 , 2005 ; bhoj and chen , 2009 ) , recent research has shown the involvement of another clearance system , autophagy , in the innate immune response . 
 autophagy is a bulk degradation system that delivers cytoplasmic constituents into lysosomes ( fig . 1 ; 
 this process enables the reuse of intracellular constituents and supplies an amino acid pool during periods of starvation . 
 indeed , mice deficient in atg3 , atg5 , or atg7 die within 1 d of delivery , indicating that autophagy is essential for survival during neonatal starvation ( kuma et al . , 2004 ; komatsu et al . 
 autophagy is also involved in the clearance of old / damaged organelles , long - lived proteins , insoluble protein aggregates , and lipid droplets , thus regulating cellular homeostasis , cell death / survival , and lipid metabolism ( yoshimori and noda , 2008 ; mizushima , 2009 ; czaja , 2010 ) . 
 several essential components of the autophagic machinery have been identified by yeast genetic screening , the so - called autophagy - related proteins ( atgs ; he and klionsky , 2009 ; nakatogawa et al . , 2009 ) . 
 recent studies have identified mammalian counterparts of yeast atgs , such as ulk1 ( atg1 ) , atg35 , beclin ( atg6 ) , atg7 , lc3 ( atg8 ) , atg9a , atg10 , atg12 , atg13l , atg14l , atg16l1 , fip200 ( atg17 ) , and wipi-1 ( atg18 ) , indicating that atgs are phylogenetically highly conserved between yeast and mammals ( mizushima et al . 
 , 2003 ; proikas - cezanne et al . , 2004 ; yamada et al . , 2005 ; 
 young et al . , 2006 ; hara et al . , 2008 ; itakura et al . , 
 ; chang et al . , 2009 ; ganley et al . , 2009 ; hosokawa et al . , 2009 ; jung et al . , 2009 ; matsunaga et al . , 2009 ; 
 the coordinated action of atgs mediates the membrane trafficking required for autophagosome formation ( fig . 
 autophagy , a bulk degradation system , is induced after sensing various types of stress , such as nutrient starvation , the accumulation of protein aggregates , and invasion by microbes . 
 this system enables the reuse of cellular components and the clearance of unfavorable substances , thus contributing to the maintenance of cellular homeostasis and the prevention of disease . 
 atgs are recruited to the isolation membrane , a source membrane of autophagosomes , and play a vital role in driving the membrane trafficking necessary for the generation of autophagosomes . 
 autophagosomes then fuse with lysosomes to become autolysosomes , leading to the degradation of the constituents trapped inside . 
 autophagy plays a critical role in host defense responses by promoting the elimination of pathogens and the induction of acquired immunity ( deretic , 2005 ; deretic and levine , 2009 ; virgin and levine , 2009 ) . 
 the rab7-dependent formation of bacteria - containing autophagosomes is critical for the killing of invading bacteria , such as streptococcus pyogenes and coxiella burnetii , in nonphagocytic cells ( bern et al . , 2002 ; 
 , 2004 ; romano et al . , 2007 ; yamaguchi et al . , 2009 ) . 
 bacteria , such as shigella flexneri , escape from the autophagic machinery via expression of the virg protein , which is capable of inhibiting atg5 function in nonphagocytic cells ( ogawa et al . , 2005 ) . 
 immunity - related gtpase family m , an ifn-inducible gtpase , promotes autophagy , and the autophagic machinery effectively eliminates mycobacteria in macrophages ( gutierrez et al . , 2004 ; singh et al . , 2006 ) . 
 sequestosome 1 ( sqstm1)/p62 , which interacts with both lc3 and ubiquitinated proteins , is recruited to bacteria - containing ubiquitinated vacuoles upon infection and promotes the killing of invading pathogens by autophagy ( bjrky et al . , 2005 
 ; komatsu et al . , 2007 ; yoshikawa et al . , 2009 ; ponpuak et al . , 
 in addition to the formation of autophagosomes , atgs are responsible for the transport of irga6 , an immunity - related gtpase , from the er  golgi to the vacuoles containing invading microbes , which is a function required for the elimination of toxoplasma gondii by macrophages ( zhao et al . , 2008 ) . 
 these findings clearly demonstrate that atgs are critical for the direct elimination of infectious agents . 
 autophagy also plays an indispensable role in antigen presentation to antigen - specific t cells ( a process essential for the induction of acquired immunity ; gannag and mnz , 2009 ) . 
 major histocompatibility complex ( mhc ) class ii molecules are localized on autophagosomes , and the autophagic machinery promotes the presentation of viral and self - antigens by mhc class ii molecules to antigen - specific cd4 t cells ( nimmerjahn et al . , 2003 ; dengjel et al . , 2005 ; paludan et al . , 2005 ; zhou et al . 
 autophagy controls the mhc class i  dependent presentation of viral antigens to cd8 t cells ( english et al . , 2009 ) . 
 in addition to these functions , recent studies reveal that atgs are important for innate immune responses provoked by the engagement of prrs with microbial components . in this review 
 recent genome - wide association studies have identified atg16l1 as a candidate gene responsible for susceptibility to the inflammatory bowel disease , crohn s disease ( hampe et al . 
 atg16l1 forms an 800-kd protein complex with atg12-atg5 conjugates , and the self - multimerization of atg16l1 via its coiled - coil domain is required for the recruitment of this conjugate into an isolation membrane ( a source membrane of autophagosomes ; fig . 1 ; mizushima et al . , 2003 ; fujita et al . , 
 . the atg12-atg5atg16l1 complex recruits an atg3-lc3 intermediate to the isolation membrane , and in doing so , defines the site at which lc3 is conjugated to phosphatidylethanolamine ( fujita et al . , 2008 ) 
 consistently , cells lacking atg16l1 are deficient in the conjugation of lc3 to phosphatidylethanolamine , an essential process for the elongation of autophagosomes ( saitoh et al . , 2008 ) . 
 this phenotype is similar to that observed in other atg - deficient mice ( kuma et al . , 2004 ; komatsu et al . , 2005 ) 
 thus , atg16l1 is an essential component of autophagy under both nutrient - rich and nutrient - starved conditions . using atg16l1-deficient mice 
 , we have shown that the loss of atg16l1 enhances endotoxin - induced inflammatory immune responses ( fig . 
 commensal bacteria are thought to be one of the major causative agents of bowel disease . 
 when intestinal epithelial cells are damaged , commensal bacteria are able to pass through the epithelial layer and stimulate prrs , leading to the induction of intestinal inflammation . 
 toll - like receptors ( tlrs ) , one family of prrs , detect microbial components and induce the production of inflammatory cytokines such as tnf- , il-1 , il-6 , il-12 , and il-18 by macrophages / dendritic cells ( dcs ) , resulting in the infiltration of activated lymphocytes into the intestine ( beutler , 2009 ; kawai and akira , 2009 ) . 
 macrophages lacking atg16l1 produce high amounts of il-1 and il-18 in response to lps , a ligand for tlr4 . 
 macrophages from atg7-deficient mice also show enhanced production of il-1 , indicating the importance of atg in the regulation of the inflammatory response . 
 stimulation of the other tlr family members ( except for tlr3 ) fails to induce this enhanced production of il-1 by atg16l1-deficient macrophages . 
 trif ( toll / il-1 receptor domain  containing adaptor inducing ifn- ) , the adaptor protein involved in tlr3/4 signaling pathways , also mediates il-1 production in atg16l1-deficient macrophages . 
 it was shown that atp , uric acid crystals , silicas , and asbestos induce the generation of reactive oxygen species ( ros ) , leading to the activation of caspase-1 , an essential protease for il-1 production ( dostert et al . , 2008 ; stutz et al . , 2009 ; zhou et al . , 2010 ) . 
 in atg16l1-deficient macrophages , the enhanced il-1 production is induced by trif - dependent generation of ros and is blocked by ros scavengers . 
 mice with hematopoietic cells lacking atg16l1 are highly susceptible to dextran sulfate sodium  induced colitis , which is relieved by the injection of anti 
 these findings indicate that the loss of atg16l1 , an essential component of the autophagic machinery , results in the production of inflammatory cytokines in response to endotoxin . however , the source of the ros has not yet been identified . 
 other possible sources of ros include mitochondria because autophagy is required for the clearance of old / damaged mitochondria . 
 recent studies have shown that loss of the atg results in the accumulation of ros in immune - competent cells , such as t cells , as a result of the disruption of mitochondrial turnover ( pua et al . 
 further studies are needed to elucidate the source of ros in macrophages deficient in atgs . 
 the ikk-ikk-nemo complex mediates the activation of the transcription factor nf-b , which in turn induces the transcription of proinflammatory cytokines and pro  il-1. the tbk1ikk - i complex mediates the activation of the transcription factor irf3 , which then induces the transcription of type i ifns and ifn - inducible genes . in autophagy - deficient cells , 
 high levels of ros are generated , which mediate trif - dependent caspase-1 activation , resulting in the processing of il-1. however , in wild - type macrophages , limited amounts of il-1 are induced by lps as the result of a lack of ros generation . 
 after detection of the fungal cell wall component zymosan , tlr2 induces the maturation of phagosomes , leading to the elimination of the fungus . 
 atgs such as atg5 , atg7 , and pi3k are involved in the fusion of phagosomes with lysosomes . 
 ( c ) ligands for tlr7 such as ssrna and imiquimod induce the formation of autophagosomes via myd88 , an essential adaptor molecule , and promote the elimination of bacillus calmette - guerin . 
 lps , a ligand for tlr4 , induces the formation of autophagosomes via the trif - p38 signaling axis , leading to the elimination of mycobacterial bacilli . 
 atgs such as atg5 , beclin , and pi3k are required for the formation of autophagosomes by tlr stimulation . 
 recent studies have demonstrated that tlr signaling induces the maturation of phagosomes after exposure to bacteria and promotes mhc class ii 
 ( 2007 ) assessed the involvement of atgs in the maturation of phagosomes after exposure to microbial components and found that zymosan particles ( cell wall components of fungi ) stimulate tlr2 , thus promoting the fusion of gfp - lc3positive phagosomes with lysosomes in macrophages ( fig . 
 surprisingly , myd88 ( myeloid differentiation primary response gene 88 ) , an essential adaptor molecule for cytokine production via tlr2 stimulation , is not necessary for the recruitment of gfp - lc3 to phagosomes by zymosan , suggesting that tlr2 triggers an as yet  unidentified pathway to induce the maturation of phagosomes . 
 atgs such as atg5 , atg7 , and pi3k are required for the efficient maturation of phagosomes induced by tlr2 . 
 ( 2007 ) also mentioned that pam3csk4 , a soluble tlr2 ligand , fails to increase the number of gfp - lc3positive phagosomes , but pam3csk4 fused with latex beads induces the maturation of phagosomes in primary macrophages . 
 thus , tlr2 signaling is necessary but not sufficient for the induction of phagosome maturation . 
 although atgs are important for the maturation of phagosomes after tlr2 stimulation , subsequent characterization by electron microscopy shows that the gfp - lc3positive phagosomes do not have the morphological characteristics of autophagosomes , indicating that tlr2 stimulation induces the atg - dependent , but autophagy - independent , exclusion of fungi in phagolysosomes . 
 it would be of interest to examine how zymosan activates the function of atgs via a tlr2-dependent but myd88-independent pathway and how atgs promote the fusion of phagosomes with lysosomes in macrophages . because the receptor dectin-1 , which recognizes the -glucan structure in zymosan , can activate the tyrosine protein kinase syk - dependent signaling pathway and promote uptake of zymosan ( brown and gordon , 2001 ; brown et al . 
 , 2003 ; gantner et al . , 2003 ; rogers et al . , 2005 ) 
 , it will be important to assess the involvement of this receptor in the atg - dependent maturation of phagosomes after infection by fungal pathogens . the tlr - dependent induction of autophagy in macrophages / monocytes has been reported by other groups . 
 delgado et al . ( 2008 ) reported that the engagement of tlr7 induces autophagy and promotes the elimination of bacillus calmette - guerin in autolysosomes ( fig . 
 2 c ) . both atg5 and beclin are required for the induction of autophagy in macrophages after tlr7 stimulation . 
 although myd88 is not necessary for the formation of gfp - lc3 dots after stimulation by zymosan , it is involved in the formation of gfp - lc3 dots after stimulation of tlr7 ( sanjuan et al . 
 it would be of interest to examine the reasons why myd88 is differentially involved in the formation of gfp - lc3 dots after the engagement of tlrs and how myd88 signals atgs to induce the formation of autophagosomes . 
 however , xu et al . ( 2007 ) reported that tlr4 stimulation results in the pi3k - dependent formation of gfp - lc3 dots and enhances the elimination of mycobacteria in macrophage cell lines ( fig . 
 lps stimulation increases the number of autophagosomes in primary human monocytes , although it fails to induce autophagy in primary mouse macrophages ( xu et al . 
 the signal transducers , trif , ripk1 ( receptor - interacting protein kinase 1 ) , and p38 , are required for the tlr4-induced formation of gfp - lc3 dots in macrophage cell lines . 
 further studies are required to explain the cell type or species - specific induction of autophagy by lps and to identify the target of p38 , whose phosphorylation is responsible for the activation of atgs . 
 nod1 ( nucleotide - binding oligomerization domain 1 ) and nod2 are intracellular sensors that recognize the unique bacterial polypeptides ie - dap ( -d - glutamyl - meso - diaminopimelic acid ) and muramyl dipeptide ( mdp ) , respectively ( franchi et al . , 2009 ) . 
 thus , nod1 and nod2 detect invading bacteria and play a central role in the production of cytokines and antimicrobial peptides . 
 importantly , a strong relationship has been found between nod2 mutation and the development of crohn s disease ( cho and weaver , 2007 ) . it has also been shown that nod1 is a candidate gene responsible for susceptibility to crohn s disease ( mcgovern et al . , 2005 ) . 
 indeed , immune responses to bacterial polypeptides and microbial infection are impaired in cells isolated from patients harboring nod2 mutations ( franchi et al . , 2009 ) . 
 in mouse models , nod1 or nod2 deficiency results in enhanced intestinal inflammation upon bacterial infection as a result of the disruption of innate immune responses . furthermore , nod2 mutant mice , whose nod2 locus harbors the homologue of the most common crohn s disease susceptibility allele 3020insc , are highly sensitive to chemical - induced colitis because of the enhanced production of proinflammatory cytokines such as il-1 and il-1 ( maeda et al . , 2005 ) . because a single nucleotide polymorphism in the atg16l1 gene is also associated with the development of crohn s disease ( hampe et al . 
 , 2007 ; rioux et al . , 2007 ) , the involvement of nod1 and nod2 in the induction of autophagy was assessed . 
 ( 2010 ) found that stimulation of nod2 by mdp induces the formation of autophagosomes , thus promoting antigen presentation on mhc class ii molecules by human dcs ( fig . 
 atgs such as atg5 , atg7 , and atg16l1 are required for the induction of autophagy in human dcs and the subsequent promotion of antigen presentation by mdp ( cooney et al . , 2010 ) . 
 ripk2 , one of the downstream regulators of the nod2 signaling pathway , is also involved in mdp - induced formation of autophagosomes . 
 importantly , dcs expressing crohn s disease  associated nod2 or atg16l1 variants fail to induce autophagosome formation and antigen presentation in response to mdp . 
 ( 2010 ) also found that stimulation with both nod1 and nod2 triggers the induction of autophagy in mouse embryonic fibroblasts ( mefs ) , mouse macrophages , human lymphoblasts , and human cell lines ( fig . 
 however , nod1 and nod2 are not necessary for the formation of autophagosomes induced by nutrient starvation and rapamycin treatment , indicating that nod1 and nod2 are specifically involved in the formation of bacteria autophagosomes . in mefs , nod1 , but not the downstream signaling molecules ripk2 and ib kinase-/nemo , is required for the generation of s. flexneri  containing autophagosomes and promotes the elimination of the invading bacteria . 
 interestingly , both nod1 and nod2 associate with atg16l1 and recruit it to the bacterial entry sites after bacterial infection . 
 the most common nod2 mutation associated with crohn s disease fails to induce the movement of atg16l1 and the formation of autophagosomes . 
 these studies indicate a role for both nod1 and nod2 in the formation of bacteria autophagosomes and in the regulation of antimicrobial responses . 
 however , the signaling pathways downstream of nod1 and nod2 that are responsible for the induction of bacteria autophagosomes remain unclear . 
 nod1 and nod2 are composed of n - terminal cards , a centrally located nucleotide - binding oligomerization domain ( nod ) , and multiple c - terminal leucine - rich repeats ( lrrs ) . 
 the leucine - rich repeats of nod1 and nod2 detect ie - dap and mdp , respectively . after sensing these ligands , nod1 and nod2 induce the formation of autophagosomes , leading to the promotion of antigen presentation and the enhancement of bactericidal responses . 
 atgs such as atg5 , atg7 , and atg16l1 are involved in nod1- and nod2-mediated formation of autophagosomes . 
 for host defense against rna viruses , type i ifns ( ifn- and ifn- ) induce the expression of a series of antiviral factors and play crucial roles in the establishment of an antiviral state ( beutler , 2009 ; kawai and akira , 2009 ) . 
 plasmacytoid dcs ( pdcs ) detect the single - stranded rna ( ssrna ) of rna viruses via tlr7 , which is expressed on endolysosomes . 
 tlr7 stimulates irf7 ( ifn regulatory factor 7 ) , a transcription factor that induces ifn stimulation - responsive element - dependent transcription via the adaptor molecule myd88 and mediates the production of type i ifns by pdcs . in cell types retinoic acid 
 inducible gene i ( rig - i ) and mda-5 , caspase recruitment domain ( card)containing rna helicases called rig - like receptors ( rlrs ) sense the cytoplasmic double - stranded rna ( dsrna ) of rna viruses and mediate signals to ips-1 ( ifn- promoter stimulator-1 ; also called mavs / visa / cardif ) . 
 ips-1 , a card - containing mitochondrial protein , activates the transcription factors irf3 and nf-b via the ib kinase family , leading to the production of type i ifn and the expression of ifn - inducible genes . 
 recent studies have shown an important role for atgs in both tlr - dependent and rlr - dependent antiviral innate immune responses ( jounai et al . , 2007 ; lee et al . 
 two models have been proposed for the involvement of atgs in rlr - mediated innate immune responses . 
 ( 2007 ) found a possible role for atg12-atg5 conjugates in the disruption of the rlr 
 4 a ) . the atg12-atg5 conjugate associates with the card domains of rlr and ips-1 , and ectopic expression of atg12 or atg5 prevents rlr- or ips-1mediated activation of the type i ifn promoter . consistently , 
 loss of atg5 enhances the production of type i ifn by vesicular stomatitis virus ( vsv ) infection and dsrna treatment and limits the replication of vsv in mefs . 
 loss of atg7 , an essential requirement for the conjugation of atg12 with atg5 , also results in enhanced type i ifn production by dsrna ( jounai et al . , 2007 ) 
 2009 ) showed that the disruption of cellular homeostasis by a deficiency in autophagy affects rlr  ips-1 signaling pathways ( fig . 
 damaged mitochondria accumulate because of the loss of autophagy , resulting in the elevated expression of the ips-1 protein . 
 rlr stimulation triggers an elevation in ros production by the accumulated mitochondria , leading to the activation of ips-1dependent innate immune responses in atg5-deficient cells . 
 further analysis will be required to reveal the mechanisms responsible for the ros - dependent activation of rlr signaling . 
 ( a ) rig - i and mda-5 , card - containing rna helicases , sense viral dsrna and signal to ips-1 , a card - containing adaptor molecule , to induce the expression of type i ifn / ifn - inducible genes and proinflammatory cytokines . 
 atg12-atg5 conjugates are recruited to the card domains of rig - i , mda-5 , and ips-1 and suppress the dsrna - induced innate immune response . 
 ( b ) tlr7 detects viral ssrna and induces innate immune responses . targeted disruption of atg5 or pharmacological inhibition of pi3k results in the impairment of tlr7-mediated expression of type i ifn / ifn - inducible genes and proinflammatory cytokines . 
 ( c ) sting , a multispanning membrane protein , and tbk1 , an irf3 kinase , mediate the induction of innate immune responses by dsdna . after sensing dsdna , sting moves from the er to the golgi apparatus and finally reaches the cytoplasmic punctate structures to assemble with tbk1 , resulting in the irf3-dependent expression of type i ifns / ifn - inducible genes . sting colocalizes with atg9a in the golgi apparatus after dsdna stimulation . 
 the loss of atg9a greatly enhances the dsdna - induced assembly of sting and tbk1 , leading to aberrant activation of the innate immune response . 
 ( 2007 ) reported that autophagy is necessary for the tlr7-dependent production of type i ifns and cytokines in pdcs after rna virus infection ( fig . 
 loss of atg5 , or pharmacological inhibition of pi3k , severely impairs the production of both type i ifn and cytokine il-12 p40 by pdc infected with vsv and sendai virus . 
 the number of autophagosomes in pdcs does not increase after tlr7 stimulation , indicating that basal autophagy , which constitutively occurs under nutrient - rich conditions , is responsible for the tlr7-dependent induction of innate immune responses in pdcs . 
 these findings suggested that the viral ssrna from replicating vsv or sendai virus is generated in autophagosomes , or autolysosomes , in pdcs , resulting in the tlr7-dependent production of type i ifns and proinflammatory cytokines . 
 ( 2007 ) also reported that the production of type i ifn but not il-12 p40 by a / d - type unmethylated cpg oligonucleotides ( a ligand for tlr9 ) is severely impaired in atg5-deficient pdcs . because tlr7 and tlr9 share the signaling pathway that induces type i ifn , autophagy or atgs might be involved in signaling events required for tlr7/tlr9-dependent type i ifn production in pdcs . 
 it is known that hyperactivation of b cells by nucleic acid  containing antigens results in the development of autoimmune diseases such as systemic lupus erythematosus ( marshak - rothstein , 2006 ) . 
 although b cell receptors ( bcrs ) on the cell surface and tlr7/tlr9 in the endolysosomes are involved in hyperimmune responses to nucleic acid  containing antigens , it is unclear how nucleic acid  containing antigens induce the aberrant activation of b cells via these receptors . 
 ( 2008 ) found that stimulation of bcr by dna - containing antigens results in the translocation of both the bcr and tlr9 to autophagosome - like compartments where the dna - containing antigens are accumulated ( fig . 
 inhibition of pld , but not diacylglycerol kinase , blocks the translocation of tlr9 after it engages the bcr , suggesting that bcr stimulation induces the pld - dependent generation of phosphatidic acid to promote the recruitment of tlr9 to autophagosome - like compartments . 
 the movement of tlr9 from the endosome into the autophagosome - like compartments on bcr stimulation depends on a functional microtubular network . 
 treatment with nocodazole , an inducer of tubulin depolymerization , inhibits the translocation of tlr9 and the subsequent hyperactivation of mapk by dna - containing antigens . 
 thus , the movement of bcr and tlr9 induced by dna - containing antigens and the colocalization of the receptors and dna - containing antigens in autophagosome - like compartments facilitates the efficient engagement of the receptors , resulting in the synergistic activation of downstream signaling cascades . 
 study of primary b cells lacking atgs is needed to elucidate the requirement for autophagosomes in the bcr- and tlr - dependent activation of b cells by dna - containing antigens . 
 dna - containing antigens stimulate bcrs , leading to the translocation of tlr9 from the endosomes to autophagosome - like compartments . 
 dna - containing antigens also promote the movement of bcrs from the cell surface to autophagosome - like compartments . in these autophagosome - like compartments , 
 dna - containing antigens trigger both bcr and tlr9 signaling pathways , resulting in the synergistic activation of mapk . functional pld and microtubules 
 are required for the translocation of these receptors and play a critical role in the hyperresponse to dna - containing antigens . 
 microbial dna induces the expression of type i ifns and proinflammatory cytokines , leading to the potent induction of innate immunity ( stetson and medzhitov , 2006a ; kawai and akira , 2009 ) . 
 furthermore , synthesized dna stimulates the innate immune system and acts as a good adjuvant to induce the efficient induction of acquired immune responses ( ishii et al . , 2008a ) . 
 indeed , tlr9 , the receptor for single - stranded dnas containing unmethylated cpg motifs , is involved in the protection of hosts suffering dna virus infection , and the ligands for tlr9 efficiently induce acquired immune responses upon vaccination . 
 however , dsdna derived from bacteria and dna viruses , as well as host genomic dna from dying cells , could induce the expression of both type i ifns and ifn - inducible genes via a tlr - independent pathway ( okabe et al . 
 , 2005 ; ishii et al . , 2006 ; stetson and medzhitov , 2006b ; stetson et al . , 2008 ) . 
 although the specific sensors involved in dsdna - induced innate immune responses are still unclear , recent studies have revealed that tbk1 ( tank - binding kinase 1 ) and stimulator of ifn genes ( sting ; also called mpys / mita / eris ) , function as mediators in dsdna - induced production of type i ifn ( ishii et al . , 2006 
 ; stetson and medzhitov , 2006b ; ishikawa and barber , 2008 ; jin et al . , 2008 
 are also required for the efficient induction of acquired immune responses by dna - based vaccines and play a key role in host defense against the dna virus herpes simplex virus 1 ( ishii et al . , 2008b ; ishikawa et al . , 2009 ) . 
 upon stimulation by dsdna , tbk1 phosphorylates the transcription factor irf3 , leading to the activation of ifn - stimulated response element  dependent transcription of type i ifn / ifn - inducible genes . sting is a multispanning membrane protein that associates with tbk1 to mediate dsdna - induced signaling pathways . 
 recently , we and other groups reported that the translocation and assembly of these essential signal transducers ( sting and tbk1 ) are required for dsdna - triggered innate immune responses ( fig . 
 after stimulation with dsdna , sting moves from the er to the golgi apparatus , finally reaching the cytoplasmic punctate structures to assemble with tbk1 . 
 exoc2/sec5 , a component of the exocyst complex responsible for targeting exocytic vesicles to specific docking sites on the plasma membrane , colocalizes with sting and regulates innate immune responses to dsdna . 
 thus , it is clear that a membrane trafficking system mediates the dynamic movement of sting , leading to the efficient induction of innate immune responses to dsdna . 
 we assessed involvement of atgs in the translocation of sting and found that , after dsdna stimulation , sting colocalizes with atg9a in the golgi apparatus and with lc3 in cytoplasmic punctate structures but not with other atgs ( fig . 
 characterization by electron microscopy revealed that the sting - positive puncta induced by dsdna stimulation did not have the morphological characteristics of autophagosomes but were unidentified membrane - bound compartments , suggesting a unique function of atg9a or lc3 in the regulation of innate immune responses . 
 atg9a is the only multispanning membrane protein identified as an atg in mammals and localizes to the golgi apparatus and late endosomes ( yamada et al . , 2005 ; webber and tooze , 2010 ) . 
 atg9a does not reside at one site , but rather , it dynamically cycles between these organelles under starvation conditions , mediating membrane transport to generate autophagosomes . 
 mice lacking atg9a can not survive neonatal starvation periods , and cells lacking atg9a are deficient in the generation of autophagosomes under both nutrient - rich and nutrient - starved conditions , indicating that atg9a is an essential component of autophagy ( saitoh et al . 
 interestingly , the loss of atg9a greatly enhances the assembly of sting and tbk1 triggered by dsdna , leading to aberrant activation of the innate immune response ( fig . 4 c ) . 
 however , both the localization of sting and cytokine production are almost normal in dsdna - stimulated mefs lacking either atg7 or atg16l1 . 
 thus , deficiency of atg9a , but not of autophagy , affects the movement of sting and tbk1 after dsdna stimulation . 
 further studies are needed to reveal the unique function of atg9a involved in the regulation of the membrane trafficking responsible for the assembly of sting and to assess importance of atg9a in host defense against dna viruses and pathogenic bacteria . 
 recent advances in the study of the involvement of atgs in innate immunity have clearly demonstrated the importance of membrane trafficking systems to host defense 
 . however , in some cases , it is still unclear whether autophagy or atgs are involved in the control of prr - mediated innate immune responses . 
 furthermore , it is difficult to judge whether the observed phenotypes represented by a deficiency of atgs are caused by the inhibition of transiently induced autophagy because autophagy is constitutively induced , even under normal conditions , and maintains cellular homeostasis , the disruption of which often affects the regulation of innate immune responses . 
 for example , sqstm1/p62 , accumulated through a deficiency in autophagy , might indirectly affect innate immune responses by inhibiting the function of the ubiquitin ligase keap1 , leading to the activation of nf - e2related factor 2 , a transcription factor involved in the regulation of prr - mediated innate immune responses ( bjrky et al . 
 , 2005 ; thimmulappa et al . , 2006 ; komatsu et al . , 2007 , 2010 
 in future studies , it will be important to identify the precise molecular mechanisms underlying the disruption of prr - mediated innate immune responses because of the loss of atgs . 
 although the role of atgs in host defense responses is extensively investigated in vitro , it is important that future studies assess its role in vivo . 
 this is because certain cell types are difficult to maintain in vitro and because atgs might be required for the development of unique but important cell types in vivo . indeed , cadwell et al . 
 ( 2009 ) reported an essential role for atgs in the development of paneth cells , intestinal epithelial cells that produce the antimicrobial peptides defensin via nod2 ( kobayashi et al . 
 interestingly , t300a mutation of the atg16l1 protein disrupts the development of paneth cells in patients suffering from crohn s disease , suggesting that atg16l1 is important for the development of this inflammatory disorder ( cadwell et al . , 2008 ) . 
 furthermore , it is possible that the loss of atgs by a particular cell type in vivo indirectly affects innate immune responses . 
 for example , hepatocyte - specific deletion of atgs results in compromised lipid metabolism , which controls the level of triglycerides and cholesterol ( shibata et al . , 2009 ; 
 singh et al . , 2009 ) . altered lipid storage in the liver might indirectly affect the innate immune response because oxidized lipids and cholesterol crystals are potent inducers of inflammation ( febbraio et al . 
 , 2000 ; ricci et al . , 2004 ; duewell et al . , 2010 ; stewart et al . , 2010 ) . 
 specific knockout mice are required for the detailed analysis of atg - dependent regulation of the innate immune response in vivo . 
 recent advances in the study of atgs provide strong evidence that atgs influence both the host defense response and the regulation of inflammation . 
 consistently , it has been proposed that the compromised function of autophagy regulators results in the development of immune - related disease such as crohn s disease ( hampe et al . 
 . therefore , enhancement of autophagic activity is an attractive approach to protect the host against infectious diseases and inflammatory disorders . 
 it would be of interest to assess the therapeutic effects of strategies to increase autophagic function , such as a hypocalorific diet and autophagy - inducible drugs , in the treatment of these diseases .","pattern recognition receptors detect microbial components and induce innate immune responses , the first line of host defense against infectious agents . 
 however , aberrant activation of immune responses often causes massive inflammation , leading to the development of autoimmune diseases . 
 therefore , both activation and inactivation of innate immune responses must be strictly controlled . 
 recent studies have shown that the cellular machinery associated with protein degradation , such as autophagy , is important for the regulation of innate immunity . 
 these studies reveal that autophagy - related proteins are involved in the innate immune response and may contribute to the development of inflammatory disorders .",pubmed
"the association of this habit with oral cancer was noted over a century ago ; yet the chewing of betel quid in india was considered to be a comparatively innocuous practice with complete socio - cultural acceptance . in recent years it has emerged as a significant public health problem in india 
 . the normal cultural practice of betel nut ended up to a health hazard when tobacco was introduced by the european settlers four centuries ago . 
 in addition to its use for smoking , as the europeans demonstrated , tobacco somehow got mixed with betel quid and prepared to be chewed . in india 
 tobacco was culturally accepted as it was used with a culturally accepted betel quid substance . during the second half of the 20 th century , 
 the causal association between chewing of betel quid with tobacco and oral cancer was well established , so it could no longer be regarded as a completely innocuous practice . 
 the major culprit was clearly tobacco in the quid ; however , probably because of its ' exotic ' appeal , areca nut was erroneously thought of as playing a more important role . 
 betel nut was reported safe in many studies where only tobacco in the quid was held responsible for oral cancers . 
 later it was clear that betel nut chewing was responsible for oral submucous fibrosis ( osmf ) , an established precancerous condition with trismus . 
 osmf was a rare disease in the early 80 s , occurring generally among older age groups who chew betel quid frequently . 
 a later incidence of osmf increased amongst the indian young population as a result of increasing gutkha chewing . 
 the frequency of oral cancer has increased among young adults as a result of increasing in number of osmf ( a precancerous condition ) . 
 the increasing incidence of osmf and oral cancers during the last two decades was due to industrial manufacturing and marketing of these chewable mixtures gutkha . 
 the incidence of oral cancer is 12.5/100,000 and the largest numbers of oral cancer patients are present in india . in india , 
 this epidemic proportion of oral cancer in india is due to the rampant use of betel nut , betel quid , and gutkha and pan masala chewing which are proven to be carcinogens . 
 oral cancer is uncommon disease in the west but continue to be the major cause of cancer related death in indian men . 
 over last few decades , large funds have been spent to educate public , implement laws effectively , rehabilitate tobacco growers , build cessation facility , create health care infrastructure , etc to reduce the betel nut , betel quid , gutkha and other forms of chewable tobacco usage . 
 this is a retrospective study of 63 cases of oral cancer patients who were managed in kvg medical college sullia . 
 sixty three patients , 38 males and 25 females , were included in our study . 
 the youngest patient was 47 years old and the oldest was 73 . a detailed history of the patients was taken plus oral and oropharyngeal examination . 
 chest x - ray along with ap neck x - ray , lateral view and panoramic view were taken ( figure 1 ) . 
 the biopsy of the ulceroproliferative mass was taken ; histopathology of all specimens showed squamous cell carcinoma . 
 tnm staging ( tumor size , lymph nodes , metastasis ) showed most of the cases were in advanced stages . 
 all of 63 patients were managed according to the oral cancer management protocol and all signed written informed consent . 
 the mean age in males was 61.20 years and female median age was 59.92 years . 
 the mean duration of betel chewing in males was 18.56 years and in females was 20 years ( figure 2 ) . 
 all patients in our study changed to chewing packaged panmasala and gutkha which were easily available . 
 the mean duration of panmasala and gutkha chewing in males was 8.66 years and in females was 7.36 years ( figure 3 ) . 
 thirteen males ( mean 11.2 years ) in our study were tobacco smokers and 12 males ( mean 9.4 years ) were occasional alcohol drinkers . 
 twelve males ( mean 10.3 years ) and 14 females ( mean 8.4 years ) were workers in tobacco processing industry . 
 ischemic heart disease and hypertension were seen in 22 patients ( 16 males and 6 females ) . 
 oral submucous fibrosis and leukoplakia were excluded from the study after histopathological examination ( figure 4 ) . out of 63 cases , 14 had lip carcinoma , 41 with carcinoma of the buccal mucosa and 7 had carcinoma of tongue and there was 1 case of carcinoma of upper alveolus . 
 out of 14 cases of carcinoma of lip , 2 cases were in stage1 , 3 cases in stage 2 , 7 cases in stage 3 , and 2 cases in stage 4 . 
 out of 41 cases of buccal mucosa carcinoma , 4 were in stage 1 , 11 in stage 2 , 18 in stage 3 , and 8 cases in stage 4 ( figure 5 ) . 
 out of 7 cases of tongue carcinoma , 2 were in stage 1 , no cases in stage 2 , 2 cases in stage 3 , and 3 cases in stage 4 ( figure 6 ) . 
 the case of carcinoma of upper alveolus refused treatment and was lost in follow up ( figure 7 ) . 
 all stage 1 oral cancer patients in our study were treated by wide excision only and followed up for 6 months to 2 years . 
 three cases of lip carcinoma with stage 2 were treated by wide excision and postoperative radiotherapy ( figure 8) . 
 two cases of buccal mucosa carcinoma with stage 2 were widely excised and were sent for postoperative radiotherapy , 8 patients were sent for radiotherapy as they refused surgery and 1 refused radiotherapy ( table 1 ) . out of 7 cases of lip carcinoma with stage 3 , 3 patients refused treatment and 4 had wide excision with neck dissection and postoperative radiotherapy . 
 out of 18 cases of carcinoma of buccal mucosa stage 3 , 7 patients underwent wide excision with neck dissection plus radiotherapy , 11 refused surgery so they were referred to radiotherapy . 
 one case of carcinoma stage 3 had wide excision with radiotherapy and the other refused surgery but took radiotherapy . 
 two cases of lip carcinoma with stage 4 refused surgery so they were given radiotherapy . 
 all buccal mucosa carcinoma patients with stage 4 refused surgery but only 2 of them received radiotherapy . 
 a direct relation between oral cancer and duration of betel quid , gutkha and panmasala chewing was seen in our study . 
 betel nut may be consumed alone or as a betel quid in combination with betel leaf and slaked lime and may contain other substances like tobacco , catechu , spices or sweeteners [ 9 , 10 ] . 
 betel leaf that is commonly consumed with this nut contains a known carcinogen named safrole [ 9 , 10 ] . 
 the lime in the betel quid enhances the extraction of the betel nut alkaloids by salivary degradation and promotes carcinogenesis [ 9 , 10 ] . 
 the alkaloids in betel nut which are responsible for the biological actions in humans are arecoline , arecaidine , guvacine , and guvacoline [ 9 , 10 ] . asians and indians even after immigrating to western countries still continue the habit of betel nut chewing which show an addiction among these populations [ 9 , 10 ] . 
 the betel nut derived alkaloids are cholinergic agonists and they affect nervous system via acetylcholine . 
 prolonged use of betel nut causes peculiar facial and systemic features , staining of teeth , attrition of teeth , and creation of wear facets with higher incidence of periodontitis . 
 betel nut for consumption is obtained from areca catechu which is the fourth most common addictive substance after tobacco , alcohol , and caffeine . 
 it is possibly the second most consumed carcinogen after tobacco in the indian subcontinent . in india 
 , betel nut is chewed for variety of reasons such as stress reliever , mouth freshener , improving concentration , and for digestion after food . 
 addiction withdrawal symptoms are mood swings , anxiety , and irritability , loss of concentration , sleep disturbance and craving which were seen in most cases in our study . 
 betel nut psychosis has been reported in heavy users following sudden cessation of the habit . 
 india is the greatest producer of betel nut something like 330,000 million tonnes per year ( nearly half of global betel nut production ) . 
 the total area under betel nut cultivation in india is estimated to be around 372,000 hectares and total production is 340,000 tonnes . 
 it is estimated that nearly 10 million people depend on betel nut industry for their livelihood in india . 
 most of the betel nut production is consumed within india because it has limited export potential . 
 betel nut products ( gutkha , panmasala ) are being advertised as mouth fresheners which are misleading indian youth . 
 gutkha and panmasala are more dangerous than betel nut alone because they are kept in mouth for a longer time . 
 the other promoting factors in this industry are lack of statutory warning , low cost , easy availability , attractive packaging and aggressive marketing . 
 panmasala is basically a preparation of betel nut , catechu , cardamom , lime and a number of natural , artificial perfuming and flavoring materials . 
 gutkha is a variant of panmasala , in which in addition to these ingredients flavored chewing tobacco is added . 
 both products are often sweetened to enhance the taste .betel leaf is perishable and preparation of betel quid is somewhat complex or requires visits to shops selling pan / bq . with the emergence of commercial pan masala and gutkha about three decades ago , not only did the indian market witness massive growth in the sales of smokeless tobacco and betel nut products , but also a huge worldwide export market developed . 
 all our patients earlier chewed betel quid but turned to gutkha , pan masala chewing over the last two decades . just like tobacco , 
 the cancer causing properties of betel nut have been well - reported in animal , human and epidemiologic studies . 
 cancers caused by betel nut chewing also shows a strong dose - response relationship for frequency and duration of chewing . 
 n - nitroso compounds converted from betel nut alkaloids are responsible for oral and pharyngeal cancer . also betel nut chewing ( with or without tobacco ) 
 is associated with hepatocellular carcinoma , cholangiocarcinoma , cancers of the larynx , stomach , lung and cervix in humans . 
 betel nut is a synergistic carcinogen along with tobacco with which it is commonly consumed in india . 
 all patients in our study had betel quid and gutkha panmasala chewing as the etiology with smoking , alcohol consumption and working in tobacco processing factories as other etiologies . the who and international agency for research on cancer classified betel nut as a group 1 human carcinogens with sufficient evidence of increased risk of osmf ( precancerous oral lesion ) and cancers of the oral cavity , pharynx and esophagus [ 17 - 20 ] . 
 cancers caused by betel nut chewing also shows a strong dose - response relationship for frequency and duration of chewing . 
 it is postulated that betel nut - specific n - nitroso compounds converted from alkaloids are responsible for oral and pharyngeal cancer . 
 there are several other reports that have linked betel nut chewing ( with or without tobacco ) with hepatocellular carcinoma , cholangio - carcinoma , laryngeal cancers , stomach , lung and cervix in humans . 
 studies in mice have shown that , betel quid extract given by gavages produced carcinoma of lung , stomach , liver and salivary gland . 
 fibrosarcomas at the injection site were observed by subcutaneous injections of betelnut extracts in mice . 
 animal research has shown that panmasala in the diet led to tumors in various organs , most frequently adenocarcinoma of lung . in another experiment , 
 arecoline was given as gavage and it produced carcinoma of lung , stomach and hemangiomas of liver . 
 esophageal cancers increased 195 times , pharyngeal cancers 96.9 times and laryngeal cancers 40.3 times when betel quid chewers had habits of smoking and drinking . 
 betel quid chewing resulted in a statistically significant increase in the risk of overall mortality and cerebrovascular deaths in the elderly population [ 22 , 23 ] . 
 betelnut addiction has also been found to be associated with heart attacks , arrhythmia , metabolic syndrome and diabetes [ 24 - 27 ] . 
 chronic betel nut chewers women give birth to low weight babies similar to smoker women [ 29 , 30 ] . 
 betel nut and betel quid chewing has been a popular oral habit in india and many other southeast asian countries for a long time [ 31 , 16 ] . 
 betel nut , betel quid and gutkha chewing habit has become the top five oral habits ( including smoking , alcohol , tea , coffee , and betel chewing ) in the world . 
 there are about 200 - 600 million betel nut , betel quid and gutkha chewers in the world [ 28 , 29 ] . 
 they are proven carcinogens resulting in oral cancer , oral precancerous lesions , hepatocarcinoma , diabetes mellitus ( dm ) , cardiovascular diseases and other systemic diseases [ 31 , 16 ] . 
 india accounts for 1/3 of an estimated 3 million betel and tobacco related deaths in the world per year . in 2001 , the sale of cigarettes was banned to people under 18 . in view of high usages of betel nut , betel quid and other tobacco containing products such as gutkha and their proven role as a carcinogen , it is necessary to look into the hazardous effects of these addictions and their impact on health and society . 
 so a ban on betel nut , betel quid and tobacco in any form is immediately necessary to eradicate the menace of the morbidity . 
 panmasala and gutkha have flooded the indian market as cheap and convenient betel quid substitutes and become popular across all age groups wherever betel nut is abundantly used . 
 there is sufficient evidence that chewing of betel nut with or without tobacco and lime are carcinogenic in humans . 
 some restrictions on the manufacture and sale of these products are being implemented but because of seriousness of the situation , a permanent ban on gutkha and panmasala is necessary as they are well - established oral cancer causing agents . 
 an emphasis on health education aiming to reduce and later eliminating betel nut and tobacco products should be placed . 
 panmasala and gutkha have flooded the indian market as cheap and convenient betel quid substitutes and become popular across all age groups wherever betel nut is abundantly used . 
 there is sufficient evidence that chewing of betel nut with or without tobacco and lime are carcinogenic in humans . 
 some restrictions on the manufacture and sale of these products are being implemented but because of seriousness of the situation , a permanent ban on gutkha and panmasala is necessary as they are well - established oral cancer causing agents . 
 an emphasis on health education aiming to reduce and later eliminating betel nut and tobacco products should be placed .","backgroundbetel nut and betel quid chewing are from major etiological factor for oral cancer . 
 they also increase the risk of systemic diseases such as asthma , diabetes mellitus , metabolic syndrome , myocardial infarction , hypertension , and other cardiovascular diseases . 
 methodssixty three patients of oral cancer in our institution during jan . 
 2007 to may 2011 were included in our study . 
 this study included 14 cases of lip carcinoma , 41 cases of buccal mucosa carcinoma , 7 cases of tongue carcinoma , and 1 case of carcinoma of upper alveolus . 
 duration of use of betel quid , pan masala , and gutkha were studied as well as management.resultsall patients in our study have been chewing betel - quid for 6 - 31 years ( mean19.42 years ) . 
 all of them quit betel quid and used easily available panmasala and gutkha for 4 - 13 years ( mean 8.28 years ) . 
 nine cases of lip carcinoma , 13 cases of buccal mucosa carcinoma and 3 cases of tongue carcinoma were treated with surgery . 
 seven cases of lip carcinoma , 30 cases of buccal mucosa carcinoma and 5 cases of tongue carcinoma were treated with post operative or palliative radiotherapy.conclusionbetel nut chewing with or without tobacco and lime are proven to be carcinogens in human . direct relationship between oral cancer and betel quid , gutkha , and panmasala use 
 has been shown in our study . as betel quid , panmasala and gutkha chewing 
 were proven to be carcinogens , a permanent ban on manufacturing and sale of these products should be implemented .",pubmed
"the concept of patient blood management is gaining increased attention . during the last decade numerous reports have emphasised the need for reductions in transfusions of blood and blood products as allogeneic red blood cell ( rbc ) transfusions are associated with increased morbidity and mortality [ 110 ] , increased risk of severe postoperative infections , adverse effects or risk of transferring pathogens [ 11 , 12 ] , relatively high costs , and shortage of blood bank products [ 6 , 1214 ] . 
 postoperative severe bleeding is relatively common following cardiac surgery compared to other surgical specialties and is considered a serious complication associated with increased morbidity and mortality [ 1421 ] . within the cardiac surgery population , 
 patients with advanced age and long cardiopulmonary bypass ( cpb ) are especially at risk of postoperative bleeding [ 18 , 19 ] . 
 moreover , it is well known that excessive bleeding may be caused by surgical factors and impaired haemostasis due to enhanced fibrinolysis , platelet dysfunction , haemodilution , acidosis , hypothermia , and consumption of coagulation factors as well as the surgical trauma alone [ 22 , 23 ] . during recent years 
 several cardiac surgery studies have reported on the long term mortality after transfusion with blood and blood products [ 610 ] . 
 although not all , the majority of studies report a higher long term mortality after blood transfusion [ 6 , 9 , 10 , 24 ] ( figure 1 ) . 
 however , the reports are mainly from designated types of surgery [ 79 ] or single centre studies [ 6 , 8 , 9 ] . in some studies 
 the kaplan - meyer plots tend to run parallel after the first month when including the immediate postoperative mortality , indicating less impact on the long term survival [ 6 , 9 , 10 ] . 
 one of the last published evidence based guidelines for the transfusion of rbc concluded that  red blood cell transfusions should not be dictated by a single haemoglobin ( hb )  transfusion trigger  , but instead should be based on the patient 's risk of developing complications of inadequate oxygenation .  furthermore , that rbc transfusion would generally be indicated , but not mandatory , at hb levels lower than 6.0  g / dl ( 3.7  mmol / l ) and rarely indicated in patients with haemoglobin higher than 10.0  g / dl ( 6.1  mmol / l ) . in many types of major surgery 
 these recommendations have been followed and the use of transfusions reduced . however , in cardiac and vascular surgery the use of blood and blood products is still common clinical practice . from a survey of cardiac procedures covering monitoring , 
 anaesthesia and transfusions carried out in 2005 , it was found that almost 40% of european institutions used blood or blood products in more than half of their cardiac surgery patients . 
 when calculating the number of surgical procedures in each institution and number of patients not receiving blood or blood products , 55.7% of european cardiac surgery patients received blood or blood products in the perioperative period . 
 however , the difference in transfusions from less than 10% of patients to 100% that do receive blood or blood product transfusion ( figure 2 ) is interesting and indicates that transfusions are guided by local policies and not evidence based practice . 
 patients referred for coronary artery bypass graft ( cabg ) are more commonly treated with antiplatelet agents especially aspirin and oral adenosine diphosphate ( adp ) receptor antagonists together with newer alternative low molecular heparin drugs , like fondaparinux . 
 long term aspirin therapy is the standard of care in patients with coronary artery disease , while concomitant treatment with oral adp receptor antagonists is recommended for patients with recent acute coronary syndrome ( acs ) or percutaneous coronary intervention ( pci ) [ 2729 ] . 
 studies have found both reduced mortality and no difference in bleeding   or no difference in bleeding complications in patients treated with antiplatelet drugs [ 31 , 32 ] . 
 this is in contrast to other studies reporting higher transfusion requirements without effect on mortality   or increased bleeding and transfusion as well as increased risk of myocardial infarction . in the crusade study following established guidelines a significant increase in transfusion requirements in patients continuing on clopidogrel was observed compared to patients where the drug was discontinued more than five days before surgery . 
 similarly , large cohort studies found significant increases in major bleeding and reoperation rates in clopidogrel - exposed cabg patients [ 33 , 36 ] . 
 the use of antiplatelet agents at the time of cabg carries both benefits and risks . 
 the drugs are effective in reducing ischemic events in high - risk patients awaiting surgery . 
 after surgery drugs may prevent graft occlusion and recurrence of ischemic episodes . however , antiplatelet drugs also aggravate perioperative bleeding followed by increased perioperative blood loss , increased transfusion requirements , and hemodynamic instability [ 35 , 37 , 38 ] . 
 as the indications for oral antiplatelet drugs expand , cardiac surgeons frequently have to make decision about the timing of surgery in patients exposed to these drugs . 
 current international guidelines recommend discontinuing aspirin two to ten days before elective cardiac surgery , while the adp receptor antagonist clopidogrel should be withdrawn at least five days before elective cabg [ 39 , 40 ] . 
 the basic role of blood is to transport and deliver oxygen to the organs to maintain organ function . 
 it is evident that clinical and some hemodynamic measurements are imperative to gain the required information for decision making . 
 even with the most extensive invasive haemodynamic monitoring the obtained information is not fully adequate , as individual organ monitoring is not possible or available in clinical practice . 
 we do have some intervention possibilities for blood pressure , heart rate , haemoglobin level , and volume status and it is possible to operate influence factors such as oxygen saturation , vascular resistance , preload , and cardiac output , while organ blood flow and organ oxygenation are difficult to influence directly . 
 the correlation between blood flow , oxygen delivery , and consumption together with haemoglobin can easily be visualised ( figure 4 ) . 
 the figure demonstrates that oxygen delivery in principle is determined by only three variables , cardiac output ( co ) , haemoglobin ( hb ) level , and oxygenation ( sao2 ) . 
 . if oxygen delivery is out of balance with oxygen consumption , the compensation mechanisms are relatively simple , as a decrease in haemoglobin must result in a compensatory increase in either cardiac index or oxygen extraction or a combination . 
 furthermore , the relation is independent of the fact that co changes with age   and that the ratio of co to total blood volume changes from roughly 3  :  1 in small children to 2  :  3 or lower in the elderly cardiac patient . 
 a valuable parameter in evaluating need for inotropic support or rbc transfusion is whether oxygen consumption is adequately covered by oxygen delivery . in general , 
 when oxygen consumption is high ( i.e. , during exercise ) the increased oxygen requirement is usually provided by an increased cardiac output . however , low cardiac output , low haemoglobin concentration ( anaemia ) , or low haemoglobin o2 saturation will result in an inadequate delivery of oxygen , unless a compensatory change occurs in one of the other factors . alternatively , 
 if oxygen delivery falls relative to oxygen consumption , the tissues extract more oxygen from the haemoglobin and the saturation of mixed venous blood ( svo2 ) falls below 70% ( a - b in figure 5 ) . a reduction below point 
 c  in figure 4 can not be compensated by an increased oxygen extraction and results in anaerobic metabolism and lactic acidosis , and oxygen consumption becomes totally dependent on oxygen supply . 
 oxygen is carried in the blood in two forms : primarily by haemoglobin but a very small amount is dissolved in the plasma . when fully oxygen saturated ( po2 > 13.3  kpa ) only 3  ml of oxygen will be dissolved in every litre of plasma . if the po2 of oxygen in arterial blood ( pao2 ) is increased significantly ( breathing 100% oxygen ) , a small amount of extra oxygen will dissolve in the plasma at a rate of 0.023  ml o2/100  ml blood / kpa po2 . 
 normally , there will be no significant increase in the amount carried by haemoglobin if oxygen saturation is already higher than 95% . in the literature 
 there is some indications that patients with coexisting cardiac diseases may have a particular risk of developing impaired oxygenation at lower haemoglobin levels , but the documentation for this is not that convincing . in patients undergoing cardiac surgery 
 studies of cardiac surgery patients have shown increased mortality after rbc transfusions [ 42 , 43 ] . 
 further , others have found that lowering of the hb threshold for transfusion in patients undergoing coronary artery bypass surgery ( cabg ) from 9 to 8  g / dl ( 5.5 to 5.0  mmol / l ) was neither followed by a higher mortality nor followed by an increase in adverse effects by accepting the lower postoperative haemoglobin . 
 this indicates that patients with coexisting cardiac diseases , including coronary artery disease ( cad ) , tolerate moderate anaemia . 
 another study showed that patients may even benefit from a restrictive transfusion regimen followed by a lower morbidity and mortality . 
 the mean haemoglobin at transfusion in this study was 8.4  g / dl ( 5.1  mmol / l ) and both icu and overall mortality rate were higher in transfused patients . 
 the findings were further supported in an icu study where patients with known cardiovascular disease had equal level of mortality and less organ dysfunction if in a transfusion restrictive group . 
 in contrast , one study indicated that rbc transfusions may decrease mortality in elderly patients with acute myocardial infarction and an admission haematocrit below 30% . 
 another study shows increased in - hospital mortality in patients with lower preoperative haemoglobin levels . 
 however , the first study is not without methodological challenges and needs careful analysis as patients with normal haemoglobin levels seemed to be treated more intensely . 
 previously , we have demonstrated a correlation between preoperative haematocrit ( hct ) and 30-day mortality . 
 however , when compensated for euroscore [ 4850 ] the 30-day mortality was independent of haemoglobin level and only correlated with the euroscore . 
 the data showed a strong relationship between actual rbc transfusion and residual euroscore ( euroscore minus age factor ) and interestingly also the lack of correlation between age higher than 74 years and rbc transfusion ( table 1 ) . 
 the first priority of perioperative fluid therapy in surgical patients is to achieve optimal filling of the heart with crystalloids and colloids to optimise cardiovascular function , decrease postoperative morbidity , and shorten the length of hospitalisation [ 51 , 52 ] . in cardiac surgery patients and many intensive care patients with pao2 
 > 95% , optimisation of arterial saturation only adds little to oxygen delivery , leaving only increasing co or hb as possible means to increase oxygen delivery . 
 infusion of colloids reduces the hb level and thus , despite unchanged total haemoglobin content , oxygen delivery in theory . 
 however , the impact on co normally compensates for that and in hypovolaemic patients especially the overall impact may be substantially higher co and thus oxygen delivery . following infusion of rbc in this type of patients 
 may have a double effect on oxygen delivery as both hb and co are increased . 
 in contrast , in severely overloaded patients where further compensation in cardiac output is compromised or even deteriorated after infusion of colloid or rbc , there might be no effect on the oxygen delivery . in these cases 
 many cardiac or critically ill patients are not monitored with pulmonary artery catheters and hence information of co and svo2 is not available . 
 central venous oxygen saturation ( scvo2 ) obtained from the superior vena cava has been proposed as a surrogate for svo2 and may reflect the balance between oxygen supply and demand . 
 studies have shown that the difference between scvo2 and svo2 is consistently about 5% across a wide range of cardiorespiratory conditions in both animals and humans [ 53 , 54 ] . 
 however , scvo2 obtained from a central venous catheter is not fully interchangeable with mixed svo2 , but it can give some indication of oxygen balance in patients with peripheral saturation , co and hct within normal limits . 
 one of the principal differences in cardiac surgery compared to other types of major surgery is the use of cardiopulmonary bypass ( cpb ) . 
 this leads to a 2030% haemodilution and when weaning from cpb a relatively big portion of red blood cells ( rbc ) are left in the heart lung machine ( hlm ) ( figure 6 ) . 
 although the surgeon is very careful during the procedure , a smaller portion of blood is always lost . 
 using either cell - saver or retransfusing of suctioned blood will , however , limit the total loss . if using autotransfusion in the postoperative phase the perioperative blood loss during cardiac surgery can be diminished to approximately one unit of rbc ( scenario a ) and there is thus no need for transfusion . 
 however , if none of these procedures are used , the overall result will be a considerable blood loss and a low postoperative haematocrit level ( scenario b ) . 
 consequently , the patient will inevitably receive 13 units of rbc . if the patient continuously has problems with bleeding or haemodynamic instability , the risk of adding plasma or platelets is reasonably high . 
 some studies have found low haemoglobin levels during cpb to be associated with increased postoperative morbidity and mortality [ 56 , 57 ] while other studies have not been able to find such a correlation [ 43 , 58 ] . 
 patients with coronary artery disease ( cad ) may have an increased risk of myocardial ischemia in combination with low haemoglobin levels . 
 however , experimental and clinical data indicate that persons with cad tolerate moderate normovolaemic haemodilution well [ 5962 ] . 
 interestingly , data from our database show that patients with cabg received less blood and blood products ( table 2 ) . regional changes in 
 myocardial systolic and diastolic contractile dysfunction [ 63 , 64 ] and ecg signs of myocardial ischemia [ 6568 ] may develop in areas supplied by a compromised coronary artery at low haemoglobin levels . additionally , haemoglobin levels below 6.0  g / dl ( 3.7 
 mmol / l ) have been associated with increased postoperative mortality in patients with coexisting cardiovascular disease , including cad . during 
 both haemodilution   and blood transfusion   patients with a left ventricular ejection fraction between 25% and 85% responded similarly . 
 this indicates that also patients with a reduced left ventricular ejection fraction are able to compensate with increased cardiac output in response to haemodilution if normovolaemia is maintained . 
 however , the number of patients with low ejection fractions was limited and the findings should not necessarily be extrapolated to the low range of patients with left ventricular ejection fraction . 
 patients above 65 years without known cardiac disease tolerated haemodilution to a haemoglobin of 8.8  g / dl ( 5.4 
 beta - blocked cad patients between 35 and 81 years responded adequately during haemodilution with an increase in cardiac output and oxygen extraction . 
 similarly , during blood transfusion the compensatory changes in cardiac output , oxygen delivery , and oxygen consumption seemed to be independent of age ( 32 to 81 years ) . 
 however , this study had relatively few patients above 75 years , stressing that these findings may not apply to the very old patients . 
 patients with significant mitral insufficiency , including those with atrial fibrillation , tolerate moderate haemodilution to a haemoglobin level of 10  g / dl ( 6.1  mmol / l ) . 
 tolerance of acute haemodilution or anaemia in patients with other valve abnormalities is less well known . 
 theoretically , patients with aortic and pulmonary stenosis may be less tolerant to haemodilution as the increase in cardiac output during haemodilution may be limited due to the valvular stenosis . 
 however , in the postoperative phase these patients may most likely be treated as patients without coexisting cardiovascular diseases . in patients with coexisting cardiovascular diseases refusing rbc transfusions for religious reasons , postoperative haemoglobin levels below 6.0  g / dl ( 3.7 
 mmol / l ) were associated with increased mortality and morbidity and an increasingly greater difference in mortality and morbidity between patients with and without coexisting cardiovascular diseases . in order to avoid or minimize transfusions in patients with low haemoglobin 
 it is evident that the 2030% haemodilution during cpb might be the primary concern if the patient does not have cardiac capacity to tolerate very low post - cpb haemoglobin or that a needed high htlm cardiac output during cpb may increase perioperative complications . 
 the overall conclusion is that even patients with coexisting cardiac diseases tolerate moderate haemodilution or acute anaemia well if normovolaemia is maintained . 
 however , an excessive and aggressive haemodilution may cause myocardial ischemia which is reversible with a blood transfusion , even in normovolaemic patients [ 64 , 68 , 73 ] , and the lowest acceptable haemoglobin in patients with coexisting cardiovascular diseases is approximately 6.0  g / dl ( 3.7  mmol / l , haematocrit 0.180.20 ) . 
 patients where pre - cpb calculations indicate a lower level during or right after cpb might be the target for preoperative haemoglobin adjustment with erythropoietin with or without autologous blood transfusion . the haemoglobin level at which regional cardiac ischemia may occur varies considerably and depends on both the degree of coronary stenosis   and whether it is a single- or a multivessel cad . 
 patients with or at risk of cad should thus not automatically receive a transfusion at a specific haemoglobin level but only if oxygenation is inadequate as suggested by the american society of anaesthesiologists ( asa ) . 
 the question is whether anaemia - related myocardial ischemia is reversible by blood transfusion , which seems to be the case in some experimental   and clinical [ 68 , 73 ] settings . 
 patients with progressively lower haemoglobin levels also have other risk factors such as diabetes , preoperative congestive heart failure , prior coronary artery bypass operation , low left ventricular ejection fraction , and a higher frequency of emergency surgery . 
 a high correlation has been found between such risk factors and a low haemoglobin level   and the independent contribution of a low perioperative haemoglobin level to mortality is difficult to assess . after adjusting for other risk factors , 
 only a haematocrit below or equal to 14% during cpb remained an independent predictor for increased mortality . 
 only a prospective randomised study design can definitively determine whether varying haemoglobin transfusion triggers affect morbidity and mortality . in a study of icu patients 
 it was demonstrated that a haemoglobin transfusion trigger of 7  g / dl ( 4.3 
 mmol / l ) did not negatively impact on mortality and morbidity , neither in general icu patients nor in patients with coexisting cardiac diseases [ 1 , 45 ] . 
 several studies have shown the importance of controlling haemodynamics during cardiac [ 76 , 77 ] as well as noncardiac surgery . 
 therefore , episodes of tachycardia may occur , which may be associated with st - segment depression , in particular in patients with t low haemoglobin levels . 
 preventing tachycardia and following st - segment depression is thus imperative   in patients with or at risk of cad . 
 in addition , postoperative oxygenation is generally lower than during peroperative mechanical ventilation with a high inspiratory oxygen concentration . 
 therefore , higher postoperative haemoglobin levels of 7 to 8  g / dl ( 4.34.9  mmol / l ) may be justified in patients with coexisting cardiac diseases . 
 the question of when transfusion is appropriate in a patient with a coexisting cardiac disease thus remains unanswered . at a haemoglobin level lower than 6.0  g / dl ( 3.7 
 mmol / l ) blood transfusions may be indicated in most patients and in particular in patients with coexisting cardiac diseases [ 25 , 69 , 80 , 81 ] . 
 furthermore , it has been shown in cardiac surgery patients that oxygen consumption only increased following a blood transfusion when oxygen consumption was very low before blood transfusion . 
 as this remains impossible to determine also in patients with coexisting cardiac diseases , the haemoglobin level , at which a transfusion would generally be indicated , should follow the guidelines from the asa , stating that  red blood cell transfusions should be based on the patient 's risk of developing complications of inadequate oxygenation  . 
 the immediate question is then , what are the signs of a beginning inadequate oxygenation in patients with coexisting cardiac diseases ? 
 inadequate oxygenation may become globally manifested in the form of general haemodynamic instability with a tendency to hypotension and tachycardia despite normovolaemia , oxygen extraction higher than 50% [ 8082 ] , or myocardial ischemia detected by continuous 5-lead ecg monitoring , ideally with automatic st - segment analysis [ 65 , 68 , 83 ] and by new wall motion abnormalities in transoesophageal echocardiography . 
 st - segment depressions above 0.1  mv or new st - segment elevations above 0.2  mv during more than one minute generally are regarded as a marker of myocardial ischemia . during progressive haemodilution , primarily st - segment depression is observed , suggesting subendocardial ischaemia . 
 such anaemia - related ischaemia may , in case of tachycardia , be reversed by decreasing the heart rate   and by increasing the haemoglobin level 1 - 2  g / dl ( 0.61.2 
 new wall motion abnormalities clinically detected by transoesophageal echocardiography may be the result of myocardial ischemia and can be treated by a minor increase in haemoglobin of 1 to 2  g / dl ( 0.61.2 
 early signs of inadequate circulation are general haemodynamic instability characterised by relative tachycardia and hypotension [ 81 , 85 ] together with an oxygen extraction fraction above 50% , a low mixed venous oxygen partial pressure ( pvo2 ) , and a decrease in oxygen consumption [ 80 , 81 ] . an oxygen extraction higher than 50% has been found to indicate exhaustion of compensatory mechanisms in several studies [ 8689 ] and might thus represent a transfusion indication . 
 oxygen consumption decreases very late and at very low haemoglobin levels during progressive normovolaemic haemodilution [ 89 , 90 ] in conditions where oxygen extraction has increased and pvo2 has already decreased . 
 therefore , any decrease in oxygen consumption of more than 10% at low haemoglobin levels   should be viewed as a potential sign of a compromised oxygenation and a blood transfusion should be considered if normovolaemia has been achieved . in patients with coexisting cardiac diseases , 
 the principles of rbc transfusion are not considerably different from healthy patients and must also largely be based on early signs of impaired oxygenation of specific organs or the entire organism . 
 impaired oxygenation may be reached at higher haemoglobin values than in healthy patients and most patients have considerable variations in haemodynamic values . 
 a previous study found considerable interpatient differences and intrapatient variation in patients monitored the night before cardiac surgery ( figure 7 ) . 
 the most pronounced intrapatient variation was found in the cardiac index , ranging from 1.9 to 5.3 l / min / m . 
 most of the patients had periodic spo2 values 92 and half of them in more than 15% of the observations . 
 overall a svo2 < 70% was found in more than 40% of observations and less than 64% in more than 20% of the observations together with drops below 50% without obvious reasons . 
 although the number of patients in the study was low the conclusion was that intrapatient variation was unexpectedly high in most hemodynamic variables . 
 this demonstrated the challenge in using hemodynamic parameters to guide treatment and indicated that goal oriented therapy using currently accepted values may result in overtreatment in some patients . 
 a restrictive rbc transfusion policy has proven equally effective and possibly even superior to a more liberal transfusion strategy . 
 although there currently is no clear explanation for this effect , infections , immunosuppression , and the age of the rbc at the time of transfusion [ 5 , 93 , 94 ] have been suggested as possible impact factors . 
 the clinical consequence of the storage lesions is a reduced survival time of red blood cells after the transfusion . whether stored rbc is also compromised in transporting oxygen to tissue is still controversial . 
 several clinical [ 93 , 96 , 97 ] and experimental animal studies [ 98 , 99 ] support the notion that rbc older than 3 weeks have a reduced ability to transport oxygen to the tissues . 
 animal studies have found a malperfused and underoxygenated microvasculature after haemodilution   and a 26% decrease in micro vascular oxygen concentration after transfusion with washed human rbcs in haemodiluted rats . in human a recent study 
 has shown increased risk of severe postoperative infections after cardiac surgery associated with the use of old rbc . 
 however , the findings are not uniform as a recent clinical study could not demonstrate an effect of stored blood on oxygenation parameters   and another did not observe an increase in morbidity in coronary artery bypass graft surgery patients receiving old rbcs   or could not find a correlation between median ages of the rbc units and clinical outcomes 
 . these differences in study outcomes may be explained by the inherent difficulty to measure tissue oxygen concentrations in the clinical setting . to estimate the effect of rbc transfusion on tissue oxygenation in patients , indirect measuring techniques have been used as surrogate endpoints . in addition , the diversity in the design of the performed studies complicates a proper comparison of their results . 
 there seems to be an agreement that when a substantial part of the units issued for transfusion is stored for more than 3 weeks , a discussion about fresh and stored rbc could be clinically relevant . 
 the evidence supports that each institution establishes its own patient blood management strategy to both conserve blood products and maximise outcome . 
 newer transfusion guidelines in patients with coexisting cardiac diseases are similar to those in patients without such comorbidities . 
 thus , rbc transfusions are most often indicated at haemoglobin levels below 6.0  g / dl ( 3.7  mmol / l ) and hardly ever at haemoglobin levels above 10  g / dl ( 6.1  mmol / l ) . in cardiac surgery , especially in cad patients , the lower limit should probably be elevated to 7  g / dl ( 4.3  mmol / l ) , though no available hard evidence . 
 institutions should evaluate how they approach transfusion and each physician should evaluate each patient for signs and symptoms of ischemia or imbalance in oxygen supply and delivery , where the major challenge is how to decide when that imbalance is occurring in certain tissues . evidently such signs may be reached at higher haemoglobin values than in healthy patients , but further studies are needed to fully confirm that transfusing these patients at a higher haemoglobin level positively is making more good than harm . following this 
 teams and institutions need to conduct open discussions about how they will approach patient blood management and instead of previous fixed haemoglobin levels the best practice would be individual haemoglobin / haematocrit triggers . 
 strategy of transfusion : make local policy and guidelines for transfusion including 
 preferably developing an algorithm for transfusioneducation and knowledge of physicians and nursespolicy on blood saving precautions , mechanical or medicalevaluation of cost of blood / blood products and cost alternativesavailability of blood and blood productstransfusion of only actual bleeding and/or unstable patientssafety related to needaffection of immune system acceptable ? 
 preoperative evaluation 
 deciding blood saving precautionscardiovascular reservedefining acceptable blood loss / haematocrit / absolute haemoglobin content . 
 evaluate perioperative 
 haematocrit look at it  do not treat it butevaluate the absolute amount of haemoglobin . 
 strategy of transfusion : make local policy and guidelines for transfusion including 
 preferably developing an algorithm for transfusioneducation and knowledge of physicians and nursespolicy on blood saving precautions , mechanical or medicalevaluation of cost of blood / blood products and cost alternativesavailability of blood and blood productstransfusion of only actual bleeding and/or unstable patientssafety related to needaffection of immune system acceptable ? 
  preferably developing an algorithm for transfusion education and knowledge of physicians and nurses policy on blood saving precautions , mechanical or medical evaluation of cost of blood / blood products and cost alternatives availability of blood and blood products transfusion of only actual bleeding and/or unstable patients safety related to need affection of immune system acceptable ? 
 preoperative evaluation 
 deciding blood saving precautionscardiovascular reservedefining acceptable blood loss / haematocrit / absolute haemoglobin content . 
  deciding blood saving precautions cardiovascular reserve defining acceptable blood loss / haematocrit / absolute haemoglobin content . 
 evaluate perioperative 
 haematocrit look at it  do not treat it butevaluate the absolute amount of haemoglobin . 
 haematocrit look at it  do not treat it but evaluate the absolute amount of haemoglobin . reevaluate cardiovascular capacity and decide transfusion need .","blood transfusion is associated with increased morbidity and mortality and numerous reports have emphasised the need for reduction . following this 
 there is increased attention to the concept of patient blood management . however 
 , bleeding is relatively common following cardiac surgery and is further enhanced by the continued antiplatelet therapy policy . 
 another important issue is that cardiopulmonary bypass leads to haemodilution and a potential blood loss . 
 the basic role of blood is oxygen transport to the organs . 
 the determining factors of oxygen delivery are cardiac output , haemoglobin , and saturation 
 . if oxygen delivery / consumption is out of balance , the compensation mechanisms are simple , as a decrease in one factor results in an increase in one or two other factors . 
 patients with coexisting cardiac diseases may be of particular risk , but studies indicate that patients with coexisting cardiac diseases tolerate moderate anaemia and may even benefit from a restrictive transfusion regimen . 
 further it has been shown that patients with reduced left ventricular function are able to compensate with increased cardiac output in response to bleeding and haemodilution if normovolaemia is maintained . in conclusion 
 the evidence supports that each institution establishes its own patient blood management strategy to both conserve blood products and maximise outcome .",pubmed
"this process requires proliferation , differentiation and migration of neurogenic stem cells into neurons . for many decades 
 , it was believed that in mammals , neurogenesis occurs only during embryonic life , but a recent work has made it evident that neuronal regeneration occurs even after birth in specific tissues like the hippocampus , dentate gyrus , subventricular zone and olfactory epithelium ( 1 ) . 
 damage to the olfactory periphery destroys the population of olfactory sensory neurons and , in the case of direct epithelial lesion , also eliminates other constituents of the epithelium . in 
 marked contrast to other parts of the nervous system , there is substantial anatomical and functional recovery of the olfactory epithelium and its projection into the cns even in the face of overwhelming injury ( 2 ) . 
 years , neuroscience researchers have searched for an ideal cell with the properties of absolute totipotency and regenerability in vitro and in vivo . 
 science has come very close to the quest , but still many unanswered questions remain . 
 recent studies indicate that transplantation of pure stem cell population is insufficient for attaining the maximum positive effect of neural regeneration . 
 more complete recovery of the structure , sensory and motor functions of injured spinal cord can be attained using a complex of cells , including , apart from olfactory ensheathing cells ( oec ) , fibroblasts , astrocytes , schwann cells , and olfactory epithelial ( oe ) multipotent stem and progenitor cells . 
 hence , the problem of the efficiency and the possibility of using various oe cells and tissues for transplantation therapy in cerebral and spinal injuries doubtlessly requires further experimental studies . 
 nevertheless , consistent efforts with increasing optimistic outcome are being made to identify a candidate ( neuron- progenitor cell ) for a reliable therapeutic intervention of central nervous system ( cns ) injury . in this venture , recent years witnessed an increasing bang on the olfactory epithelium for suitable multipotent stem cells . 
 multiple numbers of pluripotent stem cell candidates have been tried in the past for neuro - regenerative therapies with unconvincing results ( 3 ) . 
 studies involving exposure of olfactory epithelium ( and causing its lesion ) to an irritant like methyl bromide ( mebr ) have shown that the olfactory epithelium is restored its normal status that is indistinguishable from unlesioned epithelium within 68 weeks after damage with no relation to the severity of the initial damage which could be up to 90% of the epithelium being destroyed . 
 the first sign of regeneration of olfactory neurons appears on the 4 day after mebr exposure , the first mature neurons emerge during the 2 week after which there is an accelerated production of neurons which falls to normal around the 6 week after the lesion ( 2 ) . 
 the reconstitution of the epithelium is sufficiently robust and precise so that the spatial distribution and the numbers of odorant receptors ( or ) are restored to normal ( 4 ) . 
 transplantation of adult animal and human oe cells have been used in the past for experimental and clinical correction of spinal injuries ( 5 ) . 
 the adult olfactory epithelium ( oe ) is a unique ( for its capacity to renew olfactory receptor neurons throughout adult life ) and a complex tissue containing heterogeneous population of epithelial cells . 
 apart from the support cells and neruoreceptor cells , this complex is said to retain a kind of progenitor cells that are competent to make neurons ( neural stem cells ) and non- neural support cells like olfactory ensheathing cells , oligodendrocytes and schwann cells ( 6 ) . 
 these support cells , play a role in the regeneration and myelination process of normal and of injured cns . 
 recent studies identified stem like characteristics in a group of cells called globose basal cells ( gbcs ) residing in the basal compartment of the olfactory epithelium . 
 the gbcs are the small , round , morphologically non - descript and cyto - keratin negative cells that sit between the horizontal basal cells ( hbcs ) below and the immature olfactory receptor neurons above , that proliferate at a high rate in the normal oe , are limited to the oe , and are poorly characterized at the level of their molecular phenotype . 
 further , a second population of cells that are called the hbcs with stem - cell like properties has been described to reside among the gbcs . 
 therefore , it appears that the olfactory epithelium contains at least two populations of cells with possible neuropotency ( 7 ) . 
 some studies have shown that there is an increase in the population of gbcs and not the hbcs during the regenerative process of the olfactory epithelium ( 8) . 
 gbcs function as broadly multipotent progenitors capable of giving rise to neurons and all of the cell types of the epithelium , and hence may be totipotent stem cells of the epithelium . 
 marker studies using antibodies that are selective for gbcs in normal epithelium , during the acute phase in the recovery after mebr exposure , suggest that the gbcs are differentiating into non - neuronal cell types ( 9 ) . 
 intranasal infusions of gbcs derived from retroviruses , give rise to neurons and multiple types of non - neuronal cells , while other stem cell progenitors give rise only to non - neuronal cells ( 10 ) . 
 gbcs that are separated from the normal epithelium by cell cytometry using a gbc - selective primary antibody and are pure and free from other cells and contaminants , engraft easily into the mebr - lesioned epithelium after infusion into the nasal cavity and give rise to most of the neural and non - neural cells present in the oe ( 11 ) . 
 embryonic stem cells ( 3 ) , haematopoetic stem cells and other mesenchymal stem cells have enormous ability to differentiate into different types of cells in the body ; but they are still the possibilities of immune rejection , adverse effects of immunosuppressive therapy and many ethical issues which exclude them as an ideal candidate for clinical practice ( 12 ) . 
 moreover , these different varieties of stem cells do not reside in a niche similar to the neural stem cells and hence can not proliferate easily in the central nervous system . on the other hand , 
 olfactory epithelial cells share the same environmental niche as the central nervous system and also have the same developmental hierarchy as the neural cells . 
 olfactory mucosa is readily accessible , easily biopsied for autologous cell transplantation and regenerates completely without any loss of function . 
 these properties make the olfactory epithelial stem cells the ideal candidate for use in clinical research and therapy . 
 since the basal compartment contains two morphologically distinct cell types , horizontal basal cells ( hbcs ) and globose basal cells ( gbcs ) , there has been arguments regarding the identification of the neural stem cell population which can not be answered merely based on their location and mitotic activity and studies focusing on search for unique stem cell progenitor markers have to be undertaken ( 8 , 13 , 14 ) . 
 researchers in the recent years have proposed the most likely lineage of the cells in the olfactory epithelium by using specific markers , determination of various transcription factor expressions and incorporation of analytical studies using h - thymidine and bromodeoxyuridine which suggest the proliferative activity of dividing cells ( 15 , 16 ) . 
 it is now clear that immature orns are generated from one of the basal cell types and they in turn give rise to mature orns , but the identity of these cells has to be further characterised . only in limited experiments 
 , it has been shown that gbcs are the cell population to multiply and regenerate vigorously after epithelial damage . 
 there is still an uncertainty of the lineage relationship between the two basal cell populations . in this research 
 , we study different neural progenitor markers and ionic channels that are present on the gbcs and hence characterize these cells as definitive mutipotent stem cells residing in the basal compartment of rat olfactory epithelium capable of transforming into neurons and non - neuronal elements . 
 six albino wistar rats were purchased from the animal facility of christian medical college vellore after obtaining the clearance from the animal ethics committee . 
 albino wistar rats are the most commonly used laboratory animals for genetic and cell line studies as many aspects of their behaviour and physiology are similar to humans and this similarity can be easily observed in them when compared to other animals ( 17 ) . 
 this strain of rats was used in this study since their olfactory mucosa is easily accessible and their olfactory epithelium is histologically similar to that of the human . 
 all animals were weighed prior to the experimental ( 94 -120 g , with an average of 105 g ) . 
 the olfactory mucosa of the rats was obtained by surgical excision under complete , universal sterile conditions . 
 the albino wistar rats were anesthetized by intraperitoneal administration of 90 mg / kg body weight of ketamine and 10 mg / kg body weight of xylazine . 
 the nasal septum was completely excised along three lines : the arc of the perpendicular plate , the cribriform plate and the ceiling of the oral cavity to obtain the olfactory mucosa ( figure 1 ) . 
 the nasal septum along with the olfactory mucosa was placed in frozen dulbeco s modified eagle s medium ( dmem ) containing antibiotics ( penicillin , streptomycin and amphotericin ) . 
 the nasal septum along with the olfactory mucosa was then treated with 0.5 ml of dispase ii and was incubated at 37c for 45 min . 
 the olfactory epithelium was separated from the lamina propria on both sides of the nasal septum using a micro spatula under a leica ez2hd dissecting microscope . 
 the olfactory mucosa was mechanically dissociated with a scalpel blade and treated with 0.05% trypsin and an enzymatic cocktail containing collagenase 1 mg / ml , hyaluronidase 1.5 mg / ml and trypsin inhibitor  0.1 mg / ml to separate the cells . 
 the oe cells were transferred to epithelial culture medium which was modified form of the previously described medium ( 18 - 20 ) . 
 the culture medium was composed of dmem / f12 ( 1 : 1 ) 47.5 ml , 2% fetal bovine serum ( invitrogen , gibco , india ) 
 1.0 ml , n2 supplement ( invitrogen , gibco , india ) 0.5 ml , epidermal growth factor ( 25 ng / ml  invitrogen , gibco , india ) 12.5 l and l - glutamine ( invitrogen , gibco , india ) 0.5 ml . 
 a - arc of perpendicular plate , b - cribriform plate , c - ceiling of oral cavity . 
 om - olfactory mucosa ( yellowish ) , rm - respiratory mucosa ( pinkish ) ten l of gbc iii antibody was added to the epithelial cell pellet under dark conditions and was left in ice for 45 min . 
 gbc - iii is a mouse monoclonal igm antibody which recognizes a 40 kda surface antigen which is a laminin receptor surface protein . 
 it is highly specific as a marker for gbcs , unlike the earlier antibodies used like gbc - i which were nonspecific markers for gbcs and showed positive reaction even with hbcs , sus and duct cells ( 21 ) . 
 gbc - iii in powdered form was reconstituted into the recommended volume of 250 l . 
 ten l of r - phycoerythrin - conjugated affinipure ( fab)2 fragment goat anti - mouse igm ( secondary antibody ) was added to the cell pellet and left under dark conditions in ice for 10 min . 
 fluorescence assisted cell sorting ( facs ) was used to separate the gbcs from other epithelial cells . 
 finally , 99% pure gbcs were obtained using facs and gbc - iii antibody and were plated in the epithelial medium . 
 gbc cell suspension in the epithelial medium was washed 3 times with hanks balanced salt solution . 
 cells were trypsinised using 100 l of 0.1% trypsin / edta and incubated at 37c for 5 min . 
 epithelial medium was added immediately after incubation to neutralize the enzymes and the cell suspension was centrifuged at 1000 rpm for 8 min . 
 a poly d - lysine plated cover slip was placed in a 35 mm petridish . 
 the cell suspension was then plated on this cover slip and incubated for 2 hr . 
 micropipettes were prepared using f-500 fine point microforge to have the tip diameter of 1 micron . 
 the cover slip coated with the cells was washed with ecf ( extra cellular fluid ) and was placed in a 35 mm petridish filled with ecf . 
 composition of the neuron solution ecf was nacl  135 mm , kcl  4 mm , nah2po4  1 mm , cacl2 1.2 mm , mgcl2  0.5 mm , hepes  10 mm and glucose  10 mm . 
 all the micropipettes were filled with icf ( intra cellular fluid ) before being attached to the pipette electrode . 
 composition of the neuron solution icf was kcl  140 mm , egta  1 mm , mgcl2  1 mm , hepes  10 mm and glucose - 10 mm . 
 the petridish was placed on the stage of a phase contrast microscope and the bath electrode was introduced into the ecf . 
 the micropipette tip was gently approximated to the cell membrane of the gbc and a negative suction was given to form a seal . 
 the average seal resistance for the gbcs was between 1.2 m ohm and 2 m ohm . 
 multiple sweeps were taken to calculate the current flow and analysed using the clampex scope episodic software . 
 immunostaining of pure gbcs was done with multiple numbers of neural progenitor stem cell markers to characterize gbcs as neural progenitor cells . 
 primary antibodies used for immunostaining were anti - ncam , anti - nestin , anti - sox-1 , anti - sox-2 , anti - cd29 , anti - cd54 and anti - cd73 . 
 secondary antibodies used were goat anti - mouse igg 1 and goat ant - rabbit igg ( percp - conjugated affinipure f(ab)2 fragment ) . 
 cover slips ( coated with poly d - lysine ) plated with the gbcs were washed in pbs 3 times . 
 cover slips were placed on the slides and blocked using 2% goat serum ( blocking agent ) mixed with 0.1 % triton x ( permeating agent ) for 2 hr at room temperature . after blocking , the slides were washed 3 times with pbs and treated with the primary antibodies and incubated overnight at 4c . 
 dilutions used for different primary antibodies were as follows ; anti ncam- 1:50 , anti nestin- 1:20 , anti sox 1- 1:100 , anti sox 2- 1:5 , anti cd29- 1:50 , anti cd54- 1:50 and anti cd73- 1:50 . 
 six albino wistar rats were purchased from the animal facility of christian medical college vellore after obtaining the clearance from the animal ethics committee . 
 albino wistar rats are the most commonly used laboratory animals for genetic and cell line studies as many aspects of their behaviour and physiology are similar to humans and this similarity can be easily observed in them when compared to other animals ( 17 ) . 
 this strain of rats was used in this study since their olfactory mucosa is easily accessible and their olfactory epithelium is histologically similar to that of the human . 
 all animals were weighed prior to the experimental ( 94 -120 g , with an average of 105 g ) . 
 the olfactory mucosa of the rats was obtained by surgical excision under complete , universal sterile conditions . 
 the albino wistar rats were anesthetized by intraperitoneal administration of 90 mg / kg body weight of ketamine and 10 mg / kg body weight of xylazine . 
 the nasal septum was completely excised along three lines : the arc of the perpendicular plate , the cribriform plate and the ceiling of the oral cavity to obtain the olfactory mucosa ( figure 1 ) . 
 the nasal septum along with the olfactory mucosa was placed in frozen dulbeco s modified eagle s medium ( dmem ) containing antibiotics ( penicillin , streptomycin and amphotericin ) . 
 the nasal septum along with the olfactory mucosa was then treated with 0.5 ml of dispase ii and was incubated at 37c for 45 min . 
 the olfactory epithelium was separated from the lamina propria on both sides of the nasal septum using a micro spatula under a leica ez2hd dissecting microscope . 
 the olfactory mucosa was mechanically dissociated with a scalpel blade and treated with 0.05% trypsin and an enzymatic cocktail containing collagenase 1 mg / ml , hyaluronidase 1.5 mg / ml and trypsin inhibitor  0.1 mg / ml to separate the cells . 
 the oe cells were transferred to epithelial culture medium which was modified form of the previously described medium ( 18 - 20 ) . 
 the culture medium was composed of dmem / f12 ( 1 : 1 ) 47.5 ml , 2% fetal bovine serum ( invitrogen , gibco , india ) 
 1.0 ml , n2 supplement ( invitrogen , gibco , india ) 0.5 ml , epidermal growth factor ( 25 ng / ml  invitrogen , gibco , india ) 12.5 l and l - glutamine ( invitrogen , gibco , india ) 0.5 ml . 
 a - arc of perpendicular plate , b - cribriform plate , c - ceiling of oral cavity . 
 ten l of gbc iii antibody was added to the epithelial cell pellet under dark conditions and was left in ice for 45 min . 
 gbc - iii is a mouse monoclonal igm antibody which recognizes a 40 kda surface antigen which is a laminin receptor surface protein . 
 it is highly specific as a marker for gbcs , unlike the earlier antibodies used like gbc - i which were nonspecific markers for gbcs and showed positive reaction even with hbcs , sus and duct cells ( 21 ) . 
 gbc - iii in powdered form was reconstituted into the recommended volume of 250 l . 
 ten l of r - phycoerythrin - conjugated affinipure ( fab)2 fragment goat anti - mouse igm ( secondary antibody ) was added to the cell pellet and left under dark conditions in ice for 10 min . 
 fluorescence assisted cell sorting ( facs ) was used to separate the gbcs from other epithelial cells . 
 finally , 99% pure gbcs were obtained using facs and gbc - iii antibody and were plated in the epithelial medium . 
 gbc cell suspension in the epithelial medium was washed 3 times with hanks balanced salt solution . 
 cells were trypsinised using 100 l of 0.1% trypsin / edta and incubated at 37c for 5 min . 
 epithelial medium was added immediately after incubation to neutralize the enzymes and the cell suspension was centrifuged at 1000 rpm for 8 min . 
 a poly d - lysine plated cover slip was placed in a 35 mm petridish . 
 the cell suspension was then plated on this cover slip and incubated for 2 hr . 
 micropipettes were prepared using f-500 fine point microforge to have the tip diameter of 1 micron . 
 the cover slip coated with the cells was washed with ecf ( extra cellular fluid ) and was placed in a 35 mm petridish filled with ecf . 
 composition of the neuron solution ecf was nacl  135 mm , kcl  4 mm , nah2po4  1 mm , cacl2 1.2 mm , mgcl2  0.5 mm , hepes  10 mm and glucose  10 mm . 
 all the micropipettes were filled with icf ( intra cellular fluid ) before being attached to the pipette electrode . 
 composition of the neuron solution icf was kcl  140 mm , egta  1 mm , mgcl2  1 mm , hepes  10 mm and glucose - 10 mm . 
 the petridish was placed on the stage of a phase contrast microscope and the bath electrode was introduced into the ecf . 
 the micropipette tip was gently approximated to the cell membrane of the gbc and a negative suction was given to form a seal . 
 the average seal resistance for the gbcs was between 1.2 m ohm and 2 m ohm . 
 multiple sweeps were taken to calculate the current flow and analysed using the clampex scope episodic software . 
 immunostaining of pure gbcs was done with multiple numbers of neural progenitor stem cell markers to characterize gbcs as neural progenitor cells . 
 primary antibodies used for immunostaining were anti - ncam , anti - nestin , anti - sox-1 , anti - sox-2 , anti - cd29 , anti - cd54 and anti - cd73 . 
 secondary antibodies used were goat anti - mouse igg 1 and goat ant - rabbit igg ( percp - conjugated affinipure f(ab)2 fragment ) . 
 cover slips ( coated with poly d - lysine ) plated with the gbcs were washed in pbs 3 times . 
 cover slips were placed on the slides and blocked using 2% goat serum ( blocking agent ) mixed with 0.1 % triton x ( permeating agent ) for 2 hr at room temperature . after blocking , the slides were washed 3 times with pbs and treated with the primary antibodies and incubated overnight at 4c . 
 dilutions used for different primary antibodies were as follows ; anti ncam- 1:50 , anti nestin- 1:20 , anti sox 1- 1:100 , anti sox 2- 1:5 , anti cd29- 1:50 , anti cd54- 1:50 and anti cd73- 1:50 . 
 the basal cells in the olfactory epithelium are believed to be the easily accessible autologous stem cells in the adults . 
 however selection and isolation of the specific basal cells which are progenitors for neural cells are still being studied . 
 the present research work helped us to isolate and decipher numerous new facts about the globose basal stem cells in the olfactory epithelium of rats . 
 culture medium used for growing and obtaining maximum confluency of the olfactory epithelial cells was standardized . 
 the epithelial medium was composed of chemicals , enzymes and factors essential for epithelial cell proliferation and neurosphere formation . 
 the desirable viable cell density for obtaining optimum growth of epithelial cells on the media was calculated to be 10  10 . 
 phase contrast image showing 2 generation of neurospheres on the 14 day in epithelial medium as it is shown in figure 3 , 99% pure gbcs were obtained using facs and gbc - iii antibody . 
 the highest number of gbcs was found in the area with the total cell density of 4  10 ( figures 4 and 5 ) . 
 immunofluorescent microscopic picture showing gbc stained with gbc - iii antibody facs diva 6.0 graph showing p1 and p2 areas . 
 green- gbc - iii positive cells ; red- gbc - iii negative cells facs diva 6.0 of unstained cells used as negative control . green- 
 gbc - iii positive cells ; red- gbc - iii negative cells gbcs sorted by facs were immunohistochemically characterised using various neural progenitor cell markers . 
 immunostaining of gbcs was positive with anti - ncam ( figure 6 , va ) , anti - nestin ( figure 6 , vb ) and anti - sox 2 ( figure 6 , vc ) , hence confirming that these cells contain intracellular proteins like ncam , nestin and sox 2 and are neural progenitor cells . 
 gbcs staining was also positive for cd-29 ( figure 6 , vd ) , cd-54 ( figure 6 , ve ) and cd-73 ( figure 6 , vf ) which are cell surface markers present on a variety of cells including neural progenitor cells . 
 marker study with sox-1 was negative probably because of reduced number of gbc in that respective well . 
 electrophysiological characterisation of the isolated gbcs was done using patch clamping ( figure 7 ) . 
 whole cell voltage - clamp recording from gbc was obtained using 60% series resistance compensation , following correction . 
 also , + 1 mv increments were applied starting from -40 mv to -10 mv to check for opening of any voltage gated na ( sodium ) channels . 
 voltage gated na channels were completely absent , hence proving the unexcitable nature of gbcs ( figure 8) . 
 then , + 10mv increments were applied starting from -150 mv to + 150 mv to check for opening of any voltage gated k ( potassium ) channels . 
 a smooth i - v curve was not obtained and only leaky k channels were found to be present on the gbc which was of no significance ( figure 9 ) . 
 va : immunofluorescent image of a gbc stained with anti - ncam antibody ( 10x ) vb : immunofluorescent image showing nestin positive gbcs ( 10x ) vc : immunofluorescent image showing sox-2 positive gbcs ( 20x ) vd : immunofluorescent image of cd-29 positive gbcs ( 10x ) . 
 nuclei is stained with dapi ve : immunofluorescent image of cd-54 positive gbcs ( 40x ) vf : immunofluorescent image of cd-73 positive gbcs ( 20x ) . 
 29 0f 30 sweeps are seen left arrow- na channels ( closed ) ; middle arrow- leaky k channels ; right arrow- holding voltage i - v curve of potassium current showing leaky channels . 
 culture medium used for growing and obtaining maximum confluency of the olfactory epithelial cells was standardized . 
 the epithelial medium was composed of chemicals , enzymes and factors essential for epithelial cell proliferation and neurosphere formation . 
 it further enhanced the capability of the cells to propagate and form neurospheres . the desirable viable cell density for obtaining optimum growth of epithelial cells on the media was calculated to be 10  10 . 
 as it is shown in figure 3 , 99% pure gbcs were obtained using facs and gbc - iii antibody . 
 the highest number of gbcs was found in the area with the total cell density of 4  10 ( figures 4 and 5 ) . 
 immunofluorescent microscopic picture showing gbc stained with gbc - iii antibody facs diva 6.0 graph showing p1 and p2 areas . 
 green- gbc - iii positive cells ; red- gbc - iii negative cells facs diva 6.0 of unstained cells used as negative control . green- 
 immunostaining of gbcs was positive with anti - ncam ( figure 6 , va ) , anti - nestin ( figure 6 , vb ) and anti - sox 2 ( figure 6 , vc ) , hence confirming that these cells contain intracellular proteins like ncam , nestin and sox 2 and are neural progenitor cells . 
 gbcs staining was also positive for cd-29 ( figure 6 , vd ) , cd-54 ( figure 6 , ve ) and cd-73 ( figure 6 , vf ) which are cell surface markers present on a variety of cells including neural progenitor cells . 
 marker study with sox-1 was negative probably because of reduced number of gbc in that respective well . 
 electrophysiological characterisation of the isolated gbcs was done using patch clamping ( figure 7 ) . 
 whole cell voltage - clamp recording from gbc was obtained using 60% series resistance compensation , following correction . 
 also , + 1 mv increments were applied starting from -40 mv to -10 mv to check for opening of any voltage gated na ( sodium ) channels . 
 voltage gated na channels were completely absent , hence proving the unexcitable nature of gbcs ( figure 8) . 
 then , + 10mv increments were applied starting from -150 mv to + 150 mv to check for opening of any voltage gated k ( potassium ) channels . 
 a smooth i - v curve was not obtained and only leaky k channels were found to be present on the gbc which was of no significance ( figure 9 ) . 
 va : immunofluorescent image of a gbc stained with anti - ncam antibody ( 10x ) vb : immunofluorescent image showing nestin positive gbcs ( 10x ) vc : immunofluorescent image showing sox-2 positive gbcs ( 20x ) vd : immunofluorescent image of cd-29 positive gbcs ( 10x ) . 
 nuclei is stained with dapi ve : immunofluorescent image of cd-54 positive gbcs ( 40x ) vf : immunofluorescent image of cd-73 positive gbcs ( 20x ) . 
 29 0f 30 sweeps are seen left arrow- na channels ( closed ) ; middle arrow- leaky k channels ; right arrow- holding voltage i - v curve of potassium current showing leaky channels . 
 neurogenesis begins in the early embryonic period ( 4 week ) and continues 10 to 12 years after birth ( 22 ) . 
 the nervous system development is completed only during the adolescent period when myelination of the neurons of major tracts is completed ( 23 ) . 
 early researchers believed the nervous system to be fixed and incapable of proliferation and regeneration based on the their opinions that neurons and non - neuronal cells in the nervous system could not divide actively via mitosis and they lacked the potential to transform from simple to more complex forms of neuronal morphologies . 
 another reason for such belief was inability of researchers in the past to demonstrate progenitor stem cells residing in the nervous system niche by specific markers . 
 cells in the nervous system could be demonstrated only by negative staining which was very non - specific ( 18 , 22 , 23 ) . 
 the earliest demonstration of neurogenesis in adults was done by joseph altman in 1962 in the cerebral cortex ( 24 ) followed by demonstration of neurogenesis in the dentate gyrus of the hippocampus in 1963 ( 25 ) . 
 later , it was evident that neurogenesis also occurred in the subgranular zone and subventricular zone of the lateral ventricles ( 26 ) . 
 now , researchers also believe that neurogenesis continues in the cerebellum during adulthood ( 27 ) . 
 the most recent development in this field of research is the demonstration of neurogenesis in the olfactory system including the olfactory bulbs and more importantly in the olfactory mucosa . 
 this research work studies one such population of stem cells residing in the olfactory epithelium which has the niche required to get accepted in the cns environment and is derived from the same germ line as the neuronal cells . 
 this gives a hint that these might be the ideal neural progenitor cells which can be easily harvested and used for autologous transplantations . 
 olfactory epithelial- mesenchymal stem cells have been isolated in the past from the olfactory mucosa using enzymatic dissociation with collagenase ia ( 28 ) in an impure form . 
 isolated cultures of gbcs and horizontal basal cells have been grown in earlier experiments by immunoadhesion ( 29).this research work standardizes chemical methods to isolate these stem cells from the olfactory epithelium in a pure form . 
 epithelial cells were harvested on the culture media after calculating the feasible cell densities for optimal growth and neurosphere formation . 
 densities higher than this resulted in overcrowding of cells and loss of cells due to unwanted cell death 
 . densities lower than this did not provide us with adequate number of cells and hindered neurosphere generation . 
 the self renewal potential of olfactory stem cells was evident from the formation of multiple generations of neurospheres . 
 egf and n-2 supplement seem to play a significant role in providing an optimum external niche for formation of multiple generations of neurospheres from epithelial stem cells . 
 egf is a potential stem cell mitogen allowing cell proliferation through activating signalling pathway and inducing the production of beta fibroblast growth factor ( fgf ) from the progenitor cells . 
 olfactory mucosa in cultures has been shown to produce certain soluble factors which help with proliferation , regeneration and neurosphere formation ( 30 ) . 
 the area p1 was chosen with relatively higher percentage of gbc - iii stained cells and a graph was plotted showing the gbc density to be 65.3% of the total number of cells . 
 p2 , a part of p1 , with the highest cell density was chosen and a similar graph was plotted which showed the gbc density to be 79.9% of the total ( figure 4 ) . 
 . an insignificant number of 41 positive cells ( accounting for 0.4% of the total ) were sorted from the negative control proving the efficacy of the procedure ( figure 5 ) . 
 gbcs have been isolated in the past from other progenitor cells of the olfactory epithelium using facs by tagging them with less sensitive and specific antibodies like gbc - i ( 9 ) and gbc - ii ( 29 ) . 
 the complexities of the phenotypical stages of transformation of the gbcs into olfactory neurons upon neuroablation of the olfactory bulb have been explained using enhanced green[t6 ] fluorescent protein tagged with markers like sox2 and neurog1 ( 31 ) 
 . further research on gbcs and other potential neural progenitor cells present in the olfactory mucosa will help us to understand their biology and effectiveness in neurogenesis . 
 it will also help to apply these results to medical use for therapeutic treatment of different diseases . 
 few areas on which special attention can be drawn in future are : 
 isolation of gbcs from the oe using magnetic assisted cell sorting ( macs ) . 
 hence , its usage will help us to compare its efficacy with facs and a better purity of cells can be obtained ( 32).electrophysiological studies to find out more about different ion channels present on the gbcs will help us to have a better understanding of the functional properties of these cells and their drug interactions.animal studies using gbcs to repair the spinal cord injuries and other nervous system disorders should be the ultimate aim to propagate and effectively utilize the present knowledge on these cells in human trials . 
 hence , its usage will help us to compare its efficacy with facs and a better purity of cells can be obtained ( 32 ) . 
 electrophysiological studies to find out more about different ion channels present on the gbcs will help us to have a better understanding of the functional properties of these cells and their drug interactions . 
 animal studies using gbcs to repair the spinal cord injuries and other nervous system disorders should be the ultimate aim to propagate and effectively utilize the present knowledge on these cells in human trials . 
 this research work has been able to throw some light on the prevailing ambiguity of the olfactory stem cells and has derived some definite conclusions about the nature and behaviour of these cells . 
 non - excitable nature of gbcs has been documented in the present study . positive expression of all the neural progenitor cell markers has proved substantially about the neuro - transformation capability of the gbcs . 
 now , after undertaking this study , we can conclude with concrete explanations that gbcs are the colony of neural stem cells residing in the basal compartment of the olfactory epithelium responsible for the ongoing neurogenesis in the oe throughout adult life .","objective(s):in the past few decades , variety of foetal , embryonic and adult stem and progenitor cells have been tried with conflicting outcome for cell therapy of central nervous system injury and diseases . 
 cellular characteristics and functional plasticity of globose basal stem cells ( gbcs ) residing in the olfactory epithelium of rat olfactory mucosa have not been studied in the past by the neuroscientists due to unavailability of specific markers for gbcs . in the present research , we standardized some techniques to isolate gbcs from rat olfactory epithelium in pure form using a highly selective gbc - iii antibody passaged through fluorescence activated cell sorter ( facs ) . 
 we also characterized these cells immunohistologically using various pluripotent stem cell markers . 
 this work also throws some light on ionic channels present on these stem cells which are responsible for their neuron induction potential.materials and methods : globose basal stem cells were isolated from rat olfactory epithelium using gbc - iii antibody and were characterized as multipotent stem cells using various neural progenitor markers . 
 ionic channels on gbcs were studied with voltage clamping.results:gbcs could be isolated in pure ( 99% purity ) form and were found to be stained positive for all neural progenitor cell markers . 
 voltage gated na+ channels were completely absent , which proves the unexcitable nature of gbcs . 
 leaky k+ channels were found to be present on the gbc which was of no significance.conclusion:this research work can be helpful in understanding the nature of these stem cells and utilising them in future as potent candidates for neuro - regenerative therapies .",pubmed
"in the past , the central nervous system ( cns ) was considered a site of immune privilege . 
 microglia , first described by achcarro and subsequently thoroughly characterized by his successor , rio - hortega , are now known to play a pivotal role in innate immunity within the cns . 
 in addition , emerging evidence demonstrates the presence of a conduit that enables communication between the brain parenchyma and cervical lymph nodes via the glymphatic system ( an interstitial fluid ( isf)/cerebrospinal fluid ( csf ) exchange system located in the perivascular space ) ( figure 1 ) . along with brain microglia , this exchange system facilitates immune surveillance of brain isf . 
 these findings suggest that the cns immune system is more constitutively active than previously envisioned . 
 a currently supported view concerning the origin of microglia postulates that they are derived from erythromyeloid progenitors in yolk sac that enter the brain during different developmental stages in the brain . 
 yolk sac erythromyeloid progenitors express the chemokine receptor cx3cr1 . to closely analyze the fate of erythromyeloid progenitors in the cns during embryogenesis , kierdorf et  al . 
 used cx3cr1gfp / wt mice , which contain a gfp knock - in on one allele of the cx3cr1 gene . 
 they discovered that mouse microglia were derived from primitive c - kit erythromyeloid precursors present in the yolk sac of embryos at embryonic age of 7.5 to 8.0 days ( e7.5e8.0 ) ( figure 2(a ) ) . 
 these precursors develop into cd45 c - kit cx3cr1 immature ( a1 ) cells and mature into cd45 c - kit cx3cr1 ( a2 ) cells . 
 microglial maturation is accompanied by a reduction in cd31 expression and concomitant upregulation of the macrophage marker , f4/80 , and macrophage colony stimulating factor receptor ( mcsf - r ) . 
 proliferating a2 cells develop into microglia and invade the developing brain at e9.5 , a process that requires activity of matrix metalloproteinase . after entering the cns , 
 inhibition of microglia - mediated synaptic pruning results in aberrant brain circuitry with excessive dendritic spines and immature synapses . within the postnatal brain , microglia 
 are maintained through cellular division or expansion of progenitors that colonized the brain during cns development . 
 microglia form an autonomous population of cells within the brain that demonstrate activities independent of circulating monocyte activity . 
 resident microglia are distributed in the brain parenchyma , and use motile processes to survey the microenvironment ( left ) . 
 the brain parenchyma is equipped with the glymphatic system , which clears interstitial solutes through the exchange of isf and csf along the perivascular space between the wall of the veins and astrocytic endfeet . 
 subsequently , csf is absorbed into the meningeal lymphatic vessels , which are located in the vicinity of the venous sinus . 
 figure 2.(a ) schematic representation of microglial development : microglia are derived from c - kit - positive erythromyeloid precursors ( emps ) in the yolk sac at e7.5e8.0 . 
 these precursors develop into cd45 c - kit cx3cr1 immature ( a1 ) cells and mature into cd45 c - kit cx3cr1 ( a2 ) cells . 
 proliferating a2 cells develop into embryonic microglia . before completion of the blood  brain barrier ( bbb ) , they invade the developing brain in a manner requiring matrix metalloproteinase ( mmp ) activity at e9.5 . 
 unless the bbb is disrupted , microglia and monocytes are independent of each other in the postnatal stage . 
 ( b ) multiple roles of microglia in the postnatal brain : microglia in the postnatal brain are multifunctional . 
 primarily , they are involved in immune surveillance , and when necessary , they initiate an inflammatory response . 
 although this contributes to the elimination of pathogens , an excessive inflammatory response may result in bystander tissue damage . 
 microglia also phagocytose pathogens and apoptotic neurons and exert scavenging actions on tissue debris under pathological conditions . 
 microglia recognizes apoptotic cells by phosphatidylserine and other apoptotic signals . with regard to synaptic refinement 
 in addition , microglia - derived tnf can exert neuroprotective as well as neurodestructive action within different contexts . 
 microglia also play a role in angiogenesis through by synthesizing vascular endothelial growth factor ( vegf ) . 
 resident microglia are distributed in the brain parenchyma , and use motile processes to survey the microenvironment ( left ) . 
 the brain parenchyma is equipped with the glymphatic system , which clears interstitial solutes through the exchange of isf and csf along the perivascular space between the wall of the veins and astrocytic endfeet . 
 subsequently , csf is absorbed into the meningeal lymphatic vessels , which are located in the vicinity of the venous sinus . 
 ( a ) schematic representation of microglial development : microglia are derived from c - kit - positive erythromyeloid precursors ( emps ) in the yolk sac at e7.5e8.0 . 
 these precursors develop into cd45 c - kit cx3cr1 immature ( a1 ) cells and mature into cd45 c - kit cx3cr1 ( a2 ) cells . 
 proliferating a2 cells develop into embryonic microglia . before completion of the blood  brain barrier ( bbb ) , they invade the developing brain in a manner requiring matrix metalloproteinase ( mmp ) activity at e9.5 . 
 unless the bbb is disrupted , microglia and monocytes are independent of each other in the postnatal stage . 
 ( b ) multiple roles of microglia in the postnatal brain : microglia in the postnatal brain are multifunctional . 
 primarily , they are involved in immune surveillance , and when necessary , they initiate an inflammatory response . 
 although this contributes to the elimination of pathogens , an excessive inflammatory response may result in bystander tissue damage . 
 microglia also phagocytose pathogens and apoptotic neurons and exert scavenging actions on tissue debris under pathological conditions . 
 microglia recognizes apoptotic cells by phosphatidylserine and other apoptotic signals . with regard to synaptic refinement 
 in addition , microglia - derived tnf can exert neuroprotective as well as neurodestructive action within different contexts . 
 microglia also play a role in angiogenesis through by synthesizing vascular endothelial growth factor ( vegf ) . in the postnatal brain , microglia have multiple functions ( figure 2(b ) ) . 
 as the powerhouse of local innate immunity , microglia protect the postnatal brain from infection . in response to pathogens invading the cns , microglia act as a front - line defense system by phagocytosing pathogens , secreting humoral substances , such as proinflammatory cytokines , and producing reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) . 
 microglia - producing proinflammatory cytokines include interleukin-1  ( il-1 ) , il-6 , and tumor necrosis factor  ( tnf ) . 
 the activation pattern of microglia in response to invading pathogens is typical of the classical proinflammatory phenotype of macrophages ( m1 polarization ) . 
 for instance , cerebral ischemia and amyloid  ( a ) stimulate il-1 synthesis in microglia through the activation of the inflammasome . 
 they tend to retract their processes and under extreme conditions , assume an amoeboid shape . 
 dead neurons expose phosphatidylserine and other apoptotic  eat - me  signals on their cell surface , which prompts microglia to carry out phagocytosis . 
 such microglia - mediated clearance of unwanted neurons is an important process of proper brain tissue remodeling . with regard to microglia function under the physiological condition , 
 recent in  vivo imaging experiments have uncovered that these resident microglia constantly survey their surrounding microenvironment with their motile processes , which is likely relevant to immune surveillance . however , such motion has been observed even in the absence of offending microorganisms . 
 increasing evidence has revealed that microglia engulf presynaptic and postsynaptic elements by extending their processes to contact dendritic spines , axons , and synapses as observed in the developing brain . 
 microglia - mediated synaptic pruning is dependent on regional neuronal activity . in the visual cortex , 
 visual experience causes microglial processes to change their morphology and motility ; these processes alter distributions of extracellular space , display phagocytic structures , appose synaptic clefts more frequently , and envelope synapse - associated elements more extensively . 
 intriguingly , synaptic pruning occurs preferentially in response to less active inputs , which apparently contributes to activity - dependent synaptic plasticity and learning . 
 microglia express cr3 , the high - affinity receptor for c3 , which is required for the precise execution of synaptic pruning . 
 moreover , microglia are involved in synapse formation by secreting brain - derived neurotrophic factor ( bdnf ) . 
 consistent with these data , genetic inhibition of microglia results in derangement of both synapse formation and elimination of dendritic spines . 
 cortical spreading depression ( csd ) is defined as a slowly propagating ( 25  mm / min ) wave of rapid , near - complete depolarization of neurons and astrocytes followed by a period of electrical suppression of a distinct population of cortical neurons ( figure 3 ) . by a strict definition 
 , csd refers only to the electrical silence of brain electrical activity following spreading depolarizations . 
 csd is accompanied by secondary changes of cerebral blood flow ( cbf ) , which comprised the following four distinct phases : ( i ) an initial , brief hypoperfusion , ( ii ) a marked , transient hyperemia , ( iii ) a later , smaller hyperemia , and ( iv ) a long - lasting oligemia . 
 experimentally , csd can be elicited by chemical stimulation ( high potassium exposure , atpase inhibitors , endothelin-1 , n - methyl - d - aspartate ( nmda ) receptor agonists ) , pinprick stimulation , and electrical stimulation of the intact cerebral cortex . 
 the most clinically convincing evidence for this came from an mri study by hadjikhani et  al . 
 , which demonstrated the clinico - radiological correlation of visual percept of aura symptoms and propagation of cortical blood oxygenation level - dependent ( bold ) signals in the visual cortex of a patient experiencing a migraine . 
 in addition , csd has been recorded by electrocorticography in patients with ischemic and hemorrhagic stroke and brain trauma . in most cases , however , electrical activity of brain tissue is already compromised before the development of spreading depolarizations . as a result , the characteristic temporal profile of csd , in which rapidly evolving depolarizations of neural cells were followed by suppression of electrical activity , may not necessarily be observed . 
 because of this ,  spreading depolarization  is now regarded as a more precise and preferred term to describe such conditions than csd . spreading depolarization can be elicited by cerebral ischemia or traumatic brain injury experimentally . 
 detailed electrophysiological analysis using hippocampal slices revealed that the apical dendrites of neurons were depolarized earlier than the somata during csd . 
 as compared to action potentials , csd induces a greater magnitude of extracellular potential shift ( typically 1020 millivolts ) which continues for much longer ( at least several minutes ) . 
 the initiation of csd entails a rapid increase in [ k]e from 4  mm to 3060  mm and a rapid decline in [ na]e and [ cl]e from 140  mm to 5070  mm and [ ca]e from 1.52.0  mm to 0.20.8  mm . 
 for example , glutamate , the excitatory amino acid , is released into the extracellular space and binds to nmda - type glutamate receptors to further promote the influx of ca and na . in response to these environmental changes , 
 consistent with these results , a loss - of - function mutation in the gene encoding the 2 isoform of the glial na , k - atpase ( atp1a2 ) causes familial hemiplegic migraine type 2 ( fhm2 ) , which is clinically characterized by prolonged aura episodes . 
 hence , clearance of potassium and glutamate from the extracellular space appears to be a critical event that determines predisposition to csd inductions . 
 activation of the glial na , k - atpase is required to establish neuronal resting membrane potential and correct the csd - induced derangement of the ionic environment . since this process is accompanied by elevated atp consumption . 
 as expected , multiple consecutive csd episodes robustly increase the cerebral metabolic rate of glucose ( cmrglu ) , as compared to a single csd episode . 
 csd - associated depolarizations occur first in the dendrites and then propagate to the soma in cortical neurons . 
 the initiation of csd entails a rapid increase in [ k]e from 4  mm to 3060 mm and a rapid decline in [ na]e from 140  mm to 5070  mm ( black arrows ) . 
 na , k - atpase activity then rectifies the resultant abnormal ion distribution ( red arrows ) . 
 however , csd - associated oligemia may have a negative impact on na , k - atpase activity . 
 csd - associated depolarizations occur first in the dendrites and then propagate to the soma in cortical neurons . 
 the initiation of csd entails a rapid increase in [ k]e from 4  mm to 3060 mm and a rapid decline in [ na]e from 140  mm to 5070  mm ( black arrows ) . 
 na , k - atpase activity then rectifies the resultant abnormal ion distribution ( red arrows ) . 
 however , csd - associated oligemia may have a negative impact on na , k - atpase activity . 
 microglia express voltage - sensitive ion channels , including nav1.1 , kv1.3 , and kv1.5 , and are thought to sense electrical activity pertaining to csd . 
 csd initially elevates extracellular ph , which subsequently leads to a gradual decrease in tissue ph . 
 accumulation of protons may be detected by microglial transient receptor potential cation channel subfamily v , member 1 ( trpv1 ) . 
 a recent study revealed that microglia exhibit increased nmda - dependent inward rectifying potassium conductance after csd , which can be interpreted as a compensatory mechanism for elevated extracellular k concentration . 
 reported that the number of major histocompatibility complex ( mhc ) class ii antigen - positive microglia significantly increased in the rat cerebral cortex between 16 and 24  h after csd . 
 moreover , it has been shown that csd can increase the proliferation and migration of microglia , both of which are well - recognized essential features of the immune response . 
 biochemically , there is evidence that csd induces ros production in microglia that are located in the affected brain tissues . 
 in addition , csd stimulates microglial secretion of il-1 and tnf. collectively , these data suggest that csd can initiate inflammatory activation of microglia . 
 interestingly , ros and tnf have been shown to lower the threshold for csd induction , thus forming a positive feedback mechanism that favors the perpetuation of csd induction . 
 on the other hand , tnf has been shown to reduce csd amplitudes in a dose - dependent manner . 
 in addition , il-1 is also known to attenuate csd amplitudes via the gabaa receptor activity at lower concentrations . however , at a high dose , il-1 did not alter the magnitude of csd amplitudes . since these cytokines promote vascular permeability , as a whole , they are likely to have a deleterious effect on cns tissue in csd pathophysiology . 
 moreover , in hippocampal slice culture studies , selective depletion of microglial cells with clodronate conferred resistance to csd induction . 
 conversely , restoration of microglial cells to previously depleted cultures restored the susceptibility to csd . a similar phenomenon has been reported in an in  vivo neuroimaging study . 
 an in  vivo calcium imaging technique demonstrated that microglia depletion led to a perturbation of neuronal calcium response . 
 hence , it is inferred that a certain microglia - regulated neuronal calcium response may be required for csd occurrence . 
 furthermore , evidence shows that microglial csd - generating activity can be inhibited by insulin - like growth factor-1 ( igf-1 ) treatment and environmental enrichment , the latter of which promotes m2-polarization of microglia ( figure 4 ) . 
 it has been demonstrated that igf-1 antagonizes tnf. these data were obtained in experimental settings where csd was induced in cluster . 
 clinically , successive csd / spreading depolarizations have been demonstrated in patients with stroke and traumatic brain injury . 
 experimental evidence demonstrates that csd / spreading depolarizations contribute to the expansion of infarct volume by several mechanisms . 
 first , a limited supply of atp due to ischemia renders neurons unable to reestablish the resting membrane potential , which may lead up to the occurrence of terminal depolarization . 
 igf-1 treatment and other therapeutic interventions that promote m2-polarization of microglia may ameliorate secondary brain damage resulting from cerebral ischemia . 
 resultant production of tnf and ros enhances the susceptibility of brain tissue to csd development . 
 in addition , microglia polarized to the m2 activation mode by environment enrichment exhibit much less csd susceptibility . 
 the action of environmental enrichment can be mimicked by nasal administration of interleukin-11 ( il-11 ) . 
 resultant production of tnf and ros enhances the susceptibility of brain tissue to csd development . 
 in addition , microglia polarized to the m2 activation mode by environment enrichment exhibit much less csd susceptibility . the action of environmental enrichment can be mimicked by nasal administration of interleukin-11 ( il-11 ) . 
 as mentioned above , multiple csd inductions are likely to activate microglia . consequently , to determine if microglial activation occurs after a single csd event , as seen in the usual migraine aura , we compared morphological changes of microglia in response to single and multiple csd episodes . 
 we found that a single csd induction led to only subtle morphological changes in microglia , whereas multiple csd inductions caused marked enlargement of microglia after 24  h. this morphological change normalized by 72  h after csd inductions . 
 our data suggest that a single episode of csd is  innocuous  to the brain in terms of microglial activation . 
 we did not detect activation of caspase-3 or dna fragmentation detectable by the tunel assay ( unpublished data ) . 
 nedergaard and hansen reported that csd was not associated with neuronal injury in the normal brain . 
 this is also consistent with the traditionally held belief that a single attack of migraine with aura is a benign condition that does not cause any clinically relevant structural brain damage . 
 we sought to clarify the events upstream of the multiple csd - induced microglial enlargements . 
 reported that csd causes the release of hmgb1 ( high - mobility group box 1 ) from neurons via pannexin 1 channels . 
 although hmgb1 is primarily located in the nucleus , upon injurious stimuli , it is released into the extracellular space , where it serves as a damage - associated molecular pattern ( damp ) . 
 we found that neuronal hmgb1 release was dependent on the number of csd inductions such that only multiple csd events were able to cause significant hmgb1 release from neurons ( figure 5 ) . 
 in addition , transcriptional activity of the hmgb1 gene in cortical neurons was enhanced in response to multiple csd events , which may reflect an attempt to replenish the cellular pool of the hmgb1 protein . 
 in general , damp molecules bind to their corresponding receptors , thus transmitting a danger signal to surrounding cells . 
 the ligation of hmgb1 with these receptors initiates intracellular signaling cascades that involve myd88 and irak4 . 
 we found that multiple csd inductions enhanced the transcriptional activity of the tlr2 , tlr4 , myd88 , and irak4 genes in brain tissue . 
 moreover , application of anti - hmgb1 antibody to the cortical surface attenuated the morphological alterations of microglia caused by multiple csd inductions . 
 these data indicate that the hmgb1tlr2/4 axis plays a crucial role in the microglial activation caused by multiple csd events . 
 we observed that the majority of hypertrophic microglia displayed prominent immunoreactivity for cathepsin d , a lysosomal acid hydrolase ( figure 6(a ) ) . 
 nevertheless , we did not observe any apoptotic changes in the brain tissue subjected to multiple csd episodes , making it unlikely that activated microglia are involved in the execution of apoptosis or phagocytosis of dead neurons . 
 an alternative concept is that activated microglial phagocytotic activity is involved in synaptic pruning ( figure 6(b ) ) . 
 a recent in  vivo multiphoton microscopy study disclosed that csd causes dramatic structural alterations of synapses between axons and the dendritic spines of cortical neurons . 
 several minutes after csd , axonal bouton density increased by 20% and bouton size decreased by 25 to 40% compared to the resting state . 
 concomitantly , there was a morphological shift from predominantly stubby spines to thin or mushroom spines after csd , which implies enhancement of synaptic excitability . 
 although single csd events cause abnormalities in synaptic morphology and functionality , these alterations are short - lived and reversible . however 
 , successive csd episodes prevent the recovery of synaptic abnormalities , indicating that the accumulated stress of prior csd events lead to irreversible dendritic injury . 
 thus , it is plausible that activated microglia engage in the repair of damaged synapses through synaptic pruning and formation after multiple csd episodes . 
 figure 5.microglial activation is influenced by the number of csd episodes . a single csd event , 
 as seen in the usual aura of migraine patients , does not cause significant release of hmgb1 from cortical neurons . 
 conversely , clustering of csd , as seen in stroke and brain trauma , induces a robust hmgb1 release from cortical neurons . 
 subsequently , hmgb1 acts on the tlr2/4 on the surface of microglia , which is followed by microglial activation , as demonstrated by their hypertrophic morphological alterations . 
 they may contribute to neuroprotection , for example , through the synaptic repair on the dendritic spines of multiple csd - affected cortical neurons . 
 figure 6.upregulation of cathepsin d , a representative lysosomal acid hydrolase , in activated microglia subjected to multiple csd episodes . 
 ( a ) as compared to cortical microglia in untreated mice ( upper row ) , those subjected to multiple ( five times ) csd inductions ( lower row ) exhibit increased cathepsin d immunoreactivity ( arrow ) . 
 bar  =  20  m . ( b ) microglia activated by multiple csd events may be recruited to damaged dendritic spines for synaptic repair . 
 microglial activation is influenced by the number of csd episodes . a single csd event , 
 as seen in the usual aura of migraine patients , does not cause significant release of hmgb1 from cortical neurons . 
 conversely , clustering of csd , as seen in stroke and brain trauma , induces a robust hmgb1 release from cortical neurons . 
 subsequently , hmgb1 acts on the tlr2/4 on the surface of microglia , which is followed by microglial activation , as demonstrated by their hypertrophic morphological alterations . 
 they may contribute to neuroprotection , for example , through the synaptic repair on the dendritic spines of multiple csd - affected cortical neurons . 
 upregulation of cathepsin d , a representative lysosomal acid hydrolase , in activated microglia subjected to multiple csd episodes . 
 ( a ) as compared to cortical microglia in untreated mice ( upper row ) , those subjected to multiple ( five times ) csd inductions ( lower row ) exhibit increased cathepsin d immunoreactivity ( arrow ) . 
 bar  =  20  m . ( b ) microglia activated by multiple csd events may be recruited to damaged dendritic spines for synaptic repair . 
 although aforementioned data suggest that microglia are required for the occurrence of csd , the mechanisms by which microglia induce csd remain unknown . 
 provided that microglia raise extracellular k concentration before csd occurrence , they would have to follow through in a voltage - independent manner . 
 microglia have been shown to express potassium intermediate / small conductance calcium - activated channels , subfamily n , member 4 ( kcnn4/kca3.1 ) . 
 this voltage - insensitive and calcium / calmodulin - regulated potassium channel has been implicated in lipopolysaccharide- and ischemia - induced brain damage . 
 therefore , the involvement of kcnn4/kca3.1 in the initiation of csd may be worthy of investigation . 
 another unresolved issue is whether multiple csd - induced microglial activation is an adaptive phenomenon or a harmful event to brain tissue . 
 in - depth analysis of the molecules produced in these cells would be required to solve this problem . 
 if microglia are indeed involved in synaptic pruning , the identification of synaptic components , like psd95 , within enlarged microglia should be an important confirmatory finding . 
 lastly , it remains uncertain whether immunological and inflammatory activation of multiple csd - subjected microglia can influence systemic immunological activity . 
 as mentioned above , the recently discovered lymphatic system makes possible immunological communication between the brain parenchyma and the peripheral lymphatic system . in this paradigm , 
 fhm is characterized by prolonged migraine aura , and neuroimaging data support repeated inductions of csd . 
 it has been reported that fhm attacks often cause high fever and in a fewer cases , csf pleocytosis . 
 it may , therefore , be interesting to explore whether a potential crosstalk between activated microglia and the csf / lymphatic system is really relevant to csd pathophysiology . 
 the publication of this article was supported by a jsps kakenhi ( grant number 26460706 to ms ) and a grant from nippon zoki pharmaceutical co. , ltd . 
 the author(s ) declared no potential conflicts of interest with respect to the research , authorship , and/or publication of this article . 
 ","microglia play a pivotal role in innate immunity in the brain . during development , 
 they mature from myeloerythroid progenitor cells in the yolk sac and colonize the brain to establish a resident population of tissue macrophages . in the postnatal brain , they exert phagocytosis and induce inflammatory response against invading pathogens 
 . microglia also act as guardians of brain homeostasis by surveying the microenvironment using motile processes . 
 cortical spreading depression ( csd ) is a slowly propagating ( 25  mm / min ) wave of rapid , near - complete depolarization of neurons and astrocytes followed by a period of electrical suppression of a distinct population of cortical neurons . 
 not only has csd been implicated in brain migraine aura , but csd - like events have also been detected in stroke and traumatic injury . 
 csd causes a considerable perturbation of the ionic environment in the brain , which may be readily detected by microglia . 
 although csd is known to activate microglia , the role of microglial activation in csd - related neurological disorders remains poorly understood . in this article , we first provide an overview of microglial development and the multiple functions of microglia . 
 then , we review existing data on the relationship between microglia and csd and discuss the relevance of csd - induced microglial activation in neurological disease .",pubmed
"for a long time , transposable elements ( tes ) have been considered as pure selfish and junk elements parasiting the genome of living organism [ 1 , 2 ] . 
 these sequences are able to  move  , that is , to insert into new locations within genomes . this phenomenon is called transposition . 
 retroelements use retrotransposition , that is , the reverse transcription of an rna intermediate and integration of the cdna molecule produced , to generate new copies of themselves within genomes ( copy - and - paste mechanism ) . 
 this mechanism directly increases the copy number of the element . among protein - coding autonomous retroelements 
 , distinction is generally made between elements with long terminal repeats ( ltrs : ltr retrotransposons and retroviruses ) and retroelements without ltrs ( non - ltr retrotransposons or line elements ) . 
 retroviruses and ltr retrotransposons are mainly distinguished by the presence versus absence of an envelope gene , which encodes a protein necessary for virus entry into the target cell . 
 after germ line infection , reverse - transcribed retrovirus genomes can be integrated into the host genome and transmitted through vertical inheritance to the host progeny . 
 gain or loss of the envelope gene can transform a retrotransposon into a retrovirus , and vice versa [ 4 , 5 ] . 
 the second large category of tes , dna transposons , generally excises from their original insertion site and reintegrate into a new location ( cut - and - paste mechanism ) . for most dna transposons , transposition is catalyzed by an enzyme called transposase . finally , noncoding nonautonomous elements using for their transposition proteins encoded by autonomous sequences exist for both retroelements and dna transposons . despite the deep - rooted vision of junk dna , there is growing evidence that tes are more than simple genome parasites . 
 particularly , they have been shown to serve as a genomic reservoir for new regulatory and coding sequences allowing genetic innovation and organismal evolution . 
 a fascinating facet of the roles of tes in evolution is their ability to be  molecularly domesticated  to form new cellular protein - coding genes [ 7 , 8 ] . 
 te - encoded proteins have properties that can be of interest for host cellular pathways . 
 they can bind , copy , cut , process , and recombine nucleic acids , as well as modify and interact with host proteins . 
 there are many cases of te - derived genes fulfilling important functions in plants , fungi , and animals , including vertebrates ( for review , [ 8 , 9 ] ) 
 . we will present here several prominent examples of vertebrate genes formed from te - coding sequences during evolution , with more emphasis on gypsy integrase ( gin ) genes that we have analyzed in different fish species . 
 several multigenic families have been formed from different events of molecular domestication of the gag gene of ty3/gypsy elements , a super family of ltr retrotransposons active in fish and amphibians but extinct in mammals [ 9 , 10 ] . 
 the gag gene encodes a structural protein with three functional regions : the matrix ( ma ) domain playing a role in targeting cellular membranes , the capsid ( ca ) domain involved in interactions with other proteins during particle assembly , and the nucleocapsid ( nc ) , which binds to viral rna genomes through zinc fingers . 
 most mart genes are found on mammalian x chromosome , suggesting an initial event of molecular domestication on the x , followed by serial local duplication events that subsequently extended this gene family . 
 all mart genes have retained from the original gag sequence an intronless open reading frame . some of them still encode the ancestral gag zinc finger , suggesting nucleic acid binding properties for the protein . 
 two autosomal mart genes , peg10 ( mart2 ) and peg11/rtl1 ( mart1 ) , are subject to genomic imprinting and are expressed from the paternal allele [ 12 , 13 ] . 
 this epigenetic regulation has been proposed to be derived from a defence mechanism repressing the activity of the ancestral retrotransposon before domestication . 
 at least two mart genes , peg11/rtl1 ( mart1 ) and peg10 ( mart2 ) , have essential but nonredundant functions in placenta development in the mouse [ 15 , 16 ] . 
 peg10 and other mart genes might also control cell proliferation and apoptosis , with possible involvement in cancer ( and references therein ) . 
 another mammalian gene family derived from a ltr retrotransposon gag gene is called ma or pnma ( paraneoplastic ma antigens ) . 
 fifteen ma / pnma genes are present in the human genome , most of them being located on the x chromosome as observed for mart genes . 
 some ma proteins are expressed by patients with paraneoplastic neurological disorders and might be targeted by autoimmune response leading to progressive neurological damage . 
 several ma proteins are also involved in apoptosis , including ma4 ( pnma4/map1/maop1 ) and ma1/pnma1 [ 19 , 20 ] . 
 this family is constituted of dna binding proteins with an n - terminus region called the scan domain , which is derived from the gag protein of a gmr1-like gypsy / ty3 retrotransposon [ 2124 ] . 
 the scan family is vertebrate specific , with approximately 70 and 40 members in human and mouse , respectively . 
 several scan proteins have been shown to be transcription factors regulating diverse biological processes such as hematopoiesis , stem cell properties , or cell proliferation and apoptosis ( for review ) . 
 one of them , fv1 , is of retroviral origin and controls replication of the murine leukaemia virus in the mouse . during mammalian evolution , 
 retroviral envelope genes have been domesticated several times independently to generate genes involved in placenta development . 
 syncytins mediate the fusion of trophoblast cells to form the syncytiotrophoblast layer , a continuous structure with microvillar surfaces forming the outermost foetal component of the placenta . 
 two syncytin genes of independent origins encoding placenta - specific fusogenic proteins are present in human and other simians ( syncytin-1 and -2 , ) as well as in rodents ( syncytin - a and syncytin - b , ) . 
 independent syncytin genes are also found in rabbit , guinea pig , and carnivora , indicating multiple convergent domestication of env - derived syncytin genes in different mammalian sublineages . 
 for example , human syncytin-1 plays a role in osteoclast fusion , neuroinflammation , and possibly multiple sclerosis [ 33 , 34 ] . 
 other retroviral env - derived open reading frames are present in vertebrate genomes ; but intensive work is required to determine their functions . 
 some of them might confer resistance to viral infection , as shown for the fv-4 locus . 
 this locus , containing an entire ecotropic murine leukemia virus ( mulv ) env gene , controls susceptibility to infection by mulv . in mammals , a gene called cgin1 
 the integrase gene has been fused 125180 million years ago to a duplicate of the cellular gene kiaa0323 . 
 one of them , a gene encoding a protein called saspase , is necessary for the texture and hydration of the stratum corneum , the outermost layer of the epidermis . 
 finally , the telomerase , the reverse transcriptase extending the ends of linear chromosomes in vertebrates and other eukaryotes , might be derived from a retroelement . 
 many examples of genes derived from transposase genes from diverse subfamilies of dna transposons have been described in vertebrates and other organisms [ 8 , 39 , 40 ] . 
 one well - studied example is the recombination - activating protein rag1 , which together with rag2 catalyzes the v(d)j somatic site - specific recombination responsible for the formation and diversity of genes encoding immunoglobulins and t - cell receptors in jawed vertebrates . 
 rag1 has been formed from the transposase of a transib dna transposon , and the v(d)j recombination signal sequences recognized by rag1 might be derived from the transposon ends bound by the ancestral transposase . 
 the mammal - specific gene cenp - b encodes a pogo transposase - derived protein that controls centromere formation depending on the chromatin context . 
 interestingly , an independent event of molecular domestication of pogo transposase also led to the formation of centromeric proteins in fission yeast . in yeast , cenp - b - like proteins 
 restrict the activity of retrotransposons and promote replication progression at forks paused by retrotransposon ltrs [ 44 , 45 ] . 
 one example is the jerky gene , which encodes a brain - specific mrna - binding protein that may regulate mrna use in neurons . 
 similarly , several examples of genes derived from hat transposases have been found in mammals , some of them having been fused to zinc finger domains . 
 indeed , it has been shown that mutation in a regulatory sequence prohibiting zebd6/mgr binding leads to igf2 upregulation and enhanced muscle growth in commercially bred pigs [ 48 , 49 ] . in primates 
 , the gene encoding the metnase / setmar protein has been formed through fusion of the transposase gene of a mariner transposon with a set histone methyltransferase gene . 
 metnase / setmar is a dna binding protein with endonuclease activity that promotes dna double - strand break repair through nonhomologous end joining ( nhej ) [ 50 , 51 ] . 
 several genes derived from piggybac - like transposons have been detected in human and other vertebrates . 
 one of them , pgbd3 , serves as an alternative 3 terminal exon for the cockayne syndrome b ( csb ) gene , leading to the expression of a csb - transposase fusion protein . 
 at least one harbinger transposon - derived gene , harbi1 , encoding a predicted nuclease , is present in mammals , birds , amphibians , and fish . 
 likewise , genes derived from a new type of dna transposon called zisupton have been identified in fish and other vertebrates . 
 finally , mammalian and bird genomes possess at least one gene clearly derived from a p transposon ; additional vertebrate genes like thap9 encoding proteins with a thap domain might be also related to p - like transposases [ 8 , 40 , 5661 ] . 
 two vertebrate genes with unknown functions , gin1 and gin2 ( gypsy integrase 1 and 2 ) , encode proteins showing significant homologies to integrases encoded by ltr retrotransposons [ 62 , 63 ] . 
 further analyses showed that both genes have been formed from gin transposons , a new family of metazoan dna transposons with a transposase that shows strong similarities with ltr retrotransposon integrases . 
 gin1 , which shows similarities with gino transposons from hydra magnipapillata , is present in mammals , birds , and reptiles , suggesting a molecular domestication event at the base of the amniota ca . 
 mammalian gin1 proteins have conserved amino - acid residues necessary for integrase activity . using our own analyses 
 we provide here updated gin2 structural and phylogenetic analyses using new vertebrate sequences and present first expression data for this gene in fish . 
 gin2 is present in several fish species , as well as in cartilaginous fish ( elephant shark ) , coelacanth , amphibians , birds , reptiles , and marsupials , but neither in monotremes nor in placental mammals   ( figures 1 , 2 , and 3 ) . 
 hence , the molecular domestication event having led to the formation of gin2 might have taken place before the divergence between tetrapods / bony fish and cartilaginous fish around 500 million years ago , with subsequent loss in monotremes and placental mammals . 
 the formation of gin2 might even be older , since potentially domesticated gin - like sequences related to gin2 have been detected in the urochordates ciona savignyi and c. intestinalis . 
 phylogenetic analysis suggests that gin2 is derived from gina transposons , which are bona fide transposable elements in hydra magnipapillata ( figure 1 ) . 
 this suggests that gin1 and gin2 have been formed through two independent molecular domestication events , one at the base of amniota and the other in a more ancient vertebrate ancestor ( figure 3 ) . 
 after domestication , the hhcc zinc finger present in the ancestral integrase has been maintained , suggesting ability to bind to dna or rna ( figure 2 ) . 
 conservation of the important catalytic triad ( dde , aspartic acid / aspartic acid / glutamic acid ) of the integrase is less obvious . 
 while this motif has been proposed to be conserved in gin1 , this is not the case for gin2 based on a published alignment with sequences from gin - related transposases   ( figure 2 ) . as shown in figure 2 
 , the first aspartic acid residue is present in most species but absent from amphibians and birds . 
 however , multiple sequence alignment revealed an aspartate conserved in all gin2 and gin1 sequences ca . 
 the second aspartic acid residue is not found in gin2 but an aspartate is conserved four amino acids away in all gin2 sequences except for opossum . 
 finally , the glutamic acid residue is found only in several species and substituted by an aspartate in fish ; but a conserved glutamate is detected 16 amino acids away . 
 hence , the question of the functionality of gin2 as an integrase remains open and should be definitely answered through functional analyses . 
 a third domain with unknown function called gpy / f [ 64 , 67 ] is also detected in gin proteins , but in some cases the phenylalanine residue is replaced by a leucin . 
 gin2 contains eight protein - coding exons , with an exon - intron structure well conserved in fish and other vertebrates ( figure 4 ) . 
 some introns might be derived from the ancestral transposon ; others might be the result of events of intronization after molecular domestication . 
 gin2 is located in the same orthologous genomic region between ogfod2 and abcb9 in marsupials , birds , reptiles , and fish , confirming that this gene does not correspond to a mobile sequence ( figure 5 ) . 
 expressed sequence tag ( est ) analysis indicated that gin2 is expressed in different adult tissues and developmental stages in chicken : brain ( accession number : cn219658 ) , liver ( bg713188 ) , head ( bu225420 ) , embryonic tissue ( bu210425 ) , limb ( bu256599 ) , small intestine ( bu297502 ) , muscle ( bu437928 ) , and ovary ( bu447634 ) . only ests from the whole body are available for xenopus . 
 few ests are also found in zebrafish : muscle ( ct684014 ) , gills ( eb908574 ) , reproductive system ( bi867074 ) , and eye ( bi879358 ) . to determine more precisely gin2 expression pattern in fish , 
 quantitative real - time pcr was performed on different embryonic developmental stages in zebrafish ( danio rerio ) , as well as on adult tissues from zebrafish and platyfish ( xiphophorus maculatus ) ( figure 6 ) . during zebrafish embryogenesis , 
 gin2 expression level strongly increases from the dome stage and progressively decreases until the end of somite stages . 
 gastrulation , which is characterized by morphologic movements of involution and extension , starts at the beginning of the epiboly to finish at bud stage . in adult 
 zebrafish , the higher level of expression for gin2 was observed in brain , followed by gonads and eyes . 
 in contrast , gin2 expression was maximal in gonads in the platyfish ( figure 6 ) . to conclude , our analysis integrates data from several newly sequenced vertebrate genomes , particularly teleostean and cartilaginous fishes as well as coelacanth , in order to better understand the distribution and evolutionary history of gin genes . since gin2 is apparently not present in lamprey , 
 we propose that gin2 was formed before the divergence between cartilaginous and ray - finned fish about 500 million years ago ( figure 3 ) . 
 we also provide the first expression data for gin2 in fish particularly supporting a function in gastrulation during zebrafish embryogenesis . 
 dna with no important functions for genomes and organisms . today , nobody can deny the importance of transposable elements during evolution in terms of innovation power , particularly through molecular domestication events . 
 domesticated elements are bona fide cellular genes derived from transposable element sequences encoding for example integrases , transposases , gag proteins , or envelopes . after domestication , te - derived genes have lost their ability to transpose through the elimination of sequences such as long terminal repeats , terminal - inverted repeats , or other open reading frames and protein domains essential for transposition . 
 elimination of such sequences might occur by genetic drift or might even be selected for transposition or retrotransposition of a domesticated sequence might change its copy number and pattern of expression . 
 transposition of such a gene might have strongly deleterious consequences for the host , for instance cancer . 
 it might , therefore , be important to immobilize te - derived genes at fixed position within a genome to control their expression . in vertebrates , many te - derived genes are mammal specific , suggesting that molecular domestication probably played an important role in the evolution of this specific sublineage . 
 other te - derived genes like gin2 are present in some vertebrate sublineages but absent from mammals . in birds , reptiles , amphibians , and fish , domesticated sequences might be more difficult to identify due to the concomitant presence of active tes within genomes . 
 availability of additional genome sequences will probably allow the identification of many te - derived genes specific of these sublineages that contribute to diversification within vertebrates . 
 we focused on gin genes , a pair of ancient vertebrate domesticated genes for which no function has been identified so far . 
 both gin1 and gin2 are derived from gin transposons that themselves gained their transposase from the integrase of ltr retrotransposons . 
 gin1 was detected in mammals , birds , and reptiles , indicating that it was formed in a common ancestor of amniota ca . 
 gin2 might be even older , since it was detected in tetrapods , bony fish , and sharks , and possibly in urochordates . 
 the presence of both genes over such long periods of evolution is suggestive of important , so far unknown conserved functions in vertebrates . 
 gin2 was lost in a common ancestor of monotremes and placental mammals , suggesting that either gin2 function was not essential anymore , or that this function is fulfilled now by gin1 in these sublineages . 
 presence of conserved intron positions   suggests a unique origin followed by duplication and intron gain in a common ancestor of gin1 and gin2 ( paralogy ) . in this case , gin1 would have been lost among others in fish . 
 alternatively , gin1 and gin2 might have been generated from two independent events of molecular domestication , as suggested by the close phylogenetic relationship of bona fide gin transposons with each of both genes ( figure 1 ) . 
 presence of introns at conserved positions might in this case reflect intron conservation between ancestral gin transposons at the origin of both molecular domestication events . 
 gin1 and gin2 functions might be related to the binding to dna or rna , since both proteins have conserved the hhcc zinc finger present in the ancestral integrase . 
 conservation of the integrase activity appears possible but must be tested through functional assays . in fish , 
 gin2 is particularly expressed in brain and gonads ; its expression pattern during zebrafish embryogenesis suggests a role during gastrulation . functional analysis in fish 
 taken together , data on gin and other te - derived genes support the important role of molecular domestication as a driver of genetic innovation during evolution . 
 there is no doubt that future genome comparisons and functional gene analyses will uncover new domesticated genes and novel biological functions essential for the diversification of vertebrates and other living organisms .","due to their ability to drive dna rearrangements and to serve as a source of new coding and regulatory sequences , transposable elements ( tes ) are considered as powerful evolutionary agents within genomes . in this paper , we review the mechanism of molecular domestication , which corresponds to the formation of new genes derived from te sequences . 
 many genes derived from retroelements and dna transposons have been identified in mammals and other vertebrates , some of them fulfilling essential functions for the development and survival of their host organisms . 
 we will particularly focus on the evolution and expression of gypsy integrase ( gin ) genes , which have been formed from ancient event(s ) of molecular domestication and have evolved differentially in some vertebrate sublineages . 
 what we describe here is probably only the tip of the evolutionary iceberg , and future genome analyses will certainly uncover new te - derived genes and biological functions driving genetic innovation in vertebrates and other organisms .",pubmed
"these progressive diseases affect increasing number of people ( presently 45 million people world wide , expected to reach 76 millions in 2030 ) . 
 these devastating diseases have high societal costs and lead , directly or indirectly , to a dramatic loss in life expectancy and quality of life . 
 these diseases have in common the aggregation after misfolding and/or incorrect elimination of distinct polypeptides . 
 most important however is their ability to amplify by recruiting the soluble form of homologous polypeptides and to propagate from one cell to another . 
 thus , through their  template - driven misfolding of similar proteins over time , e.g. amplification , spread from one cell from the central nervous system to another , e.g. transmissibility and accumulation within the central nervous system , misfolded protein aggregates create a self - sustaining , exponential vicious circle leading to disease . 
 within our cells , nascent proteins fold spontaneously or following interaction with molecular chaperones in order to be functional . at 
 any given time a fraction of newly synthesized polypeptides populate non - native conformers [ 24 ] , with a sub - fraction corresponding to mis - processed peptides ( for example a after app cleavage by  and  secretases instead of the  and  secretase product p3 ) or polypeptides where translation errors occurred . 
 a fraction of these conformers is given another chance to fold correctly after full / partial unfolding by molecular chaperones   while another , never reaches the native state and is cleared or exported outside the cell . 
 thus , by opposing to protein misfolding , unfolding and aggregation , molecular chaperones , play a central role in the maintenance of normal cellular proteostasis . upon cellular stress 
 the amounts of misfolded / unfolded proteins and/or mis - processed and mutant polypeptides increase . 
 this triggers heat shock response via the activation of the heat shock transcription factor 1 leading to partial correction of misfolding / unfolding and incorrect processing [ 5 , 7 , 8 ] . as sustained activation of the heat shock response 
 is detrimental , normal cellular proteostasis is imbalanced with saturation of proteostatic networks [ 1 , 5 , 6 ] . as a consequence , 
 misfolded proteins aggregate into intra- or extracellular protein deposits , depending on whether they are within the cytosol of in the extracellular milieu . 
 it is widely accepted that the ubiquitin proteasome system degrades efficiently misfolded / unfolded or incorrectly processed proteins as long as the monomeric and aggregated forms are in dynamic equilibrium . in contrast , high molecular weight irreversible assemblies are cleared by autophagy . when the latter process fails either because of defects in autophagosome formation , transport , fusion with lysosomes or recognition of the substrate , protein aggregates accumulate with deleterious consequences . 
 misfolded proteins aggregation can cause both loss of function and gain of pathologic function [ 1214 ] . 
 this is the consequence of either i - misfolded protein aggregates - mediated permeabilization of plasma membrane and/or membranous compartments , ii - perturbation in membrane protein dynamics and distribution they may cause , iii - the formation of novel pathogenic signaling platforms they may mediate , iv - the trapping of significant amounts of molecular chaperones and other partner proteins within the aggregates and v - the ability of the misfolded protein aggregates to recruit the soluble form of the proteins they are formed of and amplify . 
 protein aggregates made of fibrillar material , in some cases straight as needles , in others curved and twisted , are the hallmarks of a variety of diseases affecting the central nervous system , in particular , alzheimer s ( ad ) , parkinson s ( pd ) , huntington s ( hd ) and creutzfeldt - jacob ( cjd ) diseases . 
 the number of deposits within the affected tissues correlates most often with disease severity [ 1517 ] . as the probability of occurrence of events such as i - misfolding and aggregation events of newly synthesized polypeptide chains , 
 ii - unfolding of native polypeptide chains following a variety of stresses , iii - errors during translation and iv - incorrect processing and elimination of proteins targeted to degradation , is dependent on time , misfolding diseases are associated to ageing . 
 until recently , the spread and transmission of disease via misfolded protein aggregates was thought to be restricted to the prion protein ( prp ) . 
 a wide range of recent experimental evidence suggest that other protein aggregates that are the hallmarks of major neurodegenerative diseases spread in a prion - like manner [ 20 , 21 ] . 
 thus , misfolded protein aggregates propagation and amplification appears to be a widespread phenomenon in neurodegenerative diseases as opposed to a curiosity limited to a unique protein : prp . 
 indications for such a process originated from the work of heiko braak and co - workers who described distinctive patterns of pathological changes over time throughout the brain for the major neurodegenerative diseases in man . indeed , based on autopsy cases , braak and 
 co - workers noticed that protein aggregates associated to pathology initiate in circumscribed areas of the brain specific to each disease and progress in a topographically predictable manner following anatomical connections [ 1517 ] . 
 these observations led them to establish a disease progression scale and to hypothesize that neurotropic pathogens were spreading through defined pathways to interconnected regions within the central nervous system . as for prp , these pathogens were first thought to be of viral nature . 
 the first evidence for the transmission of misfolded protein aggregates not involving prp in man came from the observation that lewy bodies in pd brains contaminate grafted fetal mesencephalic progenitor neurons decades after transplantation [ 22 , 23 ] . 
 injection of brain extracts containing amyloid plaques , which main constituent is the aggregated form of the amyloid -peptide ( a ) , neurofibrillary tangles , that are mainly made of the microtubule associated proteintau , and lewy bodies , which main constituent is the protein alpha - synuclein ( -syn ) , into the brain of model animal induced lesions characteristic of ad and pd [ 2434 ] . 
 the induced lesions , that were initially confined to the injected brain region , propagated to neighboring and/or axonally connected areas over time , suggesting directed spreading and amplification through neuronal transport processes [ 3235 ] . 
 the intraperitoneal injection of brain extracts containing amyloid plaques and neurofibrillary tangles also induced cerebral lesions characteristic of ad ( a and tau deposits ) [ 36 , 37 ] . 
 similarly , the injection of brain extracts and lewy bodies enriched fractions from patients developing pd into the brain of non - human primates , wild - type and model mice , induced several months after injection the formation of lesions characteristic of pd [ 3135 ] . finally , lewy bodies of human nature appeared in rodents neural grafts implanted in the brains of mice expressing human -syn that develop lewy bodies over time   and aggregated huntingtin ( htt ) with a polyq stretch length associated to hd and huntingtin n - terminal moiety ( htt - exon1 ) were shown to move between cultured cells , in organotypic slice cultures of a htt mouse model and between host and grafted tissues [ 3840 ] . 
 luckily , we can reproduce in test tubes the aggregation of a , tau , -syn , htt - exon1 with a polyq stretch length associated to hd , and cu / zn superoxide dismutase 1 ( sod1 ) point mutants associated to motor neuron death in familial cases of amyotrophic lateral sclerosis ( als ) . 
 pathologic htt - exon1 , only in its fibrillar form , binds to cultured cells , is internalized through yet an unknown mechanism , and induces the aggregation of sub - pathologic htt - exon1 within the cytosol of the recipient cells [ 38 , 41 ] . -syn 
 mega - dalton protein assemblies were also shown to be taken up   and to trigger the aggregation of endogenous cytosolic -syn in cultured primary cortical neurons and in neuronal cell lines [ 4245 ] . 
 they were shown to be transported anterogradely and retrogradely following their uptake in primary neurons and to be transmitted to secondary neurons . in vitro 
 generated tau assemblies were also shown to bind and enter cells and to transmit a misfolded state to intracellular tau [ 44 , 4749 ] . finally , mutant sod1 aggregates made in vitro were shown to penetrate inside neurons by macropinocytosis and induce the aggregation of soluble mutant sod1 in the cytoplasm . 
 they were taken up by neurons and glial cells and shown to transfer from neurons to oligodendrocytes . 
 fibrillar assemblies induced several months after injection the formation of lesions characteristic of pd and ad [ 32 , 37 ] . 
 under physiological ph , salt and temperature conditions , a , tau , -syn , htt - exon1 with a pathologic polyq stretch and mutant sod1 , self - associate into assemblies that can elongate in a cooperative manner by incorporation of monomers . 
 growth is unlimited as the incorporation of a monomer creates a new binding site for another monomer ( fig 
 metastable , prefibrillar oligomeric intermediates , considered as the precursors of mature fibrils , are populated prior or concomitant to fibrils formation [ 52 , 53 ] . in some cases , it is the binding of ligands to aggregation prone polypeptides or their modifications that yield oligomeric species that are off - assembly pathway as for example upon the interaction of -syn with dopamine . 
 the nature of the assemblies that exhibit the highest toxicity and/or seeding capacity is subject to intense debate . 
 fibrillar assemblies made under physiological conditions have been shown to be the only entities capable of recruiting the soluble form of homolog proteins in vitro , in cell cultures and in vivo , i.e. with seeding capacities , and a body of evidence demonstrates their cytotoxic potential [ 30 , 33 , 38 , 41 , 4345 , 4850 , 5558 ] . 
 nonetheless , they are considered as storage , inert form of misfolded proteins by those who consider the oligomers that form during the early stages of assembly as toxic [ 5963 ] . 
 toxicity in the latter case is thought to be the consequence of impairment of membrane integrity and permeability [ 59 , 64 , 65 ] . 
 the conflict is certainly due in some cases to non - physiological assembly conditions such as assembly after polypeptide dehydration , under high or low ph , at very low ionic strength ( e.g. distilled water ) , extreme salt conditions and temperatures that are suitable for extremophiles . 
 it is also and mostly due to i - comparison of the properties of protein particles at different concentrations , ii - difficulties in defining what is precisely an oligomer and iii- oligomers - fibrils inter - conversion . at a given concentration of monomeric a , tau , 
 -syn , htt - exon1 with a pathologic polyq stretch or mutant sod1 the concentrations of on - assembly pathway oligomers and fibrils diverge by several orders of magnitude as the molecular masses , number of constituting molecules and sizes of oligomeric and fibrillar species differ very significantly . 
 indeed , oligomeric species are made of less than 10 to 50 monomeric proteins at most while fibrils that are over 0.1 m in length are made of thousands of polypeptide chains . 
 thus , it is critical to compare the toxicity of the different assemblies at identical particle concentrations as cells interact with the particles as a whole , not the precursor monomeric protein within the assemblies . 
 measurement of the molecular weight of defined on - assembly pathway oligomeric assemblies and fibrils with homogeneous length allowed calculating the particle concentration of assemblies at any given concentration of precursor monomeric polypeptide . when the relative toxicities of monomeric , oligomeric , and fibrillar assemblies were compared , the fibrillar assemblies , with homolog polypeptide seeding and spreading propensity , were found most toxic . as fibrils and oligomers have distinct molecular weights they are frequently separated by centrifugation . 
 however as the solutions are often viscous , a significant amount of short fibrils may remain in the supernatant . 
 it is therefore important to bear in mind that one can not exclude that observations derived from such fractionation procedures are due to the presence of short fibrils . 
 finally as fibrils and precursor oligomeric species are in equilibrium   one needs to keep in mind that oligomers can form fibrils in the time course of the experiment either in solution or when associated to the cell membranes as their concentrations increase when moving from a three- ( test tube ) to a bi- ( cell membrane ) dimensional space . 
 similarly , while fibrils most often exhibit some resistance to detergents , one can not exclude their possible disassembly by cellular factors . 
 the toxicity of fibrillar and oligomeric assemblies is in part the consequence of their binding to and permeabilization of the cell membrane . 
 the binding of assemblies , besides inducing lipid rafts and the monosialoganglioside gm1 clustering , certainly impinge membrane curvature , thus further affecting cell survival . 
 furthermore , the lateral diffusion of assemblies in the plane of the plasma membrane together or independently of membranous components such as receptors , channels , adhesion molecules , integrins certainly contributes to the redistribution and/or coalescence of protein receptors at the surface of the cell . 
 misfolded protein assemblies may even selectively recruit membranous proteins thus creating novel signaling platform with undesirable properties , thus contributing to cell apoptosis . 
 once internalized , they appear to leak out of the endosomal - lysosomal compartments as fibrils in particular reach the cytosol and seed the aggregation of their soluble cytosolic counterparts [ 38 , 42 , 45 , 47 ] . by crossing these membranous compartments , through yet an unknown mechanism , they may cause leakage of the lysosomal content or rupture of lysozomes , with cathepsin b - dependent increase in reactive oxygen species within the cytosol , thus triggering pathogenesis . 
 the latter may account for the mitochondrial dysfunction and inflammation described in synucleinopathies . upon reaching the cytosol 
 , exogenous misfolded protein assemblies certainly imbalance proteostasis following the titration of the cytosolic molecular chaperones and/or saturation of the clearance machinery of the cell leading to additional deleterious effects [ 5 , 76 ] . 
 finally , toxicity may also be the consequence of the seeding capacity of the protein assemblies , e.g. the recruitment of fibrils soluble counterpart and exhaustion of the functional form of the protein . 
 as exogenous a , tau , -syn etc  ...   assemblies bind and penetrate cells , amplify by recruiting their endogenous soluble counterparts through a seeding process and propagate to other cells they can be considered as infectious   in accord with the modifications that have been proposed to koch s postulates . 
 the observation that the aggregation of non - mutant prp give rise to distinct diseases ( creutzfeldt jakob s disease ( cjd ) , new variant cjd ( vcjd ) and kuru in man , classical and nor98 scrapie in sheep , classical and atypical ( h - bse and l - bse ) bovine spongiform encephalopathies ( bse ) ) that are distinguishable by the disease incubation time , lesions type in the brain and proteolytic cleavage patterns has led to the notion that prp can adopt different conformations into the mega - dalton assemblies associated to disease . 
 indeed , each conformation an amino acid stretch adopts can establish a defined set of inter - molecular interactions . as a consequence alternative packing is possible yielding structurally unlike assemblies that expose distinct surfaces ( fig .  1 ) . 
 the lateral surfaces of these different assemblies rule their ability to interact with receptors and lipids at the surface of the cells and protein partners within the cell . 
 these distinct conformations are indeed imprinted to the newly incorporated prp upon assemblies growth either through a templating process or the selective recruitment of defined prp conformers that can establish intermolecular interactions with the well defined assemblies tips thus leading to the faithful amplification of distinct prion strains . in a manner similar to prp , 
 the deposition of -syn within the central nervous system is not only tightly associated to pd but also to distinct pathological phenotypes : multiple system atrophy ( msa ) and dementia with lewy bodies ( dlb ) . 
 , the aggregation of tau also leads to distinct tauopathies : argyrophilic grain disease , tangle - only dementia , pick disease , progressive supra - nuclear palsy and cortico - basal degeneration . 
 the involvement of the deposits made of the same protein in diseases exhibiting different phenotypic traits can be accounted for either by the existence of distinct i - -syn and tau strains , ii - areas within the central nervous system affected by neurodegeneration or iii - neuroinflammatory responses , given that neuroinflammation has been repeatedly linked to neurodegeneration . indeed , mounting evidence from epidemiological , postmortem , brain imaging and animal studies indicate that neuroinflammatoryprocesses are tightly linked to neurodegeneration [ 7982 ] . 
 nonetheless , it is still unclear whether the inflammatory process is involved in disease etiology or a secondary consequence of neurodegeneration and whether neuroinflammation affects to different extents distinct brain regions . 
 differences in the biochemical properties of aggregated -syn in different genetic backgrounds in humans have been reported suggesting that -syn aggregates may be unlike . 
 the existence of -syn strains was established upon the generation of pure -syn fibrillar polymorphs under stringent and tighly controlled assembly conditions in vitro . 
 distinct fibrillar polymorphs made of wild - type , full - length -syn , exhibited different shapes in the electron microscope , limited proteolytic and x - ray fiber diffraction patterns and were distinguishable by conformational antibodies . 
 assessment of their intrinsic structures by solid - state nuclear magnetic resonance ( nmr ) revealed that although they have high beta - sheet content , -syn molecules within the fibrillar polymorphs are packed in distinct manner . 
 as a consequence they recruit soluble -syn with different efficacy , elongate at distinguishable rates and must have different surface properties . 
 functional studies revealed that the distinct polymorphs bind and permeabilize cells to different extents ; they exhibit different toxicities , efficacy of recruitment of cellular -syn and persistence / propagation propensity . 
 they were also shown to imprint their distinct intrinsic structures to the recruited cellular -syn   ( fig .  2 ) . 
 the finding that distinct -syn strains bind to cells and are taken up with different efficiencies suggest that they may exhibit different tropism for cells from the central nervous system , in other words , bind with different affinities to neurons , glial cells or astrocytes . by targeting different cells from the central nervous system or circuits , they may be at the origin of distinct synucleinopathies . 
 as distinct strains differ by their surface properties they also may trigger the inflammatory response to different extents . 
 finally , the progression of distinct synucleinopathies may also reflect the different seeding propensity , resistance to clearance and persistence in cell cultures of distinct -syn strains . 
 baffling evidence for the existence of -syn strains in vivo were brought by the groups of s. prusiner , e. bezard and m. vila who demonstrated that semi - purified aggregated -syn from patients developing msa or pd faithfully led to msa or pd - like neuropathological disorders in recipient animals [ 34 , 84 ] . 
 additional evidence for the existence of putative -syn strains came from the lesion profiles observed after injection of either newly formed -syn fibrils orrepeatedly seeded -syn fibrils and the differential induction of tau aggregation by the two kinds of assemblies . 
 it also came from comparing the proteinase k degradation profiles of sarkosyl - insoluble protein fractions isolated from patients diagnosed pd or pd and ad because of abundant tau neurofibrillary tangles and senile plaque . while one can not rule out that the differences in the degradation profile of aggregated -syn are due to the abundance of tau neurofibrillary tangles and a - beta these findings remain striking . recently , amyloid plaques extracted from the brain of two patients developing ad , with distinct neuropathology and clinical history , were used to seed the aggregation of a in vitro . 
 although a aggregation in control brain extracts from healthy aged individuals and patients developing unrelated neurodegenerative diseases was not assessed , these observations suggest that a within the initial seeds recruit soluble a in vitro and imprint their structures to the synthetic , isotopically labeled , a. further evidence for the existence of a strains came from the use of tightly controlled assembly conditions in vitro of synthetic a140 and 142 . starting from the observation that synthetic fibrillar a140 recruits when injected into the brain of recipient animals both a140 and a142 while synthetic fibrillar a142 induce the aggregation of a142 , it was shown that slightly different assembly conditions lead synthetic fibrillar a142 to stably acquire the seeding properties characteristic of fibrillar a140 . 
 evidence for the existence of tau strains have been brought through the use of truncated tau . when exposed to the fibrillar form of truncated tau , 
 cells expressing the same truncated tau fused to a reporter fluorescent protein develop truncated tau clusters of different shapes . 
 the pattern of these clusters is stably inherited over generations and transmissible to nave cells . 
 when lysates from cells exhibiting distinct clusters were injected into the brain of recipient model animals , they induced distinguishable neuropathological patterns that could be stably propagated in mice . finally , brain homogenate from patients developing different tauopathies induced the clustering of reporter tau in cultured cell lines . 
 reporter tau inclusion morphology and limited degradation patterns revealed homogeneous for most pathological cases with exceptions suggesting that a number of patients may suffer from more than one taustrain . 
 symptomatic treatments are the only therapeutic tools available for patients developing ad and pd . in pd , for example drugs aiming at restoring dopamine levels , such as l - dopa , dopamine agonists , mao - b and comt inhibitors , although powerful , lose efficiency and occasion side effects with time [ 8890 ] . 
 invasive interventions , such as deep - brain stimulation , are implemented when drugs fail to relieve the patients . 
 they are used when neurodegeneration has become sufficiently severe to induce a clinical phenotype . the discovery 
 that a , tau and -syn fibrillar assemblies transit between cells within the central nervous system and amplify by recruitment of their soluble counterparts in recipient cells , thus contributing to disease progression , suggests that pathogenesis starts many years before the first symptoms become apparent . 
 targeting early enough protein assemblies involved in disease formation and propagation may therefore represent a novel and effective preventive therapeutic avenue . 
 thus , in compliment to the classical approaches aimed at either inhibiting the initial aggregation of proteins or favoring their clearance by boosting the level of molecular chaperones within the cells and/or the ubiquitin - proteasome clearance machinery , or both , therapeutic strategies targeting disease - associated protein assemblies propagation and seeding capacities become highly relevant . 
 small molecules such as the polyphenols rottlerin , curcumin or epigallocatechin-3-gallate have been shown to either interfere with the aggregation of -syn , stimulate proteostasis , activate the ubiquitin - proteasome clearance machinery or counteract the neuro - inflammatory response associated to -syn aggregation [ 9193 ] . a novel class of small molecules that repair proteostasis networks specifically through hsf-1 , foxo and nrf-2 and restore proteome balance has been identified . 
 these molecules have a promising therapeutic potential in a variety of protein conformational diseases as they enhance cell stress pathways and chaperone activity . 
 they protect therefore the cells in a generic manner against misfolding and/or aggregation and the resulting pleiotropic damages . 
 in addition , strategies aimed at protecting and/or stimulating neuronal cells through the use of neurotrophic factors such as brain - derived neurotrophic factor   or clearing the polypeptides associated to neurodegeneration prior to their aggregation / deposition through immunotherapy are explored [ 97 , 98 ] . 
 finally , neural stem cell - based treatments for neurodegenerative diseases are considered . in the case of pd , innovative strategies aimed at interfering specifically with -syn assemblies propagation and seeding capacities have not yet been developed to a stage where they can be used for therapeutic purposes . 
 the key step in this process is the docking of -syn assemblies to the cell . 
 one can interfere with this process either by i - perturbing the binding capacity of extrinsic membrane proteins that interact with -syn assemblies or ii - changing the surface properties of -syn assemblies so that they become incapable of binding to their receptors . as no specific receptors of -syn assemblies 
 have been identified so far , the strategy consisting of rendering those receptors unavailable for binding -syn assemblies is impossible . furthermore changing the binding capacities of these receptors may cause undesirable pleiotropic effects . designing -syn assemblies binders that change their surface properties in such a way that they either become unable to bind the cell surface or incapable of elongating by recruitment of the endogenous protein because their ends are capped appears more promising . 
 two classes of binders , molecular chaperones and antibodies , have been shown to interfere with -syn assemblies uptake by cells and/or toxicity [ 100 , 101 ] . 
 thus , identifying within these molecules what is necessary and sufficient for binding is critical for the design of smaller molecules with therapeutic potential . 
 the interaction interfaces between the molecular chaperone hsc70 client protein binding site and -syn have been identified through the use of chemical cross - linkers and mass spectrometry [ 102 , 103 ] . the same strategy can be employed to identify paratopes within antibodies exhibiting high affinity for -syn . 
 peptides reproducing hsc70 or anti--syn paratopes that bind with the highest affinity -syn assemblies represent potential therapeuticcandidates .","abstractmisfolded protein aggregates are the hallmark of several neurodegenerative diseases in humans . 
 the main protein constituent of these aggregates and the regions within the brain that are affected differ from one neurodegenerative disorder to another . 
 a plethora of reports suggest that distinct diseases have in common the ability of protein aggregates to spread and amplify within the central nervous system . 
 this review summarizes briefly what is known about the nature of the protein aggregates that are infectious and the reason they are toxic to cells . 
 the chameleon property of polypeptides which aggregation into distinct high - molecular weight assemblies is associated to different diseases , in particular , that of alpha - synuclein which aggregation is the hallmark of distinct synucleinopathies , is discussed . 
 finally , strategies targeting the formation and propagation of structurally distinct alpha - synuclein assemblies associated to different synucleinopathies are presented and their therapeutic and diagnostic potential is discussed .",pubmed
"hepatitis c virus ( hcv ) related morbidity and mortality risk is significant in patients with chronic hepatitis c ( chc ) , which affects approximately 3.2 million people in the united states ( 1% of the population).15 in patients treated with interferon ( ifn ) and ribavirin , 2560% achieved sustained virus response ( svr ) , and with the addition of first generation direct acting antivirals ( daas ) , svr was achieved in almost 80%.69 treatment with new daas led to svr rates greater than 90%.1012 african americans ( aa ) are twice as likely to be infected with hcv relative to the nonhispanic - caucasian us population and have historically had lower response rates to approved ifn based therapies.420 although fibrosis progression is less rapidly in aa compared to caucasians , it is unlikely to be a good clinical strategy to counsel aa that they are at lower risk for rapidly developing advanced liver disease.1921 thus , more specific information about the aa population is needed to better understand the progression of liver disease , especially after achieving viral clearance , and to identify optimal treatment strategies . 
 a number of publications have compared the response and outcomes in patients treated with ifn - based therapies.2227 all studies have demonstrated that patients who achieve an svr have better outcomes with respect to cirrhosis , development of hcc , and all - cause mortality . a report by cozen et al compared outcomes for treated and nontreated patients in two representative diverse populations . 
 the results suggested that patients who received ifn - based therapies but failed to clear hcv may be at increased risk for developing cirrhosis compared to untreated patients . 
 overall survival was not significantly different.28 additional studies provided conflicting results , but these did not contain a significant aa population . finally , a study utilizing electronic medical records ( emrs ) from a department of veterans affairs clinical registry confirmed the value of achieving svr and found that aa were at lower risk than caucasians for all liver events.29 the aim of this prospective study was to evaluate the effect of ifn based therapies on outcomes in aa patients . the predominant population in our urban clinic is aa , and we previously described the demographics and hcv relevant disease parameters in this patient group.30 here , we identified 346 aa first seen in 19952008 and followed them for 8 years on average . 
 our findings further the understanding of liver disease progression in aa and provide information that will specifically guide clinical management and therapy decisions in this underserved and under - investigated population . 
 in this prospective study for outcomes , we started with screened a database of 3,800 chc patients who were seen at the wayne state university physician group ( upg ) gastroenterology clinic between 1995 and 2008 . 
 all patients signed an institutional review board ( irb ) approved consent form . using the emr and an irb approved health insurance portability and accountability act ( hipaa ) exemption from upg and detroit medical center , we identified patients who were diagnosed with hcc between 2009 and 2013 and were in the original database . 
 we also identified patients were seen again between january 2012 and july 2013 in order to identify patients with a significantly long follow up time . in total , we identified 346 aa patients with hepatitis c genotype 1 that were followed for 4 to 18 years . 
 available patient information included demographic information , laboratory values , biopsies , imaging , and response to treatment . 
 first visit and  early visit  indicate that the information was relevant to the initial assessment of the patients although some data was order and obtained later as part of the initial assessment . 
 cirrhosis was diagnosed with a combination of liver biopsy , ultrasound , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and/or esophagogastroduodenoscopy evidence of portal hypertension . since repeat biopsies 
 were rarely performed , two well - studied markers of fibrosis were used to determine changes in fibrosis over time.3134 the aspartate amino transferase ( ast ) platelet ratio index ( apri ) was defined as ( ( ast(iu / ml)/40 iu / ml)/platelets ( count10/ml))100.33 fibrosis-4 ( fib-4 ) was defined as ( ( age(years))(ast(iu / ml)))/((platelets(count10/ml))(square root alt(iu / ml)).34 both assays are continuous variables and the literature cutoffs to define minimal vs significant fibrosis were 0.7 for apri and 1.45 for fib-4 . for cirrhosis , 
 the response of aa to therapy was based on intent to treat ( itt ) . 
 the sas based statistical program jmp was used for statistical analysis . to compare different groups , student s t test , analysis of variance ( anova ) , or chi - square 
 changes between early visit and recent visit were evaluated using pairwise analysis . in order to determine the relationship between multiple factors defined in univariate analysis and outcomes , multivariate analysis ( logistic regression ) was used . 
 in total , 346 aa patients , followed for an average of 8 years , were included in our study . 
 the study group was 57% male , and the mean age was 54 years with a range consistent with known infection rates of individuals born between 1945 and 1965 . 
 the mean levels of alanine aminotransferase ( alt ) and ast were elevated , but albumin and platelet levels were within normal range . the majority of patients had liver biopsy ( 257 out of 346 ( 74% ) ) , and the biopsy mean fibrosis score , as defined by the metavir , system was 1.88 . 
 significance defined by pair wise analysis ; ns , not significant , p>0.01 ; * p<0.01 ; * * p<0.001 . 
 all treated patients received ifn based therapies , and 147 patients ( 43% ) received at least one dose of treatment . 
 the majority of treated patients ( 86% ) were given dual therapy of pegylated - ifn and ribavirin . 
 the svr rate was 15% based on itt and 23% based on patients who adhered to the protocol . 
 when patients who relapsed following treatment were included , the end of treatment response ( etr ) was 24% . 
 when patients were stratified by treatment , there were minimal differences between various groups of patients at the first visit ( table 1 and fig . 
 1 ) . the largest difference , although statistically insignificant , was a reduction in hcv ribonucleic acid ( rna ) levels in patients with svr relative to other groups . 
 in addition , the values before treatment and after treatment / no treatment for liver related laboratory values as a function of response to treatment were compared . 
 the most improved patients were those who achieved an svr , had reversal of alt and ast elevation , and had no decline in albumin and platelet counts . 
 apri and fib-4 were used to assess changes in fibrosis / cirrhosis.3134 we used biopsy data to validate both the apri and fib-4 in our study population . 
 there was a strong correlation between the degree of fibrosis defined by biopsy and fibrosis as calculated by apri and fib-4 ( p<0.001 data not shown ) . 
 also the receiver operating characteristic and the area under the receiver operating characteristic curve ( auroc ) values for the aa population were between 0.56 and 0.75 ( data not shown ) . 
 these results are consistent with the literature , and confirm that both calculations are sensitive and specific for measuring differences in fibrosis in aa.3134 the fibrosis scores by apri ( a ) and fib-4 ( b ) are plotted as the average for paired patients between the initial visit ( 1 ) and follow - up visit ( 2 ) . 
 n treat , nontreated ; dis , discontinued ; nonr , nonresponders ; rel , relapsed ; svr , sustained virus response . 
 significance was defined using the paired difference test . when the apri and fib-4 were evaluated in patients at the initial visit and 
 follow - up , there was no significant change in fibrosis in patients that were not treated ( fig . 
 only the patients who achieved svr had a significant improvement in these fibrosis scores relative to the other groups . 
 statistical significance was assessed using a pair - wise analysis , and there were differences in individual patients but not group averages . 
 patients who achieved svr did not develop new cirrhosis or hcc in the follow - up period ( fig . 
 patients who were not treated , who discontinued treatment , or who were nonresponsive to treatment developed hcc and newly identified cirrhosis . 
 the nonresponding patients , however , had a lower incidence of hcc than untreated or discontinued treatment patients . 
 patients achieving an etr but not svr did not develop hcc but exhibited a similar incidence of new cirrhosis as the other groups . 
 patients were evaluated for the development of hepatocellular carcinoma ( hcc ) and new cases of cirrhosis at visits following the initial visit . 
 the majority of patients were treated with peg - ifn + ribavirin ( 86% of treated patients ) . 
 etr ( svr + relapse patients ) was 24% by itt and 36% by protocol . 
 n treat , nontreated ; dis , discontinued ; nonr , nonresponders , rel , relapsed ; svr , sustained virus response . 
 since males are more likely to develop hcc , we compared hcc development and response to therapy by gender ( fig . 
 the response to treatment was similar for both genders ( 13% f vs. 18% m ) , although the percentage of females treated ( 46% ) was greater than males ( 38% ) . 
 3 , aa males were more likely to develop hcc than females , but patients achieving an svr or etr with subsequent relapse did not develop hcc regardless of gender . 
 in addition , although males had a higher incidence of hcc , the time to development of hcc was not statistically significant between the genders ( f=9 years vs. m=8 years ) . 
 the % of patients who developed hcc was plotted by response to treatment and gender . 
 similar numbers of aa patients were treated ( 46% f and 38% m ) , and response rates based on intent to treat were also similar ( 15% ) . 
 although males were more likely to develop hcc , hcc did not develop in either the svr or etr ( relapse ) patients , regardless of gender . 
 the average time to hcc was 9 years for females and 8 years for male ( nonsignificant by chi - square ) . in order to define the natural history of the disease in aa patients 
 the time to hcc onset ranged from 1 year to 14 years after the first visit , with 4 to 6 years being the most common timeframe ( n=24 , fig . 
 4 ) . there was no difference in time to hcc diagnosis between aa patients with or without cirrhosis at the time of the first visit . 
 all patients ( n=51 ) who developed hcc were stratified by those who had cirrhosis at first visit ( hcc from cirrhotic ; n=12 ) and those who did not ( hcc - noncirrhotic ; n=39 ) . almost half 
 ( 24/51=48% ) of patients developed hcc 47 years after the first visit but a significant number of patients ( 25% ) did not receive a diagnosis of hcc for more than 11 years . 
 the risk of developing of hcc at the first visit was not predicted by the presence of cirrhosis , since the time to hcc distribution was similar for those with and without cirrhosis at the first visit . 
 untreated patients who did not develop hcc were compared to those who did develop hcc in order to determine whether particular parameters at the first visit were useful predictors of hcc development . 
 alt , ast , albumin , platelet counts , and alpha fetoprotein ( afp ) all correlated / inversely correlated with hcc development in aa ( p<0.005 , data not shown ) . fig . 
 5 presents the data related to fibrosis ( liver biopsy metavir scores , apri , and fib-4 ) , and all were significant predictors of hcc . 
 fibrosis but not cirrhosis at the first visit was a significant risk factor for hcc . 
 there was a wide range and considerable overlap in all patient parameters among patients who developed hcc and those who did not . 
 therefore , the values could not be used to predict individual risk for developing hcc . when using multivariate analysis for all risk factors ( alt , ast , albumin , platelets , afp , stage of fibrosis , apri , fib-4 , and cirrhosis ) , no parameters were identified that were independent predictors of risk for developing hcc when subjected to logistic regression analysis . when the multivariate analysis was restricted to only fibrosis relevant independent predictors ( metavir stage , apri , and fib-4 ) , the only independent predictor of hcc was stage of fibrosis ( p<0.002 ) . 
 individual patients were stratified by those who developed hcc or those who did not and their individual values for potential risk factors at first visit are plotted . for continuous variables , 
 the graphs have the mean and sem and significance was defined with a student s t test ( a , b ) . for discontinuous values ( fibrosis by metavir and presence / absence of cirrhoses ) the data are presented as mosaic plots and significance was calculated using pierson chi - square ( c , d ) . 
 the width of the plots reflect the total numbers of patients , and the various shadings represent the % of patients with each parameter ( scores of 0 - 4 or cirrhosis or no cirrhosis ) . the bar to the right of each graph reflects the % of total patients . 
 similar results were obtained for ast , albumin , platelet counts , and afp ; and the difference at entry between hcc and no hcc was significant ( p<0.001 for all , data not shown ) . 
 the wide variability and overlap between individuals in those assays was also similar to that shown in the figure . although patients who developed hcc were more likely to have cirrhosis at first visit , the difference was not statistically different . 
 in this study , we followed aa patients with chc for an average of 8 years who were infected with hcv genotype 1 . 
 chc outcomes were defined by either the development of cirrhosis , hcc , or fibrosis from the initial visit to the most recent follow up in 2012 - 2013 . as part of this study , we confirmed that a significant number of patients with early visit liver biopsy results had a good correlation with both apri and fib-4 , thus confirming them as useful surrogate markers for fibrosis in aa patients . 
 since repeat biopsies were rarely performed , fibrosis was detected using apri and fib-4.7,3134 the study demonstrated that permanent viral elimination ( defined as svr ) with ifn based treatment in aa patients with chc protected them from cirrhosis and hcc and reversed fibrosis . 
 although chc patients with etr who relapsed developed new cases of cirrhosis at rates similar to those of nontreated or treatment failure patients , they did not develop hcc . 
 in contrast , patients who failed treatment or were not treated were at high risk for the development of hcc and new onset cirrhosis . 
 this study also confirmed that aa males are at higher risk for developing hcc than aa females . 
 the response to therapy was similar and achieving an svr reduced the risk of hcc in both genders . since a significant number of patients developed hcc , we were able to determine whether hcc risk in aa was predictable based on the first clinical visit . 
 unlike many studies , we plotted individual patients rather than an average in order to assess the clinical value per patient . 
 regardless of whether serum assays ( alt , ast , afp , albumin , platelets ) or fibrosis assays ( biopsy , apri , fib-4 ) were compared between patients who did or did not develop hcc , there was no reliable predictor on an individual basis . 
 somewhat unexpected , we found that cirrhosis at first visit in aa was not predictive of hcc development . 
 when multivariate analysis was used to determine whether there was a single independent factor that was predictive of development of hcc in aa , only fibrosis , as defined by biopsy at the first visit , was even moderately predictive . 
 this lack of positive predictive value for factors was further confirmed by the finding that when patients who developed hcc were stratified by cirrhosis ( or no cirrhosis ) at first visit , there was no relationship between time to hcc and cirrhosis . 
 although the number of patients achieving an svr was sufficient for analysis , we could not rule out the possibility of the development of hcc in patients who achieved an svr . 
 there are a number of publications suggesting that the rate of hcc may be as high as 5% in patients experiencing an svr.3537 those studies are , however , primarily in asian and european populations and do not contain significant numbers of aa . 
 the primary risk factors for developing hcc in patients with svr are males with genotype 1 and cirrhosis at the time of viral clearance . 
 the majority of hcc cases occurred within 3 years , suggesting the presence of small precancerous lesions at the time of viral clearance . 
 since our population contains patients with these significant risk factors and a high incidence of hcc was present in the untreated and nonresponding population , it is possible that the development of hcc in aa after clearance of the virus is considerably less likely than in the other populations studied . to a large extent , this study is prospective in nature since patients were first seen prior to 2008 and subsequently seen until 2013 or the development of hcc . 
 patients who developed hcc were followed for 114 years , while those who did not , were seen for a minimum of 4 years and a maximum of 18 years after initial presentation . this study did not compare caucasians to aa , as the number of caucasians seen in our clinic has continued to decline and there were insufficient numbers available for statistical analyses . 
 the incidence of hcc in aa in our study was 20% in untreated patients , which was sufficient to provide statistical and clinical relevance to our study . 
 perhaps the most important observation of the study was that aa who did achieve svr were similar to other groups of svr patients who also had improved outcomes relative to untreated or nonresponding patients . with the advent of more effective daa therapies , possible differences in response rates between aa and caucasians 
 are no longer an issue , and more patients should achieve an svr.12 although there are many risk factors for the development of hcc in aa patients , if patients are not successfully treated , it remains difficult to predict the development of hcc . since identification of cirrhosis at an early visit may not be a significant risk factor in aa , surveillance in patients with significant fibrosis , rather than just cirrhosis , is warranted . unfortunately in many studies to date , it is often a challenge to provide effective hcc surveillance of this population ; and thus , elimination of the virus remains the optimal goal of therapy .","background and aims : african americans ( aa ) historically have a low response rate to hepatitis c therapies , and there is limited information available for this patient population regarding the development and treatment of chronic hepatitis c ( chc ) . 
 the aim of this study was to evaluate liver disease progression and hepatocellular carcinoma ( hcc ) development in aa with chc . 
 methods : between 1995 and 2008 , 246 aa patients with chc were identified from a database of patients and followed until 2012 - 2013 ( average 8 years ) or the development of hcc after 2008 . results : viral clearance ( intent to treat ; sustained virus response ( svr ) ) 
 was achieved in 15% of patients with interferon based therapies with or without ribavirin . 
 aa patients who achieved an svr ( n=22 ) did not develop hcc or new onset cirrhosis , whereas the hcc incidence in untreated aa patients was 23% ( 51/203 ) . 
 patients who achieved an svr also had improved fibrosis , as defined by the ast platelet ratio index ( apri ) and fibrosis-4 ( fib-4 ) score , relative to nonresponders and untreated patients . 
 conclusions : the severity of liver disease at the first visit ( except for cirrhosis ) correlated with the development of hcc , but because of the overlap in values between patients , these measurements were not useful for predicting individual risk . 
 since cirrhosis at the first visit was not a predictive factor , treatment with newer antiviral therapies is the best option for reducing the incidence of advanced liver disease and its harmful outcomes in the aa population .",pubmed
"stroke is the second common cause of death following ischemic heart disease.1 stroke has accounted for nearly 5.7 million deaths globally and 87% of these deaths take place in low and middle income nations.2 stroke is a multi - factorial disease and epidemiological and animal studies have robustly recommended genetic influences in the pathogenesis of ischemic stroke.3 the genetic influences are probably polygenic whereby multiple genes exert a small influence or risk on phenotype . 
 beta adrenergic receptors are members of a family of receptors known as g - protein coupled receptors ( gpcr ) and have a seven membrane spanning domain structure , an extracellular amino terminus , three intracellular and three extracellular loops , and an intracellular carboxyl terminus.4 these are receptors for neurohormone epinephrine and nor - epinephrine . 
 several mechanisms contribute to loss of receptor activity including uncoupling of the receptor from adenylyl cyclase activity , internalization of the receptor and phosphorylation of internalized receptors.5 several studies support the role of cyclic adenosine monophosphate ( camp ) in atherogenesis by modulating the function of vascular endothelium , the production of reactive oxygen species , the recruitment of circulating monocytes to the artery wall and their differentiation into macrophages - foam cells , by controlling the expression of pro - and anti - inflammatory interleukin , and regulating serum level of triglycerides and cholesterol.6,7 camp is also a possible target for prevention and treatment of atherosclerosis.6 the major non - synonymous snps of 2ar have been recognized at nucleotides 46 ( a > g ) ( rs1042713 ) and 79 ( c > g ) ( rs1042714 ) causing changes in amino acid residues at position 16 ( arg > gly ) and 27 ( gln > glu ) of the amino terminus respectively of the fourth intracellular loop . 
 an enhanced agonist - mediated receptor down - regulation for the gly16 variant of 2ar and a resistance to down - regulation for the glu27 variant of 2ar has been observed.8 it is hypothesized that due to the polymorphism in 2ar , functional alteration in the receptor function occurs which influences a certain intermediate mechanism for the predisposition of cardiovascular and cerebrovascular diseases.9 we conducted a meta - analysis with available published studies to precisely determine the association of 2ar with ischemic stroke in order to offer early diagnosis of the susceptible subjects . 
 the following search keywords were applied : ' bar ' or ' bar gene variant ' or 2ar , ' beta - adrenergic receptor ' or ' beta adrenergic receptor polymorphism ' and ' cerebral infarction ' or ' cerebrovascular accident ' or ' ischemic stroke ' or ' brain infarction ' . 
 inclusion criteria : ( i ) case control study studying the association between 2ar polymorphism and stroke ; ( ii ) studies published in english language with full text ; ( iii ) stroke confirmed by mri or ct . 
 exclusion criteria : ( i ) studies other than case control design ; ( ii ) studies did not report genotypic and allelic frequencies ; ( iii ) duplicate publication . 
 two investigators ( ak and mp ) independently evaluated the title , abstracts and search terms for eligibility based on the predetermined selection criteria . 
 all discrepancies were resolved after rechecking the source papers and further discussion among the two authors . 
 the relevant data from each study were independently extracted by two reviewers ( ak and mp ) using a standardized , structured form including first author 's name , year of publication , country , genotyping method , no . of cases and controls and frequency distribution of genotype and allele . 
 the following search keywords were applied : ' bar ' or ' bar gene variant ' or 2ar , ' beta - adrenergic receptor ' or ' beta adrenergic receptor polymorphism ' and ' cerebral infarction ' or ' cerebrovascular accident ' or ' ischemic stroke ' or ' brain infarction ' . 
 inclusion criteria : ( i ) case control study studying the association between 2ar polymorphism and stroke ; ( ii ) studies published in english language with full text ; ( iii ) stroke confirmed by mri or ct . 
 exclusion criteria : ( i ) studies other than case control design ; ( ii ) studies did not report genotypic and allelic frequencies ; ( iii ) duplicate publication . 
 two investigators ( ak and mp ) independently evaluated the title , abstracts and search terms for eligibility based on the predetermined selection criteria . all discrepancies were resolved after rechecking the source papers and further discussion among the two authors . 
 the relevant data from each study were independently extracted by two reviewers ( ak and mp ) using a standardized , structured form including first author 's name , year of publication , country , genotyping method , no . of cases and controls and frequency distribution of genotype and allele . 
 two studies were excluded because they did not meet the inclusion criteria for study design ( they were cohort studies ) . 
 two more studies were excluded because they studied another gene polymorphism and one study was excluded for being a pharmacogenetic study . 
 two studies11,12 reported the genotypic data , and therefore were included for genotypic association meta - analysis . 
 allelic data was reported in another study.10 a total three studies were included for allelic association meta - analysis . 
 the study characteristics , which are included in the present meta - analysis , are described in table 1 . 
 a total of three studies involving 1,642 cases and 1,673 controls , which were published from 2007 to 2014 , were subjected to meta - analysis for allelic association and 518 cases and 510 controls for genotypic association . 
 meta - analysis results did not show a significant association between arg16gly polymorphism of 2ar and ischemic stroke when assuming either recessive model of inheritance ( or 1.20 ; 95% ci , 0.92 to 1.56 ) ( figure 2a ) or dominant model of inheritance ( or 1.14 ; 95% ci , 0.68 to 1.91 ) ( figure 2b ) . 
 the allelic association also did not show statistically significant association between arg16gly polymorphism of 2ar and the risk of ischemic stroke ( or 1.04 ; 95% ci , 0.85 to 1.28 ) ( figure 2c ) . 
 meta - analysis results did show a significant association between gln27glu polymorphism of 2ar and ischemic stroke when assuming either recessive model of inheritance ( or , 2.09 ; 95% ci , 1.20 to 3.64 ) ( figure 3a ) or the dominant model of inheritance ( or , 1.47 ; 95% ci , 1.14 to 1.90 ) ( figure 3b ) . a significant allelic association was observed between gln27glu polymorphism of 2ar and ischemic stroke ( or , 1.58 ; 95% ci , 1.38 to 1.81 ) ( figure 3c ) . 
 two studies were excluded because they did not meet the inclusion criteria for study design ( they were cohort studies ) . 
 two more studies were excluded because they studied another gene polymorphism and one study was excluded for being a pharmacogenetic study . 
 two studies11,12 reported the genotypic data , and therefore were included for genotypic association meta - analysis . 
 allelic data was reported in another study.10 a total three studies were included for allelic association meta - analysis . 
 the study characteristics , which are included in the present meta - analysis , are described in table 1 . 
 a total of three studies involving 1,642 cases and 1,673 controls , which were published from 2007 to 2014 , were subjected to meta - analysis for allelic association and 518 cases and 510 controls for genotypic association . 
 meta - analysis results did not show a significant association between arg16gly polymorphism of 2ar and ischemic stroke when assuming either recessive model of inheritance ( or 1.20 ; 95% ci , 0.92 to 1.56 ) ( figure 2a ) or dominant model of inheritance ( or 1.14 ; 95% ci , 0.68 to 1.91 ) ( figure 2b ) . 
 the allelic association also did not show statistically significant association between arg16gly polymorphism of 2ar and the risk of ischemic stroke ( or 1.04 ; 95% ci , 0.85 to 1.28 ) ( figure 2c ) . 
 meta - analysis results did show a significant association between gln27glu polymorphism of 2ar and ischemic stroke when assuming either recessive model of inheritance ( or , 2.09 ; 95% ci , 1.20 to 3.64 ) ( figure 3a ) or the dominant model of inheritance ( or , 1.47 ; 95% ci , 1.14 to 1.90 ) ( figure 3b ) . a significant allelic association was observed between gln27glu polymorphism of 2ar and ischemic stroke ( or , 1.58 ; 95% ci , 1.38 to 1.81 ) ( figure 3c ) . 
 the present meta - analysis was conducted to determine the precise estimation of association between polymorphism of 2ar and risk of ischemic stroke . 
 several studies have shown an independent association of gln27glu polymorphism of 2ar gene number of diseases like obesity,13,14 dyslipidemia,14 myocardial infarction,15 and diabetes.16 thus , gln27glu polymorphism of 2ar has been suggested to be an independent risk factor for cardiovascular diseases and cerebrovascular diseases.17,18 our meta - analysis suggests a significant association of gln27glu polymorphism of 2ar with ischemic stroke . 
 our results are in agreement with earlier published study.12 however , two prospective cohort studies19,20 failed to show a significant association of 2ar variant with the incidence of stroke . a prospective cohort study including 25,225 women showed that the different haplotypic combination of beta receptor gene variant did not affect the incidence of ischemic stroke in women.20 another cohort study reported from same population which included a total of 808 black and 4,441 white participants failed to find significant association of 2ar genotypic with risk of ischemic stroke and combined cardiovascular outcome.19 these two prospective cohort studies in which significant associations were not observed , were reported from the american population19,20 while the other two studies,11,12 which showed the significant genotypic association , were reported from india and italy . 
 as the prevalence of genetics variants often varies among populations,19 this could explain the discrepancy of association of gln27glu polymorphism of 2ar and ischemic stroke across the studies . a case control study reported by zhao et al.10 in chinese population showed significant allelic association between glu allele of 2ar and risk of ischemic stroke ( figure 2c ) . 
 in all the published case control studies ( three),10,11,12 frequency of the risk allele ( glu27 ) was higher in cases as compared to controls . 
 our meta - analysis of allelic association including three studies with the risk allele versus protective allele suggested significantly higher risk of ischemic stroke with glu27 allele . the meta - analysis for genotypic association including 
 two studies also suggested a significant association between gln27glu polymorphism and risk of ischemic stroke under both dominant model and recessive model of inheritance . 
 some limitations exist in the present study , which may have affected the results of meta - analysis . 
 the haplotype analysis was not done which plays a crucial role for association studies of complex diseases . 
 the interaction between gene and environment and gene - gene interaction was not studied in this meta - analysis . 
 controls were selected from the hospital , which may have lead to the selection bias . 
 multivariate analysis for adjusting the several confounding factors that could have an effect on results was not performed as individual data from each study was not available . 
 studies were reported from different ethnic population and deviation from hwe equilibrium in control subjects in one study included in this meta - analysis may account for the heterogeneity . 
 some limitations exist in the present study , which may have affected the results of meta - analysis . 
 the haplotype analysis was not done which plays a crucial role for association studies of complex diseases . 
 the interaction between gene and environment and gene - gene interaction was not studied in this meta - analysis . 
 controls were selected from the hospital , which may have lead to the selection bias . 
 multivariate analysis for adjusting the several confounding factors that could have an effect on results was not performed as individual data from each study was not available . 
 studies were reported from different ethnic population and deviation from hwe equilibrium in control subjects in one study included in this meta - analysis may account for the heterogeneity . 
 the present study provides preliminary evidence to support the view that carrier of gln27glu polymorphism of 2ar demonstrates an increase in risk of ischemic stroke on the basis of meta - analysis of case control studies . 
 furthermore , well designed larger prospective cohort studies are required to validate this finding and to provide a higher level of evidence . 
 the underlying molecular causal pathways that confer susceptibility to ischemic stroke are warranted to be established .","background and purposethe purpose of this meta - analysis was to determine the precise association between beta-2 adrenergic receptor ( 2ar ) polymorphism and ischemic stroke.methodspublished case control studies on association between 2ar and ischemic stroke were searched from electronic databases . pooled odds ratio and 95% confidence interval were calculated by using software revman version 5.2.resultsa total of three studies involving 1,642 cases and 1,673 controls , which were published from 2007 to 2014 , were subjected to meta - analysis for allelic association and 518 cases and 510 controls for genotypic association . pooled analysis of two studies for genotypic association suggested that subjects carrying gln27glu polymorphism of 2ar had an increased risk for ischemic stroke under recessive model ( or 2.09 ; 95% ci ; 1.20 to 3.64 ) and under dominant model ( or 1.47 ; 95% ci 1.14 to 1.90 ) . 
 pooled analysis of three studies for allelic association showed a significantly higher glu27 allele of 2ar in the patients with ischemic stroke ( or 1.58 ; 95% ci ; 1.38 to 1.81).conclusionsthe present meta - analysis suggests that gln27glu polymorphism of 2ar gene is associated with increased risk for ischemic stroke .",pubmed
"methadone ( d , l - methadone hydrochloride ) is frequently used in different therapies including opioid addiction , long - lasting analgesics in cancer and neuropathic pain syndromes [ 13 ] . however , numerous reports indicate a negative impact on human cognition by chronic exposure to opioid drugs . 
 patients subjected to methadone maintenance programs show impaired cognitive abilities in aspects such as psychomotor performance , information processing , attention , problem solving , memory , decision making , reaction time , and emotional facial expression recognition [ 410 ] . 
 changes in the cytosolic free - calcium concentration ( [ ca]cyt ) are involved in control of a large number of cellular and physiological processes including neuronal excitability , synaptic plasticity , and gene transcription [ 11 , 12 ] . 
 however , the physiological ca signal can switch to a death signal when the [ ca]cyt  increases dramatically . 
 for example , excitotoxic high glutamate concentrations result in an initial transient increase in [ ca]cyt  that is followed by a delayed , irreversible rise in [ ca]cyt  known as delayed calcium deregulation  ( dcd ) . 
 although several steps preceding dcd remain to be clarified , there is evidence that dcd is the irreversible end point of a sequence involving mitochondrial ca overloading . 
 they buffer variations in ca concentrations by taking up ca when and where [ ca]cyt levels are passing a threshold level above which the mitochondrial ca uniporter is activated , and slowly release ca back to the cytosol when [ ca]cyt drop below this point . 
 mitochondrial ca overload , if large and sustained enough , may contribute to mitochondria permeability transition pore ( mptp ) formation and ultimately lead to cell death [ 11 , 15 ] . because , mitochondria may accumulate a considerable amount of ca during neurotoxic exposure , a possibility is that dcd may represent the final consequence of mitochondrial ca overload . 
 mptp is a large , proteinaceous , ca - activated , proton- and adp - inhibited voltage - dependent pore . 
 it spans the inner and outer mitochondrial membrane allowing the passage of ions and substrates less than 1.5  kda . 
 characteristically , opening of the mptp is inhibited by cyclosporin a [ 16 , 17 ] . 
 sh - sy5y cells are considered a suitable model for investigating opioid - mediated responses in neurons . 
 , we showed that sh - sy5y cells exposed to high concentrations of methadone ( 0.5  mm ) died through a necrotic - like cell death mechanism and that methadone may induce changes in the [ ca]cyt [ 19 , 20 ] . 
 however , the underlying mechanisms causing alterations of the [ ca]cyt in sh - sy5y cells in the presence of methadone remained unknown . 
 therefore , the aim of the present study was to investigate those mechanisms . a clear understanding of the factors that mediate this phenomenon might help to resolve the mechanisms that promote neuronal cell death during methadone - induced cognitive damage . 
 sh - sy5y cells ( atcc ) were plated at a density of 5.3  10  cells / cm on -dish 35  mm high ibitreat ( ibidi gmbh , martinsried , mnchen , germany ) as previously reported . 
 changes in [ ca]cyt in sh - sy5y cells were measured by loading cells with the calcium probe fura2/am and using an inverted fluorescence microscope ( nikon eclipse te2000-s ) as described elsewhere . 
 cells were perfused with a medium containing 140  mm nacl , 5  mm kcl , 1  mm mgcl2 , 2.5  mm cacl2 , 10  mm hepes , and 11  mm glucose , ph 7.35 . ratios of fluorescence emission excited at 340 and 380  nm were captured every 5 seconds . 
 methadone or other compounds were added from 1000x stock solutions to reach the appropriate final concentrations . 
 the effects of selected compounds were tested on [ ca]cyt in the absence and the presence of 0.5  mm methadone in different sets of cells . 
 cells were pooled according to the dynamics of the rises in [ ca ] .   to directly visualize mitochondrial morphology changes in intact cells overexpressing pdsred2-mito plasmid ( clontech laboratories , inc . 
 mountain view , ca , usa ) a leica sp2 confocal microscope ( 63  1.4  na objective ) was used . 
 for transfection of cells the reagent lipofectamine was used ( invitrogen , carlsbad , ca , usa ) . 
 rat liver mitochondria were isolated in msh / edta and msh media containing 210  mm mannitol , 70  mm sucrose , 5  mm hepes , with or without 1  mm edta , ph 7.4 , by differential centrifugation according to the standard procedure . 
 the mitochondrial suspensions were kept on ice and immediately used for measurements of oxygen - uptake rate . 
 the rate of oxygen uptake of isolated rat liver mitochondria was measured at 37c in a water - thermostatized incubation chamber with a computer - controlled clark - type o2 electrode ( oxygraph , hansatech , uk ) in 0.5  ml incubation buffer ( 145  mm kcl , 30  mm hepes , 5  mm kh2po4 , 3  mm mgcl2 , 0.1  mm egta , 0.1% defatted bsa , ph 7.4 ) . 
 the respiratory substrates used were complex i- or complex ii - linked , 2.5  mm glutamate/2.5  mm malate or 5  mm succinate in the presence of 2  m rotenone . 
 the following additions were applied : 250  m adp , 200  m cacl2 , 0.4  m fccp , and 0.5  mm methadone . for estimating mitochondrial ca uptake in isolated mitochondria a ca index was calculated , which denotes the ratio of oxygen - uptake rate triggered by addition of 200  m ca to previous oxygen uptake rate 
 in this study , we have investigated the mechanisms involved in methadone - induced rises in [ ca]cyt in sh - sy5y cells . 
 consistent with previous observations from our laboratory   and other data , methadone induced a rise in [ ca]cyt in most of the sh - sy5y cells . 
 analysis of the dynamics of the [ ca]cyt recordings in the absence and the presence of methadone suggested that four different types of calcium recordings can be observed in sh - sy5y cells . 
 for the first group of cells no rise in [ ca]cyt was observed during the entire period ( 30  min ) of measurements ( type 1 ) . 
 a second group of cells was unable to regulate [ ca]cyt homeostasis shortly after the addition of methadone ( type 2 ) and [ ca]cyt increased continuously . 
 a third group displayed [ ca]cyt  deregulation with a delay of 1520 minutes ( type 3 ) . 
 finally , the fourth group was able to control [ ca]cyt after an initial increase ( type 4 ) . 
 groups 1 and 4 represent , respectively , nonresponsive of cells and cells responding with transient rise in [ ca]cyt and amount 20.82 and 31.38% of the cells . 
 however , types 2 and 3 displayed short or delayed continuous rise in [ ca]cyt that may be considered as early or delayed ca deregulation according to the literature [ 2629 ] . the abundance of cells showing deregulation ( type 2 and type 3 ) amount to 32.83 and 14.92% , of the total , respectively 
 we have analyzed the relative abundance of the four types of recordings described above in the absence ( control ) and the presence ( methadone ) of 0.5  mm methadone . 
 clearly , methadone decreased type 4 ( transient responsive cells ) and increased types 2 and 3 ( deregulated cells ) . 
 these results suggest that methadone may induce a short or delayed ca deregulation in sh - sy5y cells . 
 the mechanisms underlying the observed quantitative changes in the types of ca responses mediated by methadone are unknown . 
 therefore , we performed a comprehensive pharmacological survey to study the possible contribution of different carelated mechanism to the response to methadone . 
 as illustrated in figures 1(b ) and 1(c ) and discussed below , a 5  min pretreatment of sh - sy5y cells with different conditions and drugs affected the methadone - induced [ ca]cyt response , monitored as changed frequencies of the different types of the responses . 
 we found that in the absence of extracellular calcium ( 0  ca ) no change in responses to methadone was observed ( n = 291 cells ) . 
 these results suggest that ca entry does not contribute to the reported changes in [ ca]cyt . to confirm that extracellular ca  was not involved in these responses we tested the effect of methadone in the presence of nifedipine , a specific isopropyl l - type ca channel blocker . 
 nifedipine ( 2  m ) did not modify the effects of methadone on the [ ca]cyt in sh - sy5y cells ( n = 226 cells ) . 
 so , we ruled out the involvement of this voltage - dependent channel family in methadone - induced [ ca]cyt variations . 
 cells were treated with thapsigargin ( 1100  m ) . to deplete intracellular ca stores before methadone treatment . 
 as expected , thapsigargin induced a significant release in ca from the endoplasmic reticulum , causing a transient increase in the [ ca]i that failed to returned to the basal level within a 10-minute period . a detailed observation of the methadone induced rise in [ ca]i shows that cell responses were partially affected by this treatment , suggesting that the rise in calcium is partially due to release from thapsigargin - sensitive , intracellular ca stores ( n = 146 cells ) . 
 we found that in untreated cells addition of thapsigargin induced a transient increase in [ ca ] that has been attributed to a leakage of ca from the endoplasmic reticulum . 
 this effect of thapsigargin alone interferes the interpretation of the type 2 and 3 responses after methadone addition ( data not shown ) . 
 , we treated the cells with 50  m naloxone ) , the competitive antagonist of the -opioid receptor . 
 we found that this treatment did not modify the relative abundance of any of the four types of cell populations ( n = 80 cells , 4  exp ) . 
 this result suggests that the changes in [ ca]cyt and dcd induced by methadone in sh - sy5y cells are independent of opioid receptors . in agreement with the lack of -opioid receptor participation as found here , it has been reported that methadone - toxic pathways are not mediated by  receptors [ 19 , 3032 ] .   to test the possible contribution of mitochondria we used the mitochondrial uncoupler carbonyl cyanide p - trifluoromethoxyphenylhydrazone ( fccp ; 1  m ) 
 it is well established that fccp collapses mitochondrial potential and abolishes the ability of mitochondria to take up ca . 
 we found that in untreated cells , addition of fccp induced a transient increase in [ ca ] that has been attributed to leakage of ca from depolarized mitochondria . 
 we found that fccp induced a 44% decrease in the appearance of type 2 cells whereas increased the relative abundance of cells showing a type 3 response by 2.7-fold in fccp ( figures 1(b)-1(c ) ) . 
 nevertheless , it must be taken into account that as the simple usage of protonophores does not allow a clear - cut study of the role of mitochondrial ca transport as they also may cause a lowering of the atp / adp ratio , thereby affecting atp - dependent ca pumps . 
 an approach that has been previously exploited to investigate the role of mitochondria in synaptosomal ca homeostasis involves inhibition of mitochondrial atp synthesis by oligomycin and application of glycolysis as the source of atp and independent manipulation of  
 m 
  with specific respiratory chain inhibitors [ 33 , 34 ] . 
 inhibition of atp synthase by oligomycin prevents mitochondrial oxidative phosphorylation , but unlike protonophore addition , it does not cause hydrolysis of cytoplasmically generated atp . 
 it has been reported that dcd might result from a failure in ca extrusion caused by cytoplasmic atp depletion . 
 therefore , we tested the effects of a short , 5  min , incubation with 10  g / ml oligomycin on ca responses . under these conditions , 
 oligomycin alone did not modify the [ ca]cyt responses during the 30  min recording period . 
 specifically , the abundance of sh - sy5y cells showing a type 2 response were increased whereas the pool of cells showing a dcd - related type 3 response were lost . 
 additionally , oligomycin induced a 3.3-fold increase in cells showing a type 4 response . to test further the contribution of mitochondria we investigated the possible role of the mitochondrial ca/2na exchanger . 
 mitochondrial ca efflux is normally primarily regulated by a ca/2na exchanger . to block mitochondrial ca exit we used cgp37157 ( 50  m ) , an inhibitor of the ca/2na - exchanger 
 . a five min exposure of cells to cgp37157 significantly increased the proportion of cells showing no rise in [ ca]cyt in response to methadone during the entire [ ca]cyt measurement period ( type 1 ) ( data not shown ) because cgp37157 resulted in a drastic decrease in the population of cells showing either type 2 ( 74% ) or type 3 ( 85% ) responses . 
 in addition , the relative abundance of cells showing a type 1 response returned to the value obtained in untreated cells . 
 these results support data suggesting the relevance of mitochondria in methadone - induced dcd ( figures 1(b)-1(c ) ) . to test contribution of reactive oxygen species we used the cell - permeable , small molecule compound tempol ( 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy ) to mimic superoxide dismutase activity . in the presence of tempol 
 ( 0.2  m ; n = 80 cells ) , cells responded to methadone in a different way . specifically , tempol decreased the number of cells showing a type 2 response by ~70% , whereas the abundance of cells showing a type 3 response was largely increased ( 3.5 fold ) . 
 suggested that enhanced ros is a consequence rather than a cause of dcd . in their studies , they applied a novel technique to monitor the bioenergetic status of in situ mitochondria in cultured neurons in a model of glutamate excitotoxicity . in agreement with this , a general ineffectiveness of antioxidants to decrease dcd in the presence of glutamate has been observed . finally , we tested the contribution of the mitochondrial permeability transition ( mptp ) . 
 additional efflux of mitochondrial ca can occur by induction of mptp formation , which is dependent on the mitochondrial matrix ca concentration and can be inhibited by cyclosporine a [ 16 , 17 ] . to evaluate mptp participation we administered csa ( 1  m ) . 
 csa diminished the occurrence of response type 2 by 36% ( n = 89 cells ) in cell cultures challenged with 0.5  mm methadone . 
 moreover , consistent with the hypothesized role of the pore in dcd , csa induced a 2-fold increase in cells showing type 4 response . 
 however , interpretation of these results is difficult because csa may also inhibit the mitochondrial ca uniporter in some instances . taken together , our data indicate that only drugs affecting mitochondrial handling of ca , such as oligomycin , fccp , cgp 37157 , and cyclosporine a , were able to modulate methadone - induced delayed calcium deregulation in sh - sy5y cells . 
 we therefore conclude that methadone - induced dyshomeostasis is caused by improper functioning of mechanisms that directly control mitochondrial activity rather than a participation of plasma membrane ca channels or opioid receptors . 
 next , the effect of methadone on mitochondrial morphology was studied in sh - sy5y cells transfected with pdsred2-mito . in untreated cultures 
 , mitochondria presented a long and tubular morphology ( figure 2(a ) ) , which became dramatically shorter and rounder upon three hours of methadone treatment ( figures 2(b)2(d ) ) . 
 cell counting of the different mitochondrial morphologies ( filamentous , mixed and fragmented ) indicated that methadone , in a dose - dependent manner , induced mitochondrial fragmentation ( figure 2(d ) ) . 
 this effect seems contradictory to our earlier observations that methadone failed to induce mitochondrial swelling in isolated rat liver mitochondria . 
 in fact , our data support this hypothesis , and indicate a role of dcd in methadone - induced toxicity . in agreement with this 
 , it has been reported that the mptp opens under pseudopathological conditions with relatively high ca and low atp concentrations   as was the case in our previous experiments with sh - sy5y cells . 
 the rupture of the mitochondrial membrane caused by ca overload reduces the number of  healthy  mitochondria and this will affect crucial neuronal functions including synaptic transmission and axonal transport . 
 finally , the effect of methadone on mitochondrial ca uptake was studied ( figure 3 ) . 
 we used a clark electrode and applied different respiratory substrates , namely succinate and glutamate / malate . 
 the respiratory chain is less dependent on the presence of m for succinate than for glutamate / malate . 
 therefore , as a control , 0.4  m fccp was used because this concentration of fccp resulted in an increase of oxygen - uptake rate comparable with the uptake rate calculated for 0.5  mm methadone ( figure 3(a ) ) . 
 the calculated values of the state u ( uncoupled state ) to state 4 ( resting state ) ratios ( u/4 ratios ) were as follows : for succinate 2.8  0.5 ( + fccp ) and 2.3  0.4 ( + methadone ) , and for glutamate / malate 2.1  0.5 ( + fccp ) and 2.5  0.4 ( + methadone ) . 
 then , we checked the effect of fccp and methadone on ca uptake by mitochondria ( figures 3(b ) and 3(c ) ) . 
 for this purpose a ca index was calculated , which denotes the ratio of oxygen - uptake rate triggered by addition of 200  m ca to previous oxygen uptake rate . 
 the values of the ca index were as follows : for succinate 3.0  0.7 ( control ) , 1.7  0.3 ( + fccp ) , and 1.7  0.4 ( + methadone ) , and for glutamate / malate 4.8  0.3 ( control ) , 1.8  0.4 ( + fccp ) , and 1.9  0.5 ( + methadone ) . 
 thus , the effects of fccp and methadone on ca uptake are comparable , although in the presence of glutamate / malate the effect appears to be much more pronounced . 
 this probably results from a stronger uncoupling effect of methadone on glutamate / malate access to the respiratory chain and a consecutive additional impairment of ca uptake 
 . therefore , the effect of methadone on ca uptake by mitochondria may be dependent on the respiratory substrates . 
 on the other hand , when the values of the u/4 ratios calculated in the absence and presence of ca uptake were compared ( figures 3(a ) and 3(c ) ) , a distinctive decrease was observed in traces recorded in the presence of ca uptake ( figure 3(c ) ) . 
 the calculated values of u/4 ratio decreased as follows : for succinate from 2.8  0.5 to 2.0  0.2 ( + fccp ) and 2.3  0.4 to 2.1  0.3 ( + methadone ) and for glutamate / malate 2.1  0.5 to 1.9  0.3 ( + fccp ) and 2.5  0.4 to 2.1  0.2 ( + methadone ) . 
 this could be caused by the reduction of a  component of the protomotive force as a result of ca uptake , leading to a decrease of uncoupling capacity by fccp and methadone . 
 the data presented indicate that methadone induces dcd in sh - sy5y cells by altering the capacity of mitochondria to handle calcium , and correlates with distinct changes of mitochondrial morphology . 
 these morphological changes , in turn , can be associated with mitochondrial damage and cell death . 
 interestingly , swollen mitochondria have been observed in the context of neurodegenerative diseases [ 36 , 37 ] . 
 an imbalance in mitochondrial ca homeostasis might be important for both early and late stages of the observed side effects and , perhaps account for some of the observed clinical symptoms , for example , memory impairment .","previously , we have shown that sh - sy5y cells exposed to high concentrations of methadone died due to a necrotic - like cell death mechanism related to delayed calcium deregulation ( dcd ) . in this study , we show that , in terms of their ca2 + responses to 0.5  mm methadone , sh - sy5y cells can be pooled into four different groups . in a broad pharmacological survey , 
 the relevance of different ca2 + -related mechanisms on methadone - induced dcd was investigated including extracellular calcium , l - type ca2 + channels , -opioid receptor , mitochondrial inner membrane potential , mitochondrial atp synthesis , mitochondrial ca2+/2na+-exchanger , reactive oxygen species , and mitochondrial permeability transition . only those compounds targeting mitochondria such as oligomycin , fccp , cgp 37157 , and cyclosporine a were able to amend methadone - induced ca2 + dyshomeostasis suggesting that methadone induces dcd by modulating the ability of mitochondria to handle ca2 + . 
 consistently , mitochondria became dramatically shorter and rounder in the presence of methadone . 
 furthermore , analysis of oxygen uptake by isolated rat liver mitochondria suggested that methadone affected mitochondrial ca2 + uptake in a respiratory substrate - dependent way . 
 we conclude that methadone causes failure of intracellular ca2 + homeostasis , and this effect is associated with morphological and functional changes of mitochondria . 
 likely , this mechanism contributes to degenerative side effects associated with methadone treatment .",pubmed
"risk - reducing salpingo - oophorectomy ( rrso)the removal of the fallopian tubes and ovaries to reduce the risks of breast and ovarian cancer  is a cancer prevention strategy used by many women who carry germline mutations in the brca1 and/or brca2 genes ( brca1/2 ) . 
 however , the magnitude of the risk reductions in women with brca1/2 mutations who have undergone rrso compared with those who have not is unclear . 
 a fixed - effects meta - analysis of pooled results from 10 published reports of rrso and the risks of breast and/or ovarian or fallopian tube cancer in brca1/2 mutation carriers . 
 rrso was found to be strongly associated with substantial reductions in the risks of breast , ovarian , and fallopian tube cancers among women who carry mutations in brca1 or brca2 . 
 the summary risk reduction estimates should provide guidance to women in planning cancer risk reduction strategies . 
 data were not available to evaluate the effects of birth cohort , timing of surgery , or other factors that may influence the risk reduction estimates associated with rrso . 
 risk - reducing salpingo - oophorectomy ( rrso)the removal of the fallopian tubes and ovaries to reduce the risks of breast and ovarian cancer  is a cancer prevention strategy used by many women who carry germline mutations in the brca1 and/or brca2 genes ( brca1/2 ) . 
 however , the magnitude of the risk reductions in women with brca1/2 mutations who have undergone rrso compared with those who have not is unclear . 
 a fixed - effects meta - analysis of pooled results from 10 published reports of rrso and the risks of breast and/or ovarian or fallopian tube cancer in brca1/2 mutation carriers . 
 rrso was found to be strongly associated with substantial reductions in the risks of breast , ovarian , and fallopian tube cancers among women who carry mutations in brca1 or brca2 . 
 the summary risk reduction estimates should provide guidance to women in planning cancer risk reduction strategies . 
 data were not available to evaluate the effects of birth cohort , timing of surgery , or other factors that may influence the risk reduction estimates associated with rrso . 
 to identify all reports of rrso in brca1/2 mutation carriers , we searched the pubmed database using the search terms  oophorectomy  and  brca1  or  brca2 . 
 this search yielded 346 studies that were published between january 1999 and december 2007 : 309 that included the term  brca1  in the title and 267 that included the term 
 we then evaluated the full text of these citations to identify articles presenting primary data that provided estimates of risk reduction due to rrso . 
 no publications were excluded based on quality , sample size , language of publication , or other objective criteria related to study design and analysis . 
 however , some publications that reported rrso in brca1/2 mutation carriers were not included because they did not estimate risk reduction . 
 these included case reports , psychosocial or behavioral studies , commentaries , and clinical recommendations . 
 because the number of brca1/2 mutation carriers is relatively limited and most research groups studying these women are in routine communication and collaborate with one another , we also undertook personal communications with all of the researchers or consortia that have large series of brca1/2 mutation carriers and were known to have data that could have been used to report data on this topic . 
 we undertook a fixed - effects meta - analysis using the hazard ratios ( hrs ) and/or odds ratios ( as published in the original reports ) to estimate the pooled relative risks and 95% confidence intervals ( cis ) . 
 when two or more studies had overlapping study samples , we included only one published report from each group . 
 of the studies identified here , sample overlaps were noted in the studies of rebbeck et al . 
 ( 16 ) , which had the largest sample size of these five studies , was chosen for inclusion in the meta - analysis . 
 there were no apparent overlaps among the other datasets , although we can not rule out the possibility that a few individuals had participated in more than one study . 
 we carried out separate meta - analyses in brca1 mutation carriers , brca2 mutation carriers , and among women who carried either brca1 or brca2 mutations ( denoted brca1/2 ) . 
 a chi - square test of homogeneity among the individual risk ratio estimates of the identified studies was also performed . to evaluate potential for publication bias 
 , we used the adjusted rank correlation test of begg and mazumdar ( 20 ) . 
 all analyses were conducted using stata / se v9.0 ( statcorp , college station , tx ) . 
 to identify all reports of rrso in brca1/2 mutation carriers , we searched the pubmed database using the search terms  oophorectomy  and  brca1  or  brca2 . 
 this search yielded 346 studies that were published between january 1999 and december 2007 : 309 that included the term  brca1  in the title and 267 that included the term 
 we then evaluated the full text of these citations to identify articles presenting primary data that provided estimates of risk reduction due to rrso . 
 no publications were excluded based on quality , sample size , language of publication , or other objective criteria related to study design and analysis . 
 however , some publications that reported rrso in brca1/2 mutation carriers were not included because they did not estimate risk reduction . 
 these included case reports , psychosocial or behavioral studies , commentaries , and clinical recommendations . 
 because the number of brca1/2 mutation carriers is relatively limited and most research groups studying these women are in routine communication and collaborate with one another , we also undertook personal communications with all of the researchers or consortia that have large series of brca1/2 mutation carriers and were known to have data that could have been used to report data on this topic . 
 we undertook a fixed - effects meta - analysis using the hazard ratios ( hrs ) and/or odds ratios ( as published in the original reports ) to estimate the pooled relative risks and 95% confidence intervals ( cis ) . 
 when two or more studies had overlapping study samples , we included only one published report from each group . 
 of the studies identified here , sample overlaps were noted in the studies of rebbeck et al . 
 ( 16 ) , which had the largest sample size of these five studies , was chosen for inclusion in the meta - analysis . 
 there were no apparent overlaps among the other datasets , although we can not rule out the possibility that a few individuals had participated in more than one study . 
 we carried out separate meta - analyses in brca1 mutation carriers , brca2 mutation carriers , and among women who carried either brca1 or brca2 mutations ( denoted brca1/2 ) . 
 a chi - square test of homogeneity among the individual risk ratio estimates of the identified studies was also performed . to evaluate potential for publication bias 
 , we used the adjusted rank correlation test of begg and mazumdar ( 20 ) . 
 all analyses were conducted using stata / se v9.0 ( statcorp , college station , tx ) . 
 the studies that formed the basis of this meta - analysis included case  control studies as well as prospective and retrospective cohort studies ( table 1 ) . as can be seen in this summary , limitations of the currently available data regarding rrso in brca1/2 mutation carriers include variable study designs , small sample sizes for individual studies , many of which are retrospective in nature , and short post - rrso follow - up times in prospective studies . 
 eight studies ( 8 - 10,12 - 16 ) estimated the risk of breast cancer in brca1/2 mutation carriers who were treated with rrso relative to brca1/2 mutation carriers who did not receive this treatment ( table 2 ) . as summarized in table 3 and figure 1 , 
 three nonoverlapping studies ( 1416 ) , which included 5703 participants , estimated the risk of breast cancer in brca1/2 mutation carriers who received rrso relative to brca1/2 mutation carriers who did not receive the procedure , giving a summary hr estimate of 0.49 ( 95% ci = 0.37 to 0.65 ) . 
 four nonoverlapping studies ( 12,1416 ) estimated the risk reduction associated with rrso for breast cancer in brca1 mutation carriers , giving a summary hr estimate of 0.47 ( 95% ci = 0.35 to 0.64 ) . finally , three nonoverlapping studies ( 1416 ) estimated the relative risk for breast cancer in brca2 mutation carriers , giving a summary hr estimate of 0.47 ( 95% ci = 0.26 to 0.84 ) ( table 3 , figure 1 ) 
 . published studies of risk - reducing salpingo - oophorectomy and cancer risk in brca1/2 mutation carriers * in all but one of the prospective cohort studies , women with a prior diagnosis of breast cancer were included for the ovarian endpoint . in domchek 
 et al . ( 13 ) all patients were unaffected with breast or ovarian cancer at the start of follow - up , as the study was designed to evaluate the impact of rrso on cancer incidence as well as disease - specific and overall survival . 
 b1 = brca1 mutation carriers ; b2 = brca2 mutation carriers ; b1/2 = combined brca1 or brca2 group ; pc = prospective cohort ; rc = retrospective cohort ; cc = case  control ; namc = north american multicenter cohort ; sis = single - institution study ; imc = international multicenter cohort ; ismc = israeli multicenter cohort ; emc = european multicenter cohort ; myfu = mean years of follow - up ; py = person - years ; nr = not reported ; and na = not applicable ; rrso = risk - reducing salpingo - oophorectomy . in the original publications , 
 the prospective and retrospective cohorts were analyzed by survival / failure time analysis methods ; the case  control studies were analyzed by logistic regression . 
 six studies ( 8,10,11,13,16,17 ) ( table 2 ) estimated the risk of gynecologic cancer in brca1/2 mutation carriers treated with rrso relative to brca1/2 mutation carriers who did not receive this treatment . based on data from the three nonoverlapping datasets ( 11,16,17 ) , which included 2840 participants , the summary hr was 0.21 ( 95% ci = 0.12 to 0.39 ) ( table 3 , figure 1 ) . only one study ( 16 ) estimated the risk of gynecologic cancer in brca1 mutation carriers treated with rrso relative to untreated brca1 carriers ( hr = 0.15 , 95% ci = 0.04 to 0.56 ) ( table 2 ) . 
 ( 16 ) did investigate risk reduction in 294 women with brca2 mutations , but observed no post - rrso gynecologic cancers in this sample . 
 published studies of risk - reducing salpingo - oophorectomy and cancer risk in brca1/2 mutation carriers * hazard ratios ( hrs ) , odds ratios ( ors ) ( with 95% confidence intervals ) , and sample size ( n ) are presented . 
 na = not applicable . not included in summary hr estimate because the sample set overlaps with that of other reports . 
 studies included in the summary estimate were chosen to maximize the sample size ( power ) of the meta - analysis . 
 we found no evidence of publication bias of any of our estimates based on the begg and majumder test statistics presented in table 3 . 
 no evidence of study heterogeneity was found based on the x test ( table 3 ) . 
 summary estimates for ovarian / fallopian tube cancer and breast cancer risk reduction associated with salpingo - oophorectomy in brca1/2 mutation carriers * na = not applicable ; hr = hazard ratio ; ci = confidence interval . according to begg and mazumder ( 20 ) . 
 the clinical management of cancer risk in brca1 and brca2 mutation carriers is complex and should consider patient preferences ; these preferences can be informed by accurate knowledge of the risks and benefits of the interventions considered ( table 4 ) . 
 the results of our meta - analysis suggest an 80% reduction in ovarian / fallopian tube cancer risk and a 50% reduction in breast cancer risk associated with rrso in women who carry mutations in brca1 or brca2 . 
 the consistency of these findings across the included studies confirms the strong association of rrso with reduced risks of breast and ovarian cancer in brca1 or brca2 mutation carriers . 
 in addition , modeling studies have also demonstrated that salpingo - oophorectomy has a large effect on years of life added , particularly when adjusted for quality of life ( 31 ) . 
 furthermore , in a prospective study with short - term follow - up , rrso was associated with a 90% reduction in breast cancer  specific mortality , a 95% reduction in gynecologic cancer  specific mortality , and a 76% reduction in overall mortality ( 13 ) . 
 therefore , all of the available data demonstrate the utility of salpingo - oophorectomy in this population of patients . 
 synopsis of management strategies available to brca1 and brca2 mutation carriers * rrso = risk - reducing salpingo - oophorectomy ; er = estrogen receptor ; mri = magnetic resonance imaging . despite the consistent evidence favoring rrso in women with mutations in brca1 or brca2 , 
 first , the influence of cohort effects on cancer risk over time remain unclear , despite evidence that differences in risk over time may reflect changing exposures , lifestyle , reproductive history , and use of screening or preventive surgeries ( 32 ) . 
 we lacked the data necessary to evaluate the effects of birth cohort , timing of surgery , or other factors that may influence the risk reduction estimates associated with rrso . 
 therefore , at this time it is difficult to infer whether specific cohorts , exposure groups , or other strata may experience different risk reduction effects than others . to limit the possibility that reporting bias influenced our findings 
 however , we did not include any studies that reported the association of rrso with cancer risk without providing estimates of risk reduction because these data would not contribute to pooled estimates of risk reduction . because some studies included in the this analysis were limited in sample size and statistical power , their effect estimates for rrso were large but not statistically significant , suggesting that a meta - analysis and presentation of summary statistics was appropriate . 
 two studies ( 15,17 ) were included in the summary estimates even though they used case  control designs , and therefore they yielded odds ratios rather than hazard ratios . 
 although odds ratios may slightly overestimate the risk reduction associated with rrso , the annual incidence of ovarian and breast cancer in brca1/2 mutation carriers is no more than 2%4% , with the result that odds ratios are likely to be similar to hazard ratios in this setting . 
 some of the variability in the individual study estimates reported may reflect study design differences , including the use of retrospective vs prospective samples and poorly characterized selection biases . despite these differences , we noted no statistically significant heterogeneity in the estimates of risk reduction after rrso . 
 in addition , cohort studies estimated a greater reduction in cancer risk associated with rrso ( particularly ovarian / fallopian tube cancers ) compared with the case  control studies ( table 1 ) . 
 as a result , there is some variability in the estimates obtained using case  control and cohort studies ; nonetheless , the estimates all consistently reflect risk reduction associated with rrso . 
 we have included all of the large collaborative group studies that addressed the question of reduced risk conferred by rrso and whose study populations come from and are representative of mutation carriers in north america and europe . 
 no studies of rrso in nonwhite populations have been reported , and additional data may be needed to understand the role of rrso in these groups . finally , the samples of women with brca1/2 mutations reported here represent those who have generally been identified through high - risk clinics . 
 however , they do represent the population of women who receive genetic testing and may be candidates for rrso . 
 therefore , the populations summarized here represent the most relevant group in whom rrso may be applied at this time . despite the strength and consistency of the data in the literature as reflected in our meta - analysis , a number of questions remain . 
 there are only a few estimates of the association of rrso with cancer risk in populations composed exclusively of brca1 mutation carriers or brca2 mutation carriers ( 12,1416 ) , and it is critical to understand how risk reduction may differ by gene . using a prospective cohort approach and a large consortium dataset , we recently estimated gene - specific risks and found that hormonal modulation by rrso may be associated with a greater reduction in breast cancer risk in brca2 mutation carriers than in brca1 mutation carriers ( 16 ) . 
 in contrast , the studies that used retrospective cohort ( 14 ) or case  control approaches ( 14,15 ) did not observe this difference , and therefore , there was no difference in the pooled estimates of breast cancer risk reduction reported in table 1 . 
 thus , differences in study design may influence the inferences we can make about the differences in risk reduction associated with rrso in brca1 vs brca2 mutation carriers . 
 the potentially larger risk reduction associated with rrso in brca2 vs brca1 mutation carriers is of interest , given the high proportion of estrogen receptor ( er)negative breast tumors in brca1 mutation carriers compared with brca2 mutation carriers ( 33 ) . 
 our observation of a higher risk in brca2 mutation carriers should be followed up in larger studies that specifically evaluate tumor markers . 
 in addition , attention needs to be given to the time interval between rrso and breast cancer diagnosis . 
 for example , it is possible that there is greater breast cancer risk reduction in brca2 mutation carriers , in whom the majority of tumors are er positive , given that rrso may treat some subclinical breast tumors . 
 in contrast , in brca1 mutation carriers , who have predominantly er - negative breast cancer , it is unclear whether a  treatment effect  may exist , and any primary prevention effect may require more time to emerge . 
 eisen et al . ( 15 ) reported that the breast cancer risk reduction with rrso was greater in brca1/2 mutation carriers who underwent surgery before age 50 than in women who underwent surgery after age 50 . among brca1 mutation carriers older than age 50 , 
 no statistically significant association of rrso at any age with risk reduction was observed in brca2 mutation carriers . although these findings are consistent with effects of removal of hormone exposures in premenopausal women and not in postmenopausal women , the sample sizes in this analysis ( 15 ) were relatively small . 
 the importance of understanding the optimal age at which a woman should consider rrso is underscored by a recent study ( 34 ) conducted in the general population that suggests that rrso in women younger than age 45 is associated with an increased mortality , particularly if hormone replacement therapy ( hrt ) is not used . 
 an initial report of hrt use after rrso suggests that women can undergo rrso and take hrt for a short time if needed after surgery because breast cancer risk is not substantially elevated in hrt users after rrso ( 35 ) . 
 although data on postmenopausal women do not demonstrate a cardiovascular benefit from hrt ( 36 ) , an important limitation of this study ( 36 ) was the older age of the participants . 
 more recent data have suggested that younger women going through natural menopause may indeed derive a cardiovascular benefit from hrt ( 36,37 ) , and it is possible that brca1/2 mutation carriers undergoing abrupt surgical menopause to reduce ovarian cancer risk who receive hrt may in fact derive important cardiovascular , bone health , and quality - of - life benefits . although the risk  benefit ratio of rrso is very different in brca1/2 mutation carriers than in the general population , and rrso in brca1/2 mutation carriers has been associated with improved overall survival in the short term , these studies pointing to the potentially complex relationship of rrso and hrt exposure raise important and difficult questions . 
 for example , it is not yet clear whether the long - term effects of long - term hrt in unaffected mutation brca1/2 carriers will ultimately be more beneficial in preventing noncancer mortality in these women or more harmful by increasing their risk of breast cancer ( or potentially increasing cardiovascular events ) compared with the general population . given this possibility , studies that address the type , timing , and length of administration of hrt as well as its long - term effects on the association between rrso and cancer risk and on other health factors in brca1/2 mutation carriers 
 , we provide a summary of clinical recommendations related to the detection and prevention of cancer in brca1/2 mutation carriers ( table 4 ) . 
 finally , although rrso has become the standard of care for cancer risk reduction in women who have inherited brca1/2 mutations , other options for risk reduction also exist . 
 women with brca1/2 mutations who have been treated with risk - reducing mastectomy have a substantially reduced breast cancer risk ( 30 ) . 
 furthermore , a study of breast cancer screening that added yearly magnetic resonance imaging to screening mammography suggested that combination of these modalities may also have benefit in the early detection of breast cancer in this group of women ( 28 ) . in conclusion , the summary risk reduction estimates presented here confirm that brca1/2 mutation carriers who have been treated with rrso have a substantially reduced risk of both breast and ovarian cancer . 
 therefore , additional cancer risk reduction and screening strategies are required to maximally reduce cancer incidence and mortality in this high - risk population . 
 forest plots of relative risk ( rr ) estimates for risk reduction associated with risk - reducing salpingo - oophorectomy ( rrso ) . 
 the box sizes reflect the relative sample sizes of the individual studies ; horizontal lines represent 95% confidence intervals ( cis ) . 
 estimates less than a value of 1.0 suggest a favorable reduction in cancer risk associated with rrso . 
 national institutes of health ( r01-ca083855 and r01-ca102776 to trr ) ; the department of defense breast cancer research program ( damd17 - 03 - 1 - 0375 to ndk ) ; the cancer genetics network ( hhsn21620074400c to smd ) ; the eileen genet fund and the project hope fund for ovarian cancer research and education .","backgroundrisk - reducing salpingo - oophorectomy ( rrso ) is widely used by carriers of brca1 or brca2 ( brca1/2 ) mutations to reduce their risks of breast and ovarian cancer . to guide women and their clinicians in optimizing cancer prevention strategies 
 , we summarized the magnitude of the risk reductions in women with brca1/2 mutations who have undergone rrso compared with those who have not.methodsall reports of rrso and breast and/or ovarian or fallopian tube cancer in brca1/2 mutation carriers published between 1999 and 2007 were obtained from a pubmed search . 
 hazard ratio ( hr ) estimates were identified directly from the original articles . 
 pooled results were computed from nonoverlapping studies by fixed - effects meta-analysis.resultsten studies investigated breast or gynecologic cancer outcomes in brca1/2 mutation carriers who had undergone rrso . 
 breast cancer outcomes were investigated in three nonoverlapping studies of brca1/2 mutation carriers , four of brca1 mutation carriers , and three of brca2 mutation carriers . 
 gynecologic cancer outcomes were investigated in three nonoverlapping studies of brca1/2 mutation carriers and one of brca1 mutation carriers . 
 rrso was associated with a statistically significant reduction in risk of breast cancer in brca1/2 mutation carriers ( hr = 0.49 ; 95% confidence interval [ ci ] = 0.37 to 0.65 ) . 
 similar risk reductions were observed in brca1 mutation carriers ( hr = 0.47 ; 95% ci = 0.35 to 0.64 ) and in brca2 mutation carriers ( hr = 0.47 ; 95% ci = 0.26 to 0.84 ) . 
 rrso was also associated with a statistically significant reduction in the risk of brca1/2-associated ovarian or fallopian tube cancer ( hr = 0.21 ; 95% ci = 0.12 to 0.39 ) . 
 data were insufficient to obtain separate estimates for ovarian or fallopian tube cancer risk reduction with rrso in brca1 or brca2 mutation carriers.conclusionthe summary estimates presented here indicate that rrso is strongly associated with reductions in the risk of breast , ovarian , and fallopian tube cancers and should provide guidance to women in planning cancer risk reduction strategies .",pubmed
"oral lichen planus ( olp ) is a chronic inflammatory disease affecting the oral mucosa , with a prevalence of about 1~2% of the population [ 1 , 2 ] . 
 once established , the lesions rarely undergo self - remission and , in some cases , have a malignant potency . 
 histologically , olp is characterized by a dense subepithelial infiltration of lymphocytes , increased numbers of intraepithelial lymphocytes , and the degeneration of basal keratinocytes . 
 the lymphocytic infiltrate consists predominantly of t cells [ 2 , 4 , 5 ] . 
 although the etiology of olp remains unclear , accumulating evidence supports a role of immune dysregulation in the pathogenesis of olp , especially involving a t - cell - mediated immune response and the abnormal production of various inflammatory molecules [ 2 , 4 , 6 ] . 
 previous studies have identified that a variety of proinflammatory molecules are aberrantly synthesized and secreted by both infiltrated t cells and altered keratinocytes in the lesions of olp , which contribute to the onset and persistence of the inflammatory responses [ 712 ] . 
 these findings indicated that olp lesion development is closely associated with the activation , production , and functions of these inflammatory molecules . 
 the interleukin- ( il- ) 23/il-17 axis is a newly discovered proinflammatory signaling pathway , in which il-23 and il-17 are two pivotal cytokines [ 13 , 14 ] . 
 il-23 is a heterodimeric proinflammatory cytokine , which is composed of a unique p19 subunit and a common p40 subunit shared with il-12 . 
 secreted by various cell types , such as activated dentritic cells , macrophages , and epithelium , il-23 functions as an important driving factor in the immune response . 
 notably , il-23 serves an essential role in induction and maintenance of a novel subset of cd4+th cells , namely , th17 [ 16 , 17 ] . 
 in addition , il-23 also induces th17 to produce its distinctive cytokine il-17 , which is composed of two il-17a subunits [ 18 , 19 ] . 
 subsequently , il-17 functions as a proinflammatory cytokine , which can activate different cells such as epithelial , endothelial , fibroblast , chondrocyte , and osteoblast to produce numerous inflammatory molecules including cytokines , chemokines , defensins , and mmps [ 20 , 21 ] . together with these downstream proinflammatory products , 
 the il-23/il-17 axis is extensively involved in the processes of chronic inflammation within various pathological states . 
 the il-23/il-17 axis has recently been described to play a major role in the pathogenesis of different chronic inflammatory and immune diseases , such as rheumatoid arthritis ( ra ) , psoriasis , atopic dermatitis , inflammatory bowel disease ( ibd ) , and periodontitis [ 2229 ] . in our previous study , we have detected the serum and saliva levels of il-17 in olp patients but found no significant difference compared to healthy groups . in others ' investigation , the presence of il-17 has been found in the lesions of olp , suggesting its role in the local environments . 
 however , till now , the role of il-23/il-17 axis in the pathogenesis of olp is still unclear . 
 the aim of the present study was to investigate the expression patterns and regulatory roles of il-23/il-17 axis in olp . 
 all tissue and blood specimens of olp patients and healthy volunteers were obtained from west china school and hospital of stomatology , sichuan university . 
 the olp cases were clinically diagnosed , pathologically confirmed , and subdivided as reticular or erosive forms based on the modified who diagnostic criteria of olp . 
 subjects with other oral or systematic diseases or taking corticosteroids or immunosuppressive drugs within 3 months prior to the specimen collection were excluded from the recruitment . for immunohistochemistry ( ihc ) staining , 27 olp lesion specimens , including 13 with erosive form and 14 with reticular form , were recruited from the archives of the department of pathology , and 10 normal oral mucosa ( nom ) tissues were collected from healthy volunteers receiving orthognathic surgery . for quantitative pcr analyses , 14 reticular olp and 
 10 nom tissues were obtained during the biopsy or orthognathic surgery , respectively , and subsequently snap - frozen in liquid nitrogen for the following experiment procedure . for studying the effect of il-23 on the cd4+t cells , 
 written informed consent was obtained from each subject , and the whole experiment procedure was conducted in accordance with the declaration of helsinki and approved by the scientific and ethical committee board of sichuan university . 
 paraffin - embedded sections of olp lesion specimens and nom tissues were treated in xylene and hydrated in graded ethanol , followed by blocking the activity of endogenous peroxidase with 3% hydrogen peroxide . 
 after antigen retrieval by heat and press , the sections were incubated with goat anti - human il-17 monoclonal antibody ( 1  :  100 , r&d systems , usa ) or rabbit anti - human il-23p19 monoclonal antibody ( 1  :  100 , abcam , uk ) overnight at 4c . 
 subsequently , sections were incubated with the rabbit anti - goat lgg antibody - hrp polymer or goat anti - rabbit lgg antibody - hrp polymer detection reagent ( zsgb biotechnology , beijing , china ) for 15 minutes at room temperature and then 3,3-diaminobenzidine tetrahydrochloride ( dab ) for 1 - 2 minutes . 
 the expression of il-23p19 subunit was assessed using an arbitrary scoring system as follows : 0 , no staining ; 1 , very weak staining ( 15 cells per section ) ; 2 , weak staining ( 530 cells per section ) ; 3 , moderate staining ( 30100 cells per section ) ; 4 , strong staining ( 100400 cells per section ) ; and 5 , strong staining ( > 400 cells per section ) . 
 the expression activities were assessed as previously described [ 33 , 34 ] . to evaluate the expression of il-17 , 
 five high power fields ( hpf ) at a magnification of 400 were randomly picked up and the numbers of il-17 + cells were counted separately . the average number of il-17 + cells per field was then calculated and compared . 
 five milliliters of heparinized blood was obtained from 10 olp patients , respectively . the peripheral blood mononuclear cells ( pbmcs ) 
 subsequently , cd4+t helper ( th ) cells were purified by anti - human cd4 magnetic particles ( bd biosciences , usa ) on a cell separation magnet ( bd biosciences , usa ) according to the manufacturer 's instructions . 
 the purified cd4+th cells were resuspended at a density of 1  10 cells / ml rpml 1640 medium ( thermo scientific hyclone , beijing , china ) supplemented with 10% fetal bovine serum ( fbs ; gibco , grand island , ny , usa ) . for activation of cd4+th cells , 10  g / ml of purified mouse anti - human cd3 antibody ( bd pharmingen , usa ) and 5  g / ml of purified mouse anti - human cd28 antibody ( bd pharmingen , usa ) were added . to study the effect of il-23 on the cd4+th cells 
 , the cells were cultured with or without recombinant human il-23 ( ril-23 , 20  ng / ml ; r&d systems , minneapolis , mn , usa ) for 36 hours . 
 subsequently , cells and culture supernatant were collected separately for the following intracellular cytokine staining and elisa detection . for intracellular cytokine staining 
 , collected peripheral blood cd4+th cells were resuspended at a density of 1  10 cells / ml and stimulated in rpml 1640 medium containing phorbol myristate acetate ( ( pma ) , 50  ng / ml , sigma ) , ionomycin ( 1  g / ml , sigma ) , and monensin ( 0.67  g / ml , goldistop , bd pharmingen , usa ) for 4 hours . after that , cells were stained using a cytofix / cytoperm fixation / permeabilization solution kit ( bd pharmingen , usa ) , anti - human cd4 percp - cy5.5 antibody ( bd pharmingen , usa ) , and anti - human il-17 ape antibody ( bd pharmingen , usa ) , successively , according to the manufacturer 's instructions . 
 stained cells were analyzed using the facsaria flow cytometer and bd facsdiva software ( bd biosciences , san diego , ca , usa ) . 
 the concentration of il-17 in the culture supernatants of peripheral blood cd4+th cells was measured using a human il-17 elisa kit ( boster , wuhan , china ) . according to the manufacturer 's instructions , 
 the detectable range of il-17 content was from 31.2 to 2000  pg / ml . 
 hok16e6e7 , a human immortalized oral keratinocyte cell line , was plated in 6-well plates ( 2  10/well ) in keratinocyte serum - free medium ( ksfm , gibco brl life technologies , grand island , ny , usa ) containing supplemented with epidermal growth factor and calcium . after 24 hours ' growth , medium with different doses of recombinant human il-17 ( rhil-17 , 020  ng / ml ; r&d systems , minneapolis , mn , usa ) or medium only was placed . 
 after another 24 hours ' culture , the cells were collected and kept in 70c until the rna isolation . 
 total rna was isolated from 14 reticular olp and 10 nom tissue specimens or the cultured cells using trizol reagent ( invitrogen , carlsbad , ca , usa ) . 
 cdna was synthesized from 400  ng of rna using the primescript rt reagent kit ( takara biotechnology , dalian , china ) . 
 primers purchased from takara biotechnology were listed in table 2 . to avoid the amplification of genome dna , all the primers 
 the real - time quantitative pcr on 10  ng of cdna was performed using the sybr green detection assay on an applied biosystems 7300 real - time pcr system . 
 the amplification procedure consisted of 10  s at 95c , followed by 40 cycles of the amplification procedure composed of 95c for 5  s and 62c for 40  s. each sample was run in triplicate . 
 the relative gene expression normalized to the expression of gapdh housekeeping gene and control sample were analyzed by the ct method . 
 kruskal - wallis test and mann - whitney test were used to determine differences between groups , paired t - test was used for the pair comparison , and spearman 's test was used to analyze the correlations . 
 to identify whether il-23/il-17 is involved in the local pathogenesis of olp , we firstly detected the expression and distribution of il-23 p19 , a unique subunit of il-23 , and il-17 in olp lesions and nom tissues . using ihc detection , we observed diffuse and strong expressions of il-23p19 in both erosive and reticular olp lesions . 
 the positive staining of il-23p19 predominantly concentrated on the epithelium of olp lesions and also on the extracellular matrix of the lamina propria ( figures 1(a)1(d ) ) . 
 in contrast , only a few keratinocytes in the epidermis layer of the nom tissues showed weak stain of il-23p19 ( figures 1(e ) and 1(f ) ) . 
 moreover , we found abundant il-17 positive stainings on the cytoplasm of the infiltrated lymphocytes in the lesions of both erosive and reticular olp , but only a few sporadic il-17 + cells in the normal oral mucosa ( figures 1(g)1(l ) ) . 
 the statistical data showed that both the erosive and reticular olp lesions had significantly increased immunostaining scores of il-23p19 , as well as the numbers of il-17 + cells , compared to the normal oral mucosa . 
 in addition , erosive olp lesions contained a significantly increased number of il-17 + cells compared to the reticular olp lesions . 
 however , there is no significant difference in il-23p19 staining score between erosive and the reticular olp lesions ( figures 2(a ) and 2(b ) ) . to verify the ihc results 
 , we also detected the mrna expressions of both subunits of il-23 ( il-23p19 and il-12p40 ) and il-17 in 14 reticular olp lesional tissues and 10 nom tissues and found that the mrna expressions of all the three genes in olp lesions were significantly increased compared to nom tissues ( figures 2(c ) and 2(d ) ) . 
 these data demonstrated overexpression of il-23 and il-17 in the olp lesions , indicating that the il-23/il-17 axis may be involved in the local immune network of olp . 
 considering il-23 as an important upstream inducing cytokine of il-17 , we next investigated whether the upregulation of il-23 in the progress of olp lesion is associated with the increased expression of il-17 . analyzing based on the data above 
 , we found no correlation between the il-23p19 staining scores and the numbers of il-17 + cells in the olp lesions ( figure 3(a ) ) . 
 however , in reticular olp subgroup , there was a positive correlation between the il-23p19 staining scores and the numbers of il-17 + cells ( figure 3(c ) ) , whereas no correlation was found in erosive olp group ( figure 3(b ) ) . 
 moreover , we also found that the mrna expressions of both il-23 subunits , il-23p19 ( figure 3(d ) ) and il-12p40 ( figure 3(e ) ) , are positively correlated with mrna expression of il-17 in reticular olp samples . 
 these results showed that overexpressions of il-23 and il-17 are positively correlated in the reticular olp lesion , indicating a potential regulatory role of il-23 to the expression of il-17 in the early stage of olp lesion . 
 next , we explored the potential role of il-23 in the production of il-17 in olp . 
 although il-17 has been recently reported to be produced by various cell types , a cd4+th cell subset , namely , th17 cell , is one of its main sources . on the other hand , in the local lesion of olp , 
 therefore , here we focused on the effect of il-23 to the il-17 production in cd4+th cells from 10 olp patients . 
 we observed that compared to the control group , the stimulation of ril-23 significantly increased the percentage of cd4+il-17 + cells ( identified as th17 ) in cd4+th cells from olp patients ( figures 4(a)4(c ) ) . 
 in addition , the il-17 content in the culture supernatant of cd4+th cells also increased under the stimulation of il-23 than the control group ( figure 4(d ) ) . 
 these data suggest that the overexpression of il-23 in olp lesions probably contributes to the induction of th17 and the production of il-17 . 
 we next explored the potential biological effects of il-17 , the essence effector of il-23/il-17 axis , in olp lesions . 
 it is well known that oral keratinocyte is an important component in the oral mucosa immunity and plays an important role in the pathogenesis of many chronic inflammatory oral diseases , including olp , by producing various inflammatory mediators , such as cytokines , chemokines , and defensins . 
 therefore , we investigated the effect of il-17 on the expressions of inflammatory mediators by hok16e6e7 , a human oral keratinocyte cell line . 
 first we detected effect of il-17 on the mrna expression of human -defensins ( hbd ) in hok cells . 
 we observed that il-17 significantly increased mrna expressions of hbd-2 and -3 in a dose - dependent manner , but not hbd-1 ( figure 5(a ) ) . 
 we next detected the mrna expression of 6 chemokines and found that il-17 could significantly increase the mrna expressions of il-8 , ccl-20 , but not cxcl-9 , -10 , -11 or ccl-5 , in hok cells ( figure 5(b ) ) . 
 furthermore , we found that il-17 significantly increased mrna expressions of tnf- , an important proinflammatory cytokine in the pathogenesis of olp , in hok . 
 however , mrna expression of il-6 exhibited no significant difference with or without the effect of il-17 ( figure 5(c ) ) . 
 the data revealed that the il-17 could selectively regulate the expressions of some , but not all , inflammatory mediators in oral keratinocytes , indicating the selectively regulatory role of il-23/il-17 axis in the immune network in olp lesions . 
 in recent years , the il-23/il-17 axis has been widely described to play a pivotal role in the pathogenesis of different chronic inflammatory disorders . 
 however , study about the expression and regulatory role of this new axis in olp is just at its beginning . in a recent study of our group 
 , we found no difference in the serum levels of il-23 and il-17 between the olp patients and controls . 
 the present study is focused on the expression and regulatory role of il-23/il-17 axis in the local environment of olp lesions . 
 il-23 is the upstream driving cytokine in the il-23/il-17 axis ; its importance in inflammation and autoimmunity has been widely demonstrated . to determine whether il-23 is involved in the development of olp 
 , we first detected its expression in the olp lesions compared with the nom tissues . 
 structurally , il-23 is composed of a unique p19 subunit and a common p40 subunit shared with il-12 . to avoid confusion 
 however , in the quantitative pcr assay , we examined the mrna expressions of both the il-23p19 and il-12p40 , for the expression of each gene could influence the amount of the il-23 . 
 our results revealed an upregulation of il-23p19 in both erosive and reticular olp lesions compared to the nom , indicating its involvement in the disease progress . as a driving factor of immune response 
 , il-23 is predominantly produced by the antigen presenting cells ( apcs ) including dentritic cells and macrophages , but its expression was also found in the keratinocytes from normal and psoriatic skin . 
 in addition , the epidermis in lesional psoriatic skin revealed markedly stronger il-23p19 stain than the epidermis in normal skin . 
 similarly , here we also observed a diffuse and strong expression pattern of il-23p19 in the epithelial layer of olp lesions , where keratinocytes are the major cell type . 
 these findings suggested that keratinocytes may be a major source of il-23 production under the chronic inflammatory condition in the mucocutaneous system . 
 although both the erosive and reticular olp lesions had significantly increased immunostaining scores of il-23p19 than the normal oral mucosa , the expressions of il-23p19 between the erosive and reticular olp lesions are similar . 
 this observation suggests that the overexpression of il-23 may be an early event in the pathogenesis of olp lesion and be maintained at a high level in the later progress . 
 il-17 is another key component in the il-23/il-17 axis and primarily functions as a downstream effector . 
 overexpressions of il-17 have been observed in many autoimmune and inflammatory diseases , and its pivotal roles in the pathogenesis have been profoundly identified [ 20 , 35 ] . 
 although il-17 can be secreted by a variety of innate and adaptive immune cells , t cells are still its major sources , especially the new subset of cd4+th cells , namely , th17 . considering the involvement of 
 a t - cell - mediated immune response in the pathogenesis of olp , it is not surprising that il-17 and th17 cells may be present and play a regulatory role in the local environment of the disease . 
 indeed , we observed a large number of il-17 + cells located in the subepithelial lymphocytic infiltrate in the olp lesions . besides , our data showed increased numbers of il-17 + cells and higher mrna expressions of il-17 in the olp lesions compared to the nom tissues . 
 in addition , compared to the reticular olp lesions , erosive olp lesions contained much more il-17 + cells . 
 these findings are consistent with the recent published study by piccinni and his colleagues , who also found an elevated mrna expression of il-17 , together with other th17 type molecules in the olp lesions compared to the healthy mucosa . moreover , 
 the presence of th17 was also identified in another recent study conducted by xie et al . . 
 we must admit that the overexpression of il-17 in olp lesions is attributed partly to the infiltration of lymphocytes in the local environment . 
 however , the large amount of il-17 cytokine in the local lesions of olp can not be overlooked , for its potent proinflammatory properties may induce profound biological effects and play an important role in the formation and progress of the disease . 
 on the other hand , piccinni et al . also found that the cd4+t cell clones generated from olp lesions produced significant higher levels of il-17 than those generated from the healthy oral mucosa . 
 this phenomenon indicated that the overexpression of il-17 in olp lesions attributes not only to the lymphocytic infiltration , but also to other unknown regulatory mechanisms , which is needed to be further explored . 
 since il-23 is a major upstream inducer of il-17 production , it is reasonable to further explore whether the upregulation of il-23 has any regulatory role in the il-17 production in the local environment of olp . 
 based on the data , we conducted correlation analysis of the expressions of il-23 and il-17 . although no correlation between il-23p19 staining scores and il-17 + cell numbers was found in the total olp group or erosive olp subgroup , there were positive correlations between the expressions of il-23 and il-17 at both protein and mrna levels in reticular olp subgroup , indicating that the upregulation of il-23 is associated with increased levels of il-17 in the early stage of olp lesion . 
 on the other hand , the lacking of correlation between il-23 and il-17 expressions in erosive olp lesions may be due to the persistent high levels of il-23 but various levels of il-17 , indicating the existence of other potentially regulatory mechanisms , other than il-23 , in the il-17 expressions in the erosive stage of olp . according to the recent published data by xie et al . 
 , the il-17 in the olp lesion is mainly expressed in cd4+t cells , which is identified as th17 , in the subepithelial lymphocytic infiltration , as observations by double immunofluorescence staining . 
 in addition , cd4+t cell clones generated from olp lesions exhibited an elevated activity in the il-17 production . 
 thus , we investigated the effect of il-23 on the cd4+t cells from olp patients . 
 our results showed that the stimulation of il-23 could significantly increase the percentage of th17 and the il-17 production in cd4+t cells from olp patients . 
 recent immunological findings have supported the opinion that , although il-23 has no effect on the initiation of th17 differentiation , it is crucially required for the proliferation and stability of precommitted th17 cells , their further migration into the pathological tissues , and the production of il-17 , in which way to promote the pathogenic function of th17 cells [ 37 , 38 ] . based on these findings and our data in the present study , it can be suggested that the overexpression of il-23 may be functional and contributes , at least in part , to the accumulation of th17 cells and the increased level of il-17 in the olp lesion . 
 therefore , the il-23/il-17 axis may represent a new signaling pathway in the crosstalk between the keratinocytes and cd4+t cells , which may be involved in the immunopathogenesis of olp . to investigate the potential biological effects of il-23/il17 in the olp lesion 
 , we further explored the effects of il-17 , the major effector of il-23/il-17 axis , on the production of different inflammatory mediators by the oral keratinocytes . 
 accumulated studies have demonstrated that oral keratinocyte is one of the major sources of the aberrant expressions of various inflammatory mediators in olp lesions , including cytokines , chemokines , and defensins , which interact with each other and compose the complex immune network in the olp environment [ 712 , 3941 ] . however , the mechanism of the functional alterations in the keratinocyte from olp lesions is still unclear . 
 our results showed that the stimulation of il-17 could significantly increase the mrna expressions of hbd-2 , 3 , il-8 , ccl-20 , and tnf- , but not the expression of hbd-1 , cxcl-9 , -10 , -11 , ccl-5 , and il-6 in hok cells . 
 these data revealed that the effects of il-17 on the oral keratinocyte are selective but not extensive , suggesting a unique regulatory role of il-23/il-17 axis in the local environment of olp lesions . in summary , based on the findings in the present study , we propose a novel model of interaction between the t cells and keratinocytes in the pathogenesis of olp , in which the il-23/il-17 axis is involved ( figure 6 ) . 
 firstly , keratinocytes in olp lesion produce a large amount of il-23 via an unknown mechanism . 
 next , keratinocyte - derived il-23 may contribute to the accumulation of th17 cells and the overproduction of il-17 in the local lesion of olp . 
 subsequently , il-17 reversely induces the keratinocytes to selectively produce various inflammatory mediators , which compose the complex immune network in the inflammatory environment of olp lesions . 
 our results warrant further explorations on the intrinsic mechanisms of the overexpression and the regulatory effects of il-23/il-17 axis , as well as its interaction with other signaling pathways , in the olp lesions . 
 notably , il-23/il-17 axis has been recently considered as a relevant therapeutic target in chronic inflammatory and autoimmune diseases , and several biological agents blocking il-23 or il-17 have been currently developed [ 22 , 42 ] . 
 therefore , further understanding of the role of il-23/il-17 axis in the pathogenesis may contribute to the development of novel therapeutic strategies for the prevention and management of olp in the future .","interleukin- ( il- ) 23/il-17 axis is a newly discovered proinflammatory signaling pathway and has been implicated in the pathogenesis of many chronic inflammatory and immune disorders . 
 here we investigated whether the il-23/il-17 axis was present and functional in the lesions of oral lichen planus ( olp ) , a chronic inflammatory disease affecting the oral mucosa . using immunohistochemistry and quantitative pcr , we found that the subunits of il-23 and il-17 were overexpressed in olp lesions than in normal oral mucosa tissues . 
 in addition , the expressions of il-23 and il-17 are positively correlated in reticular olp tissues . 
 results from in vitro studies revealed that exogenous il-23 could increase the percentage of th17 cells and il-17 production in the cd4+t cells from reticular olp patients . 
 furthermore , we also found that exogenous il-17 could significantly enhance the mrna expressions of -defensin-2 , -3 , ccl-20 , il-8 , and tnf- , but not -defensin-1 , cxcl-9 , -10 , -11 , ccl-5 , and il-6 in human oral keratinocytes . taken together 
 , our results revealed an overexpression pattern and selectively regulatory roles of il-23/il-17 axis in the olp lesions , suggesting that it may be a pivotal regulatory pathway in the complex immune network of olp lesions .",pubmed
"the isolation of pure populations of cells from heterogeneous cell suspensions is an essential part of clinical as well as basic research . 
 the diagnostic test for hiv infection , for example , relies on the separation of human lymphocytes from whole blood . 
 isolation of pure cell populations is an essential part of research efforts to understand fundamental aspects of the body s response to injury ( feezor et al 2004 ; cobb et al 2005 ) . 
 more recently , with major advances in stem cell biology , the isolation of rare cells has become an active area of research . 
 for example , islet 1 + cells that appear to be resident cardiac progenitor cells have been identified in mouse and human hearts ( laugwitz et al 2005 ) . 
 these cells are present in very small numbers , ~100 islet 1 + cells among 10 heart cells . 
 the progenitors are currently isolated by pre - plating of the heart cell suspension for 1 hour , after which fibroblasts and some islet 1 + cells remain attached to the tissue culture plastic . 
 after 46 days in culture , the progenitors make up only 0.5% of the total cell population found in the plates . 
 development of robust and efficient methods for isolating rare cells such as these would clearly represent a major contribution to stem cell biology . 
 miniaturized cell separation devices offer many advantages over conventional separation techniques ( eg , density gradient centrifugation ) , such as small sample volumes , portability , low cost , improved sterility , and potential for integration with analytical techniques . 
 many of these technologies are derived from the semiconductor industry or from advances in synthetic chemistry . 
 cell separation techniques can be broadly classified into two categories : techniques based on size and density , and techniques based on affinity ( chemical , electrical , or magnetic ) . in this review , rather than providing a comprehensive review of all cell separation techniques , we provide representative examples from each category with emphasis on micro- and nanotechnology . since cells are of the order of microns in size and usually handled in suspensions , nearly all of the methods described here are microfluidic 
 . however , some of these techniques , in particular the magnetic separation techniques , also incorporate nanoscale elements . 
 purity and throughput are important metrics for any separation process , and it is often impossible to attain high levels of both . 
 several novel size - based separation processes are being employed in micro- and nanoscale devices . 
 these devices are compact , simple , and usually do not require additional energy sources or additional equipment . 
 most of the devices force a fluid containing a heterogeneous particle population through a series of channels or obstacles of varied size . 
 unlike macroscale size - based separation approaches such as the use of cell strainers , the microscale geometry of the flow channels in these devices ensures that fluid flow is laminar . 
 the main advantage of the size - based approach is that it does not require the presence of cell - specific markers or proteins to achieve separation . 
 hence this approach can be used to isolate stem cells and other rare cells that do not express known markers . 
 furthermore , since the devices do not contain any biological markers or proteins , they have long shelf - life and are easily transportable 
 . however , one of the major concerns with this approach is maintaining cell viability as the cells pass through the microfluidic device . 
 huang et al ( 2004 ) describe a technique that allows for separation of micron - sized particles with a resolution of 0.1 m . in this approach 
 , individual fluid streams can be made to flow along confined lanes in a deterministic manner simply by allowing them to pass through an array of obstacles . 
 the assumption is that a fluid stream will bifurcate when it encounters an obstacle , and thereby sort the particles it brings with it ( figure 1a ) . as indicated by the arrows in the figure , the fluid streams travel around the obstacles and become confined to one of three lanes . 
 depending on the size of the particles in the stream and the obstacle parameters , the stream can enter one of two modes of displacement . 
 if the particles are smaller than the width of the lane they are flowing in , the particles zigzag back and forth around the obstacles ( zigzag mode ) , whereas if they are larger than the lane width , they collide with the obstacles and keep displacing in one direction ( displacement mode ) . by manipulating the obstacle parameters , such as their spacing and diameter , as well as the amount by which they are staggered in each subsequent row of the patterned array 
 , micron - sized particles can be sorted rapidly ( in a few minutes ) with high resolution and with an uncertainty less than 1% of the particle size , or , of the order of tens of nanometers . 
 such resolution is similar to that of other sorting techniques such as hydrodynamic chromatography and quasi - elastic laser light scattering , and considerably better than that of techniques such as cell straining and density gradient centrifugation . as a proof of concept 
 , the authors used the technique to sort fluorescent polystyrene spheres , as well as circular plasmid dna . 
 some limitations of the technique include the requirement of laminar flow and the possibility of random diffusion between adjacent lanes . 
 nanofabrication may allow for improved resolution and sorting of supramacromolecules such as viral particles and protein complexes . 
 ( a ) a stream of fluid flowing at low re ( ~10 ) is forced perpendicular to a series of obstacles of defined size and spacing ( see arrows ) . 
 if particles are smaller than the lane width , they continually zigzag between the obstacles , returning to their original lane assignment after traversing several rows of obstacles ( zigzag mode ) . 
 however , when the particles are larger than the lane width , they collide with the obstacles and displace only in one direction ( displacement mode  not shown ) , allowing for separation to occur . 
 ( b ) a series of channels of successively smaller width is microfabricated thus creating a cell sieve . 
 mohamed et al ( 2004 ) have designed a device that uses a relatively simple size - based separation approach to isolate rare cells , such as metastatic cancer cells , from peripheral blood . 
 the device contains four successively narrow arrays of parallel channels ranging from 20 m down to 2.5 m in thickness ( figure 1b ) . 
 the device essentially achieves separation by acting like a sieve , trapping larger cells such as mononuclear cells and neuroblastoma cells further upstream in the larger channels , and allowing smaller cells such as erythrocytes to pass through . 
 the authors experimented with several designs of varied channel width and height before identifying the critical dimensions required to trap the cells of interest . the channel width and height 
 were tuned to ensure that the larger , rare cells ( in this case , neuroblastoma cells or mononuclear cells ) were always trapped while allowing the smaller , bulk cells ( in this case , erythrocytes ) to always pass through . 
 thus , the approach can be applied to separation systems of mixed cell populations provided that the two cell types being separated are sufficiently different in size . to make the process more cost effective , a combination of soft lithography ( pdms , poly(dimethylsiloxane ) ) and polymers ( polyurethanes ) were used for fabrication . when cultured neuroblastoma cells were mixed with either plain medium or peripheral blood and passed through the device 
 , the larger neuroblastoma cells were consistently retained in the channels 10 m wide and 20 m deep while erythrocytes traversed through the device completely . 
 similarly , mononuclear cells in a sample of whole blood were trapped in channels 2.5 m wide and 5 m deep . while the technique is not without its limitations , including the issue of nonspecific cell adhesion to the channel walls , the technique is more effective in terms of cost and time than conventional cell sorting techniques such as fluorescence - activated cell sorting ( facs ) or magnetic - activated cell sorting ( macs ) . besides using a series of obstacles to achieve size - based cell separation , cells 
 shevkoplyas et al ( 2005 ) have developed a biomimetic microfluidic device to facilitate separation of leukocytes from erythrocytes in whole blood samples . 
 the device does not require a power source ( except for a small pressure gradient created by a water column to facilitate blood flow ) , and achieves efficient autoseparation in a single pass using low sample volumes ( ~l ) . 
 it is biomimetic in that it contains a series of bifurcating , rectangular microchannels , emulating the natural structure of blood vessels in vivo . 
 furthermore , it relies on the natural phenomenon of plasma skimming within a 2d channel to enrich the leukocyte population . 
 when the erythrocytes and leukocytes interact and collide , the erythrocytes , which are smaller and more deformable than leukocytes , preferentially migrate toward the center of the channel , forcing the leukocytes to migrate to the outer wall of the microchannel . by diverting the leukocyte - rich plasma layer through several bifurcations , 
 the authors achieved a 34-fold amplification in the leukocyte - to - erythrocyte ratio compared with the inlet concentration . 
 the device can prove particularly useful in applications where high concentrations of leukocytes or their dna and rna are required for analysis . 
 differential motility in laminar flow was used by cho et al ( 2003 ) to separate motile sperm cells from non - motile sperm cells . 
 central to their design is the assumption that viable sperm cells are motile and will therefore migrate against a laminar fluid stream . in contrast , 
 non - motile sperm simply follow the fluid stream lines along with other debris or round cells in the sample . 
 the motile sperm will move across the fluid stream and exit via a different outlet at the bottom of the device , distributing themselves along the width of this outlet , while the non - motile sperm will exit at the outlet situated at the top of the device . 
 the design does not require a power source since it utilizes a height difference between the inlet and outlet as well as surface tension of the fluid stream to create a gravity - driven pump , facilitating a constant fluid flow rate , regardless of the fluid reservoir volume . 
 the authors demonstrated that motile sperm can be purified to levels of nearly 100% . additionally , the yields of motile sperm at the outlet compared with the inlet were significantly improved and comparable with other conventional separation techniques such as direct swim - up , swim - up from centrifuged pellet , and density gradient separation . 
 cho et al have proposed several useful applications for the technique , such as home - based screening test for infertility , vasectomy , vasectomy reversal , and sperm toxicology tests . 
 further improvements to the geometry and configuration of the chip , such as serial connections , may lead to even higher yields . 
 these systems typically create a hydrodynamically focused stream of cell suspension which is interrogated using a laser beam and an array of photodetectors . 
 fluorescent labels are attached to one or more cell types in a heterogeneous suspension and the cells are sorted individually based on ( a ) how they scatter the incident laser light and ( b ) the wavelength of light that they emit . 
 a recent review by huh et al ( 2005 ) describes a number of microfluidic systems based on this principle . 
 an important advantage of microfluidic facs systems is their low cost compared with conventional systems . in the context of medicine and point - of - care diagnostics 
 furthermore , microscale facs systems have been shown to operate comparably to their macroscale analogs in terms of purity and throughput ( wolff et al 2003 ) . 
 fu et al ( 1999 ) have described a device made of silicone elastomer and glass with embedded platinum electrodes to direct the cells by electro - osmotic flow . 
 an external laser and photomultiplier tube ( pmt ) interfaced with a computer are used to excite and detect fluorescence . 
 flow of the cell suspension is controlled by changing the electro - osmotic potentials in response to the pmt signal . 
 the  forward  algorithm , cells that normally flow from the inlet to the waste reservoir are redirected to a collection channel if the fluorescence is above a preset threshold . with the  reverse 
 algorithm , cells flow at a high rate from the inlet to waste until the detection of fluorescence from a target cell induces the flow to reverse direction to allow a second detection step , followed by flow to the collection channel . 
 forward  algorithm is analogous to the mode of operation of most conventional facs systems , whereas the  reverse  is a novel algorithm that can not be implemented in conventional systems . 
 a key advantage of the  reverse  algorithm is that it can be used to make more than one measurement on individual cells . 
 this device was used to separate a mixed population of fluorescent and non - fluorescent escherichia coli cells . in a subsequent publication , 
 the same group ( fu et al 2002 ) described a more advanced version of this device which consists of integrated microvalves and micropumps . 
 the incorporation of these components  on - chip  allows for the elimination of some macroscopic components and better control of functions such as sample dispensing and recovery , and flushing . with the same sample of mixed cell populations ( fluorescent and non - fluorescent mixture of e. coli cells ) , 
 this device allowed enrichments of up to 90-fold with a throughput of 10 cells / hour . 
 a further step towards a fully integrated microfluidic facs system has been taken by krger et al ( 2002 ) . in their device , the laser and detectors are incorporated along with the microfluidic components in a single chip . 
 the only macroscopic parts of this system are a laptop computer , charge - coupled device ( ccd ) camera , and syringe pumps . 
 another example of an advanced microfluidic facs system with integrated waveguides and a cell culture chamber has been described by wolff et al ( 2003 ) . with this device , 
 the authors were able to separate fluorescent latex beads from chicken red blood cells at high throughput ( 12 000 cells / second ) . 
 this high rate was made possible by a high - speed hydrodynamic valve with a response time of 2.5 milliseconds , which was interfaced with the pmt . 
 the authors also developed a second - generation device ( figure 2 ) designed for improved hydrodynamic focusing and  on - chip  cell culture . 
 this device was used to separate normal yeast cells from yeast cells containing green fluorescent protein . 
 the detected cells were directed to the culture chamber by the high - speed valve and allowed to grow and divide for several days with continuous flow of fresh medium . 
 the incorporation of the culture chamber illustrates how microfluidic facs systems can reduce the risk of sample loss due to external handling or dead volumes , a feature that would be particularly relevant for the isolation of rare cells . 
 cells flow through a channel into an observation region . upon detection of a target cell , 
 a high - speed hydrodynamic valve switches fluid flow to send the cell into the holding / culturing chamber . 
 source : wolff a , perch - nielsen ir , larsen ud , et al . 
 2003 . integrating advanced functionality in a microfabricated high - throughput fluorescent - activated cell sorter 
 . abbreviations : pmt , photomultiplier tube ; facs , fluorescence - activated cell sorting . 
 the imag cell separation system ( bd biosciences , san jose , ca , usa ) is one example of several available separation systems . 
 when exposed to a mixed cell population , the magnetic beads attach to the surface of desired cells via antibody  antigen interaction . 
 the desired cell subpopulation can then be separated in the presence of a strong magnetic field either in separation columns or on integrated microchips . 
 magnetic separation is applicable only to cells that can be separated by specific surface antigens . for cells that are commonly distinguished by intercellular proteins ( eg 
 magnetic sorting is of particular interest since it can easily be miniaturized and utilized in applications where small sample volume is required . 
 this is the main advantage of microscale magnetic separation over the well established and efficient macroscale analogs . 
 the major challenge in the design of microscale magnetic separation systems , which is the focus of most studies , is achieving efficient separation in a continuous ( flow through ) system . 
 while several examples of microscale magnetic separation systems have been described in recent years , macroscale systems remain more attractive in terms of purity ( > 95% ) and throughput ( ~ 10 cells / hour ) ( thiel et al 1998 ) . 
 deng et al ( 2002 ) have developed a magnetic micro - filtration system for separation of magnetic beads from non - magnetic beads with up to 95% efficiency . in this 
 set - up , nickel posts 10 m in diameter were positioned in a microfluidic channel . 
 a heterogeneous suspension of beads on the order of 1 m was flowed into the channel in the presence of an external magnetic field that magnetized the nickel posts . as a result , the magnetic beads adhered to the posts while nonmagnetic beads were removed by the fluid flow . upon removal of the external magnet and 
 while keeping flow velocity unchanged , the magnetic beads were removed from the microfluidic channel . 
 a heterogeneous particle population can be separated using a combination of flow and magnetic field in the process called  magnetophoresis 
 depending on the particle size and magnetic properties , the particles were deflected into the magnetic field and carried into different outlet channels . 
 although the two systems described were not tested with cells , they appear very promising for cell separation applications . 
 the combination of magnetic microbeads and external magnetic field was utilized in a y - shaped microfluidic device by furdui and harrison ( 2004 ) to separate immunogenic t cells from whole blood with an efficiency of up to 40% ( figure 3a ) . in the first step , 
 paramagnetic beads coated with protein - a / anti - human cd3 were flowed into the micro - device and immobilized using an external magnet . 
 subsequently , whole blood was flowed over the bed of magnetic beads , resulting in the capture of t cells . upon removal of the external magnet , 
 the t cells were flushed out of the microfluidic device and utilized for pcr analysis . while this purity appears low in comparison with standard methods , such as separation using antibody - coated erythrocytes in conjunction with ficoll - hypaque density gradient centrifugation ( > 90% efficiency ; wilson et al 1976 ) , the microfluidic approach has the advantage of being easy to integrate with downstream analysis systems . magnetic cell separation and sorting . 
 ( a ) y - shaped device used to separate immunogenic t cells from whole blood in three steps . 
 paramagnetic protein a / anti - cd3-coated paramagnetic beads are flowed into the microdevice and immobilized using an external magnet . 
 subsequently , whole blood is introduced over the bed of magnetic beads , resulting in the capture of t cells . upon removal of the external magnet , 
 ( b ) combination of fluid flow ( arrows ) and ferromagnetic strips used to separate leukocytes on planar surfaces . 
 bright dots : time - lapse image of a tagged leukocyte moving along a ferromagnetic strip ( left ) . 
 untagged red blood cells are moving along the direction of the fluid flow ( right ) . 
 the matrix consists of two wire meshes superimposed at 180. source : lee h , purdon am , westervelt rm . 
 magnetic cell separation can also be achieved on planar surfaces , as demonstrated by inglis et al ( 2004 ) . 
 leukocytes were separated from red blood cells by flowing the blood over the surface coated with ferromagnetic strips . 
 the cells were labeled with anti - cd45 conjugated superparamagnetic nanoparticles ( 20100 nm diameter ) such that each positive cell carried around 5000 particles . 
 the strips were positioned at an angle of 9.6 relative to the direction of fluid flow , causing the deflection of labeled cells ( figure 3b ) . 
 magnetic labeling can also potentially enable precise manipulation and positioning of cells at the desired location . to prove this principle , lee et al ( 2004 ) 
 constructed a microelectromagnetic matrix by positioning two wire meshes on top of one another in a cross - pattern ( figure 3c ) . 
 the electric current in each wire was independently controlled via a computer , enabling precise control of the magnetic field at each point in the grid . with this set - up 
 , the authors were able to separate nonviable yeast cells from viable cells by moving the latter individually to a distant location within the grid in a controlled fashion . 
 the solution containing the live / dead - stained , magnetic bead - bound yeast cells was introduced into the microfluidic channel , integrated with the wire mesh . one viable yeast cell and two nonviable cells were initially trapped by a single magnetic field peak . 
 subsequently , the single peak was split into two smaller peaks ; one of the peaks held the nonviable cells and the other one held the viable cell . 
 the magnetic peak that held the viable cell was then moved by adjusting the current in the wires , so that the viable cell was moved away from the nonviable cells . 
 adhesion - based cell separation systems are akin to chromatography columns where a mixture is passed through a column packed with beads or other materials capable of binding to the undesired constituents of the feed . in cell separation , antibodies immobilized on surfaces are used for binding . 
 an important advantage of this technique is that it can be used to separate cell populations that have the same size and/or density , such as subpopulations of human lymphocytes . 
 another advantage of this approach is that there is no need for preprocessing incubation of the starting cell mixture with fluorescent or magnetic antibody tags . 
 conventional macroscale adhesion based systems are described by the term cell affinity chromatography ( cac ) . 
 these systems can generally provide high purity ( > 95% ) and high throughput ( 1010 cells / hour ) ( mandrusov et al 1995 ; putnam et al 2003 ) . 
 a major limitation of these systems is their packed bed design , which maximizes the surface area to volume ratio but also results in long residence times ( in the order of 12 hours ) ( mandrusov et al 1995 ; putnam et al 2003 ; ujam et al 2003 ) . 
 microfluidic cac systems provide high surface areas per unit volume but their small overall volume keeps residence times short ( order of minutes or less ) ( murthy et al 2004 ; sin et al 2005 ) . 
 microfluidic cac systems designed to separate t and b lymphocytes from mixed suspensions have been described by murthy et al ( 2004 ) and sin et al ( 2005 ) . 
 these investigations employed microfluidic chambers made of glass and pdms , whose interior surfaces were functionalized with antibodies capable of binding to surface antigens on the surface of the target cell type . 
 both investigations used t and b lymphocyte cells lines , molt-3 and raji , to create the mixed suspensions , and anti - cd5 and anti - cd19 antibodies to capture the respective cell types . in both reports , the cells captured on the surface were extremely pure ( > 97% purity ) . 
 nonspecific binding of the undesired cell type was overcome by attaching poly(ethylene glycol ) ( peg ) on the surface along with the antibody molecules ( murthy et al 2004 ) . in these approaches , 
 while the number of cells that can be captured was limited by the relatively small surface area of each device , the authors proposed that throughput can be increased by designing a device with larger surface area or by running several devices in parallel . in the above two reports , the antibodies used for separation were specific to the desired cell type . 
 anti - cd5 binds to the t cell line but not the b cell line ; and anti - cd19 binds to the b cell line but not to the t cell line . 
 but what about situations where there is no unique cell surface receptor that distinguishes the target cell type from other cells in the mixed suspension ? 
 chang et al ( 2005 ) have described an adhesion - based microfluidic separation technique that offers a way to address this challenge . 
 this technique emulates the physiological process by which leukocytes from the blood stream are captured by blood vessel walls during an inflammatory response . 
 blood vessel walls achieve this capture by presenting a variety of adhesion proteins ( such as selectins ) to the rapidly passing blood cells and selectively capturing only the cells that bear complementary ligands . 
 the authors designed microfluidic devices out of silicon and glass where the capture surfaces were square and rectangular posts arranged in square and offset arrays , respectively . in this respect , 
 their device is similar in design to that of huang et al ( 2004 ) and offers an interesting comparison even though the mode of separation is different ( adhesion - based in chang et al and size - based in huang et al ) . 
 chang et al coated the post surfaces with e - selectin igg molecules and examined cell capture using hl-60 and u-937 myeloid cell lines , both of which express ligands for e - selectin . with the help of flow experiments as well as finite volume simulations 
 , the authors observed that the movement of cells through their device occurs in three phases : ( 1 ) rolling , ( 2 ) transient capture on the trailing surface of the posts , followed by ( 3 ) detachment and subsequent flow . 
 the authors postulate that each of these three phases is influenced by the strength of bonds between the cells and the selectin molecules on the posts . in the square array device , 
 they then detached and always moved to the very next post downstream . in the offset array devices , however , the cells traveled arbitrary distances following the capture and release events , not always moving to the very next array . 
 although this report does not include experiments with mixed cell suspensions containing both cell types , the authors did measure a significant difference in transit time between the two cell types in their square array device : 1.4  0.8 mm / second for hl-60 and 2.7  1.4 mm / second for u-937 . 
 this difference could conceivably be utilized to separate cell populations that are indistinguishable in size and surface receptor expression . 
 adhesion - based separation can also be performed in a static microfabricated system , as opposed to a fluidic system . 
 revzin et al ( 2005 ) have described the fabrication of microwells made of peg for the isolation of lymphocyte subpopulations . 
 these wells are functionalized with antibodies and are capable of capturing target cells bearing complementary antigens with high selectivity . 
 cd5 + t lymphocytes ( molt-3 cell line ) adhered preferentially to microwells coated with anti - cd5 ( 95% of the available wells were occupied ) and showed only minimal adhesion to microwells coated with avidin and anti - cd19 ( well occupancies were 5% and 13% , respectively ) . with a microwell size of 15 m  15 m 
 , the authors observed that each well contained a single lymphocyte ( figure 4a ) . 
 the significance of this capability was demonstrated by the selective removal of individual t lymphocytes from an array of microwells by laser capture microdissection ( lcm , figure 4b ) . 
 lcm is technique whereby individual cells from different array locations are made to adhere to an adhesive film placed on top of the substrate surface by a pulsed and focused laser beam . 
 the adhesive film is mounted on a cap that is subsequently removed from the substrate surface . 
 the microwell platform offers a simple and effective method of isolating cell subpopulations based on antigen expression . when combined with lcm extraction , this technology 
 could potentially be employed to extract useful proteomic or genomic data from the selected cells . 
 ( a ) poly(ethylene glycol ) ( peg ) microwells containing individual t and b lymphocytes . 
 this type of capture allows subsequent extraction of individual cells by laser capture microdissection ( shown schematically in ( b ) ) . 
 source : revzin a , sekine k , sin a , et al . 2005 . 
 most electrophoretic separation processes employ a process known as dielectrophoresis ( dep ) , whereby particles with an induced electric polarization , such as dipoles , quadrupoles , and octopoles , can be trapped within a nonuniform electric field . 
 negative dep forces cause polarized objects to be repelled away from an electrode , while positive dep forces attract objects toward an electrode . 
 since dep depends on the existence of a gradient in an electric field , an alternating current ( ac ) voltage is usually employed . 
 the response of a cell to dep - mediated forces is influenced by cell type , its density , and even its metabolic and physiologic state , making these important parameters for separation . 
 there are essentially three variations of dep currently being employed for micro- and nanoscale devices : dep retention , dep migration , and dep - field flow fractionation ( dep - fff ) . 
 dep retention ( or dep affinity ) is used to hold cells against a fluid flow , so that cells only weakly influenced by dep forces are carried along with the fluid , while cells held more strongly by dep forces can be eluted later . 
 dep migration is similar to dep retention , but instead it utilizes dep to cause cells to migrate to different regions of an electrode . 
 finally , dep - fff uses dep forces to differentially levitate cells against gravity in a fluid flow profile , such as a parabolic flow profile . 
 this causes cells with different dielectric properties to travel at different velocities along the channel . like magnetic separation , 
 the primary advantage of microscale electrophoretic separation systems is the ability to handle small sample volumes . 
 voldman et al ( 2002 ) have developed a microfabricated separation system capable of trapping and sorting single cells on the basis of their response to certain stimuli . 
 the authors describe it as a microfabrication - based dynamic array cytometer ( dac ) , since it allows cells to be physically arrayed into dep - based traps , and dynamically probed and sorted thereafter . 
 an array of dep traps , each comprising four trapezoidally arranged electrodes of opposite polarity , was fabricated to create a non - uniform , quadrupole electric field ( figure 5a ) . 
 cells became electrically polarized in the presence of the quadrupole field and were physically trapped within a potential energy well against a fluid flow ( figure 5b ) . a high frequency ( ~mhz ) ac field 
 was chosen to minimize power dissipation and heat - induced damage to the cells , to prevent transmembrane polarization of cells , and also to minimize corrosion of the electrodes . 
 the cells ( hl-60 cell line ) were fed into the chip via a reservoir , and then exposed to a stimulus ( calcein ) . 
 they were then individually loaded into the traps and optically interrogated over time for their response to the stimulus ( calcein loading ) by optical microscopy . 
 each of the traps was electrically addressable , allowing for manipulation and release of a single cell or multiple cells simultaneously . 
 thus , based on their responses , the cells could be sorted by turning on or off each individual trap . 
 the authors demonstrated this capability in calcein - labeled hl-60 cells as a proof - of - concept of the technique . 
 a limitation that the authors point out in their dac system is that in some cases , more than one cell became confined in a trap . 
 ( a ) a pseudo - colored sem image showing a dioelectrophoresis ( dep ) trap consisting of four trapezoidaly arranged gold electrodes . 
 this configuration induces a dipole moment in the cell in the opposite direction to the electric field . 
 the cell is repelled from the field and stably trapped at the quadrupole s field minimum . 
 the application of higher flow rate results in the ejection of one cell ( dark grey arrow ) from the trap leaving the other cell behind ( grey arrow ) . 
 huang et al ( 1999 ) used dep - fff to separate cultured human breast cancer mda-435 cells from cd34 + hematopoietic stem cells . 
 the device consisted of three layers , including a patterned layer of eight interdigitated electrodes 50 m in width and spacing , a top glass layer , and a teflon spacer in between comprising the separation chamber . 
 a syringe pump was used to create a constant flow through the channel , forming a parabolic velocity profile . 
 since the heights attained in the fluid channel are both a function of the cell dielectric properties and the frequency of the applied voltage , the two cell populations were individually subjected to dep - fff over a range of frequencies to obtain information about their elution characteristics as a function of frequency . 
 these initial experiments showed that mda-435 cells took much longer to elute and even became trapped near the electrodes above 20 khz , while the cd34 + cells eluted more quickly and completely over a broader range of frequencies . 
 the first was a trap - and - release protocol , which utilized a fixed , 40 khz frequency to trap the mda-435 cells near the electrode , while the second was a frequency sweep between 15 and 35 khz , which allowed the mda-435 cells to be levitated slightly , allowing them to flow at a lower speed down the channel . 
 mixtures of the two cell types were then introduced into the chamber via an injection valve and allowed to equilibrate to different heights in the fluid channel for 5 minutes under an ac voltage of 10 v peak - to - peak . 
 the cells were then subjected to either of the frequency protocols described above for 7 minutes to allow the cd34 + cells to completely elute . 
 the frequency was then switched to 5 khz to allow the mda-435 cells to completely elute . 
 the results showed that in both protocols , cd34 + cells were eluted with purity greater than 99% between 3 and 5 minutes , while mda-435 cells were eluted with 96% purity between 9 and 12 minutes for the trap - and - release protocol and with 99% purity for the frequency - swept protocol . 
 these levels of purity and throughput are comparable to those of macroscale fluorescence - based and magnetic separation systems and better than that of macroscale electrophoretic systems . in another study by members of the same group ( yang et al 1999 ) , 
 the authors found that the elution time depended on both the frequency and voltage of the ac field . 
 larger voltages and frequencies resulted in larger dep forces and , accordingly , longer elution times . 
 there was no significant difference in elution time between the two cell types as a function of flow rate , indicating that the height obtained within the velocity field was mainly a function of the dep forces and not hydrodynamic lift forces created by flow within the channel . 
 in contrast to the previous study , the authors found that mda-435 cells were levitated up to 15 m higher than erythrocytes and were eluted nearly twice as fast . at a frequency of 10 khz and a flow rate of 1 ml / minute , 
 the authors obtained mda-435 cell fractions greater than 98% in purity between 10 and 12 minutes , and erythrocyte fractions greater than 99% purity between 18 and 26 minutes . 
 more recently , huang et al ( 2003 ) also developed a dep - based device to separate several strains of bacteria from erythrocytes , simulating the removal of biological warfare agents from blood . 
 the separation was achieved through dep migration , since the two cell types migrated to different parts of the microelectrode structure . 
 the device was constructed by sequential lamination of five layers : a patterned polyimide layer consisting of interdigitated dep electrodes , two pressure - sensitive acrylic adhesive layers , one of which contained microfluidic channels , a poly - carbonate substrate layer at the bottom , and a glass cover plate at the top . 
 cell suspension samples of 5 l were loaded into the device and an ac voltage of 10 v peak - to - peak at 10 khz was applied for 5 minutes . 
 the cells were then washed , at first with the ac voltage on and then with the voltage off , to collect the eluted samples . 
 the authors observed that the bacteria preferentially migrated and attached to the electrodes in the presence of a dep force . 
 in contrast , blood cells accumulated in the recessed areas between the electrodes , where field strengths were at a minimum . 
 when the three strains of bacteria were combined and mixed with blood , the dep device was able to simultaneously separate the multiple strains from blood cells . 
 pcr amplification of bacterial strain - specific genes did not reveal bands prior to separation , but revealed strong bands after separation , indicating that these pcr products were previously masked due to the presence of blood components . 
 in general , higher collection efficiencies were obtained for lower initial ratios of bacteria to erythrocytes , suggesting that the separation efficiency of the device is limited by the available surface area of its electrodes to about 1  10 bacteria per run . 
 while the above sections encompass most of the available microscale separation techniques , a number of others approaches are under development . 
 the strategy in this approach is to lyse the undesired cell populations in a mixed suspension while leaving behind the desired cells . 
 sethu et al ( 2004 ) have developed a microfluidic device for the separation of leukocytes from erythrocytes by lysis of the latter . 
 this separation is usually performed by macroscale lysis using osmotic lysing agents ( such as deionized water , sodium chloride buffer , ammonium chloride 
 sodium bicarbonate buffer ) , by density centrifugation , or some combination of macroscale lysis and centrifugation . 
 an important characteristic of macroscale lysis that the limiting step is not the actual cell lysis ( which occurs in 2030 seconds ) , but rather the diffusion of the lysing agent in the cell suspension , which can increase the time required for complete lysis to over 5 minutes . in the device designed by sethu et al , 
 blood cells are introduced into a channel that is also fed from either side by channels carrying lysis buffer ( figure 6 ) . 
 the result is that the blood cells flow in a narrow stream ( 1836 m wide ) at the middle of the channel with lysis buffer on either side . 
 this arrangement significantly reduces the time required for diffusion of the lysis buffer into the cell suspension , resulting in complete lysis within 2840 seconds , depending on the ratio of blood to lysis buffer . 
 the reduction in the total time required for this process also has the benefits of avoiding perturbation or damage to leukocytes resulting from prolonged contact with lysing agents or from centrifugation . 
 ( a ) a stream of blood cells is induced to flow along the center of the main channel by two adjacent streams of lysis buffer . 
 this narrowing , shown in ( b ) , minimizes the need for lysis buffer diffusion and allows contact with the flowing blood at nearly the single cell level . 
 sohn et al ( 2000 ) found that the capacitance of cells can be related to their dna content . using a microfluidic device fabricated from pdms and glass with embedded electrodes , 
 the capacitance was measured across a portion of the main channel with the cells flowing past one by one . 
 comparison of capacitance values with measurements of dna content obtained using conventional techniques yielded a linear graph with different cell types lysing at different points . 
 for example , this technique can distinguish between mammalian erythrocytes ( which contain no dna and therefore have zero capacitance ) and leukocytes ( whose dna content , and therefore capacitance , are nonzero ) . 
 gawad et al ( 2001 ) have described a microfluidic device that can distinguish between erythrocytes and other cells types by measurements of spectral impedance done on individual cells passing through a channel . 
 spectral impedance of an individual cell is a function of a number of variables , including cytoplasm conductance , cell size , and membrane capacitance . 
 this device was fabricated with polyimide and glass , and contains integrated channels and electrodes . 
 the broad spectrum of cell separation technologies described in this review illustrates the high level of interest and activity in this area . 
 the described size- and density - based approaches offer a great potential for separation of cell subpopulations for which specific markers are not known or can not be used ( eg , to prevent cell activation ) . 
 affinity - based approaches ( fluorescence- , magnetic- , adhesion - based , and electrophoretic ) can be employed for fast ( ~minutes ) and continuous separation with high specificity ( ~99% ) . for all of the approaches , 
 the design of the devices is such that they can be operated in a massively parallel fashion to increase scale and throughput without compromising purity and efficacy . 
 furthermore , micro - fluidic separation systems can be easily incorporated with devices that perform downstream analysis such as single - cell lysis ( irimia et al 2004 ) and proteomic and genomic analysis ( huang et al 2002 ; hashimoto et al 2005 ; parano et al 2005 ; situma et al 2005 ) . 
 given the advanced level of design , fabrication , and measurement capabilities , we expect that the focus in the coming years will shift from  proof - of - concept  prototypes to devices that can be economically produced and easily operated in applications such as point - of - care clinical diagnostics , drug discovery , and chemical  biological agent detection .","this review describes recent work in cell separation using micro- and nanoscale technologies . 
 these devices offer several advantages over conventional , macroscale separation systems in terms of sample volumes , low cost , portability , and potential for integration with other analytical techniques . more importantly , and in the context of modern medicine , 
 these technologies provide tools for point - of - care diagnostics , drug discovery , and chemical or biological agent detection . 
 this review describes work in five broad categories of cell separation based on ( 1 ) size , ( 2 ) magnetic attraction , ( 3 ) fluorescence , ( 4 ) adhesion to surfaces , and ( 5 ) new emerging technologies . 
 the examples in each category were selected to illustrate separation principles and technical solutions as well as challenges facing this rapidly emerging field .",pubmed
"a definitive diagnosis of cirrhosis is made by means of histological findings after liver biopsy . however , taking into consideration the high incidence of virus hepatitis and liver cirrhosis , a significant number of patients undergo imaging examinations before the definitive diagnosis of cirrhosis has been established . 
 several studies [ 1 - 11 ] showed that morphological signs that can be identified on routine clinical imaging examinations often accompany cirrhosis . 
 therefore , knowing these signs as well as knowing the differences of their diagnostic performance in cirrhotic liver can be useful in clinical practice . 
 different morphological signs have been described on different imaging techniques ( ultrasound , computer tomography , magnetic resonance imaging ) including the transverse diameter of segment 4 , caudate - to - right lobe ratio ( c / rl ) , the right posterior notch ( rpn ) , the right lobar atrophy , porta hepatis enlargement , and expanded gallbladder fossa ( egf ) . 
 these morphological signs are the result of fibrosis accompanied by decreased blood flow within portal veins . 
 the caudate - to - right lobe ratio is a quantitative measurement that demonstrated to have a high specificity and accuracy in diagnosing cirrhosis [ 5 , 9 ] . 
 the size of the caudate lobe has been also correlated with the hepatic functional reserve in cirrhotic patients . 
 it has been demonstrated that the caudate lobe was larger in patients with compensated cirrhosis than in patients with decompensated cirrhosis . 
 however , the main disadvantage of this quantitative assessment is represented by the fact that is time consuming since several measurements of the caudate and right lobe must be calculated . 
 therefore these measurements are rarely done in daily clinical work and have not gained widespread use . 
 the expanded gallbladder fosa ( egf ) and the right posterior notch ( rpn ) are simple qualitative visual signs of cirrhosis [ 1 , 2 ] . 
 both findings showed a high specificity and a high positive predictor value for the mr diagnosis of cirrhosis [ 1 , 2 ] . despite of their low sensitivity 
 , these signs can be routinely used in clinical practice [ 1 , 2 ] . to our knowledge , a comparison between the c / rl , egf , and rpn in cirrhotic liver has not been studied . 
 the purpose of this study was to evaluate and to compare the diagnosis performance of these signs in diagnosing cirrhosis . 
 our institutional medical database was cross - referenced to identify all consecutive patients who underwent mr imaging for suspected liver lesions or for evaluation of the severity of liver cirrhosis during a 15-month period . 
 one hundred twenty - five cirrhotic patients and 165 non - cirrhotic patients underwent mr imaging . 
 the patients with hepatocellular carcinoma larger than 2 cm and the patients who undergone hepatic resection or therapeutic interventional procedures ( e.g. ablation , chemoembolization ) were not included in this study . 
 finally , 95 cirrhotic patients were included in the cirrhosis group ( table 1 ) . 
 hepatitis c virus infection  
  hepatitis b virus infection  
  hepatitis d virus infection   in the control group were included patients who fulfilled the following criteria : no history of chronic liver diseases and no previous hepatic or biliary surgery . 
 the patients with focal liver lesions greater than 2 cm were also not included in this study since the presence of those lesions might modify the liver shape and consequently might influence the interpretation criteria . 
 the control group consisted of 57 subjects ( 26 men , 31 women , age range 18 - 83 ; mean age 51 ) . 
 institutional review board approval with waived informed patient consent was obtained for this retrospective study . 
 all mr examinations were performed using a 1.0-t mr system ( gyroscan intera , philips medical systems , netherlands ) . for optimal signal reception , 
 the imaging protocol included a transverse respiratory - triggered , non - enhanced t2-weighted turbo spin - echo ( tse ) sequence [ repetition time ( tr)/echo time ( te)= 1600ms/100ms , flip angle 90 , matrix size 256 * 512 , field - of - view 37 - 42 * 50 - 71 cm , section thickness 7 mm and intersection gap 1.0 mm ] and a breath - hold , transverse non - enhanced t1-weighted fast - field echo ( ffe ) sequence in- and out - of - phase [ tr / te=15ms/6.9ms for in - phase acquisition and tr / te=15ms/3.45ms for out - of - phase acquisition ; flip angle 25 , 1 nsa , matrix size 256x256 , field of view 37 - 42x50 - 71 cm , section thickness 7 mm and intersection gap 1.0 mm ] were acquired . 
 after acquisition of the non - enhanced sequences , gadobutrol ( gadovist , bayer schering pharma ag , berlin , germany ) was injected intravenously at a dose of 0.1mmol per kilogram of bodyweight with a flow rate of 2ml / sec followed by a 20ml saline flush at the same flow rate using a power injector ( spectris , medrad , indianola , pa , usa ) . 
 dynamic gradient - recalled mr imaging was timed to capture the arterial , portal venous and equilibrium phase and was performed with a fast - field echo ( ffe ) sequence [ tr / t 5.1ms/1.69ms , flip angle 25 , matrix size 208 * 256 , field of view 37 - 43 * 44 - 50 cm , section thickness 4.2 mm ] . 
 two radiologists ( with 6 and 4 years of experience in abdominal mri , respectively ) independently assessed data sets in two different reading sessions ( 1 , the evaluation of the presence or absence of rpn ; and 2 , the presence or absence of egf ) . to reduce any bias , 
 reading sessions were separated by 4-week intervals and data sets were analyzed in random order from both cirrhotic patients and control subjects . 
 the evaluation of both readers was made by using the non - enhanced t1 weighted images and dynamic gradient - recalled mr images . 
 both readers were blinded to all clinical patient data .   in the first reading session , 
 the readers were asked to define the presence or absence of rpn and in the second reading session , the readers were asked to define the presence or absence of egf . in both assessments 
 it was used a 2-point grading scale : 0- absence of the sign ; 1-presence of the sign . 
 the rpn was defined as a sharp indentation in the right posterior surface of the liver ( fig 1 ) and the egf is represented by the enlargement of the pericholecystic space ( fig 2 ) . 
 space is bounded laterally by the edge of the right hepatic lobe , medially by the edge of the lateral segment of the left lobe . 
 the medial segment of the lateral lobe should not be seen on the same axial image . 
 56-year - old man with liver cirrhosis child a. axial enhanced mr image ( 15/3.45 , 25 flip angle ) . 
 the c / rl is high ( 1.37 ) and the rpn is easily identified ( arrow ) . note the fact that the sharp notch that defines rpn and represents the landmark of the caudate lobe is situated on the line through the bifurcation of the right portal vein . 
 space is bounded laterally by the edge of the right hepatic lobe , medially by the edge of the lateral segment of the left lobe . 
 the c / rl ( the width of the caudate lobe / the width of the right lobe ) was calculated by one radiologist ( 6 years experience in abdominal mri ) . 
 the reader chose images for measurement on the basis of the clarity of the portal veins on enhanced dynamic gradient - recalled mr images . 
 the measurements were made by using the bifurcation of the right portal vein as the landmark of the caudate lobe   ( fig 3 ) . 
 axial contrast - enhanced ffe mr image ( 5.1/1.69 , 25 flip angle ) obtained during the portovenous phase at the level of right portal vein bifurcation shows the method of calculating c / rl . 
 line 1 , line 2 , and line 3 are parallel to the midsagittal plane . 
 line 2 is drawn through the right lateral wall of the bifurcation of the right portal vein and line 3 is drawn through the most lateral margin of the right lobe . 
 line 4 is perpendicular to line 1 and is situated at midway between the posterior wall of the main portal vein and the anterior wall of the inferior vena cava . 
 descriptive results regarding the presence or absence of rpn and egf were reported in relative numbers . 
 a kappa value of 0 indicated poor agreement , a value of 0.01 - 0.20 slight agreement , a value of 0.21 - 0.40 fair agreement , a value of 0.41 - 0.60 moderate agreement , a value of 0.61 - 0.80 good agreement , and a value of 0.81 - 1.00 implied an excellent agreement . 
 the qui - square test was used to evaluate the statistical difference of the diagnostic value of the signs between cirrhotic group and control group . 
 the diagnosis accuracy of the c / rl sign was calculated by using the roc curve . 
 the sensitivity and specificity of the c / rl sign for diagnosis of cirrhosis were calculated having a cut - off point of 0.50 . the relative risk ( odds ratio ) of c / rl 
 the statistical significance of any difference between the three signs in diagnosis of cirrhosis was calculated using friedman and wilcoxon test and the statistical significance of any difference of each sign between different   child - pugh classes of cirrhosis was calculated using kruskal - wallis and student 's t - test . 
 all statistical analyses were performed by using commercially available software ( spss 13.0 , chicago , il ) . 
 our institutional medical database was cross - referenced to identify all consecutive patients who underwent mr imaging for suspected liver lesions or for evaluation of the severity of liver cirrhosis during a 15-month period . 
 one hundred twenty - five cirrhotic patients and 165 non - cirrhotic patients underwent mr imaging . 
 the patients with hepatocellular carcinoma larger than 2 cm and the patients who undergone hepatic resection or therapeutic interventional procedures ( e.g. ablation , chemoembolization ) were not included in this study . 
 finally , 95 cirrhotic patients were included in the cirrhosis group ( table 1 ) . 
 hepatitis c virus infection  
  hepatitis b virus infection  
  hepatitis d virus infection   in the control group were included patients who fulfilled the following criteria : no history of chronic liver diseases and no previous hepatic or biliary surgery . 
 the patients with focal liver lesions greater than 2 cm were also not included in this study since the presence of those lesions might modify the liver shape and consequently might influence the interpretation criteria . 
 the control group consisted of 57 subjects ( 26 men , 31 women , age range 18 - 83 ; mean age 51 ) . 
 institutional review board approval with waived informed patient consent was obtained for this retrospective study . 
 all mr examinations were performed using a 1.0-t mr system ( gyroscan intera , philips medical systems , netherlands ) . for optimal signal reception , 
 the imaging protocol included a transverse respiratory - triggered , non - enhanced t2-weighted turbo spin - echo ( tse ) sequence [ repetition time ( tr)/echo time ( te)= 1600ms/100ms , flip angle 90 , matrix size 256 * 512 , field - of - view 37 - 42 * 50 - 71 cm , section thickness 7 mm and intersection gap 1.0 mm ] and a breath - hold , transverse non - enhanced t1-weighted fast - field echo ( ffe ) sequence in- and out - of - phase [ tr / te=15ms/6.9ms for in - phase acquisition and tr / te=15ms/3.45ms for out - of - phase acquisition ; flip angle 25 , 1 nsa , matrix size 256x256 , field of view 37 - 42x50 - 71 cm , section thickness 7 mm and intersection gap 1.0 mm ] were acquired . 
 after acquisition of the non - enhanced sequences , gadobutrol ( gadovist , bayer schering pharma ag , berlin , germany ) was injected intravenously at a dose of 0.1mmol per kilogram of bodyweight with a flow rate of 2ml / sec followed by a 20ml saline flush at the same flow rate using a power injector ( spectris , medrad , indianola , pa , usa ) . 
 dynamic gradient - recalled mr imaging was timed to capture the arterial , portal venous and equilibrium phase and was performed with a fast - field echo ( ffe ) sequence [ tr / t 5.1ms/1.69ms , flip angle 25 , matrix size 208 * 256 , field of view 37 - 43 * 44 - 50 cm , section thickness 4.2 mm ] . 
 two radiologists ( with 6 and 4 years of experience in abdominal mri , respectively ) independently assessed data sets in two different reading sessions ( 1 , the evaluation of the presence or absence of rpn ; and 2 , the presence or absence of egf ) . to reduce any bias , 
 reading sessions were separated by 4-week intervals and data sets were analyzed in random order from both cirrhotic patients and control subjects . 
 the evaluation of both readers was made by using the non - enhanced t1 weighted images and dynamic gradient - recalled mr images . 
 both readers were blinded to all clinical patient data .   in the first reading session , 
 the readers were asked to define the presence or absence of rpn and in the second reading session , the readers were asked to define the presence or absence of egf . in both assessments 
 it was used a 2-point grading scale : 0- absence of the sign ; 1-presence of the sign . 
 the rpn was defined as a sharp indentation in the right posterior surface of the liver ( fig 1 ) and the egf is represented by the enlargement of the pericholecystic space ( fig 2 ) . 
 space is bounded laterally by the edge of the right hepatic lobe , medially by the edge of the lateral segment of the left lobe . 
 the medial segment of the lateral lobe should not be seen on the same axial image . 
 56-year - old man with liver cirrhosis child a. axial enhanced mr image ( 15/3.45 , 25 flip angle ) . 
 the c / rl is high ( 1.37 ) and the rpn is easily identified ( arrow ) . 
 note the fact that the sharp notch that defines rpn and represents the landmark of the caudate lobe is situated on the line through the bifurcation of the right portal vein . 
 space is bounded laterally by the edge of the right hepatic lobe , medially by the edge of the lateral segment of the left lobe . 
 the c / rl ( the width of the caudate lobe / the width of the right lobe ) was calculated by one radiologist ( 6 years experience in abdominal mri ) . 
 the reader chose images for measurement on the basis of the clarity of the portal veins on enhanced dynamic gradient - recalled mr images . 
 the measurements were made by using the bifurcation of the right portal vein as the landmark of the caudate lobe   ( fig 3 ) . 
 axial contrast - enhanced ffe mr image ( 5.1/1.69 , 25 flip angle ) obtained during the portovenous phase at the level of right portal vein bifurcation shows the method of calculating c / rl . 
 line 1 , line 2 , and line 3 are parallel to the midsagittal plane . 
 line 2 is drawn through the right lateral wall of the bifurcation of the right portal vein and line 3 is drawn through the most lateral margin of the right lobe . 
 line 4 is perpendicular to line 1 and is situated at midway between the posterior wall of the main portal vein and the anterior wall of the inferior vena cava . 
 descriptive results regarding the presence or absence of rpn and egf were reported in relative numbers . 
 a kappa value of 0 indicated poor agreement , a value of 0.01 - 0.20 slight agreement , a value of 0.21 - 0.40 fair agreement , a value of 0.41 - 0.60 moderate agreement , a value of 0.61 - 0.80 good agreement , and a value of 0.81 - 1.00 implied an excellent agreement . 
 the qui - square test was used to evaluate the statistical difference of the diagnostic value of the signs between cirrhotic group and control group . 
 the diagnosis accuracy of the c / rl sign was calculated by using the roc curve . 
 the sensitivity and specificity of the c / rl sign for diagnosis of cirrhosis were calculated having a cut - off point of 0.50 
 . the relative risk ( odds ratio ) of c / rl , egf and rpn for the diagnosis of cirrhosis was also calculated . 
 the statistical significance of any difference between the three signs in diagnosis of cirrhosis was calculated using friedman and wilcoxon test and the statistical significance of any difference of each sign between different   child - pugh classes of cirrhosis was calculated using kruskal - wallis and student 's t - test . 
 all statistical analyses were performed by using commercially available software ( spss 13.0 , chicago , il ) . 
 the interobserver agreement between the 2 readers was excellent (  0.81 ; 95% ci : 0.92 , 1.0 ) in the evaluation of the presence or absence of rpn and egf . 
 there was a significant statistical difference of the diagnostic value of c / rl , rpn , and egf between cirrhotic patients and control group ( p<0.001 ) . 
 table 2 summarizes the sensitivities , specificities , accuracies , and the relative risk of c / rl , rpn , and egf for cirrhosis with regard to the diagnosis of cirrhosis . 
 there was no statistical significant difference between c / rl and rpn ( p=0,382 ) for the diagnosis of cirrhosis ( fig 1 ) . 
 there were statistical significant differences between c / rl and egf ( p=0,000 ) and between rpn and egf ( p=0,010 ) for the diagnosis of cirrhosis ( fig 5 ) . 
 there was a statistical significance difference between c / rl and egf and between rpn and egf for child a class of cirrhosis ( table 3 ) . 
 summary of the diagnostic performance of the c / rl , the rpn , and the egf and the relative risk of all three morphological signs in the diagnosing cirrhosis . 
 42-year - old woman with liver cirrhosis child b. axial unenhanced mr image ( 15/3.45 , 25 flip angle ) shows the right posterior hepatic notch ( large arrow ) . on the same image 
 the pericholecystic space is bounded medially by the edge of the medial segment of the left lobe ( small arrow ) and not by the lateral segment . 
 the statistical differences between c / rl , rpn , and egf in different classes of cirrhosis . 
 cross - sectional imaging has been used for the diagnosis of cirrhosis based on the quantitative evaluation of lobar changes of the liver as well as on qualitative findings such as the right posterior hepatic notch and the expanded gallbladder fossa .   in cirrhotic liver 
 there is an atrophy of the right lobe concomitant with a hypertrophy of the caudate lobe due to alterations in portal blood flow and hepatic fibrosis . 
 regardless the compensated or uncompensated type of cirrhosis the caudate lobe is larger in these patients compared with the noncirrhotic patients . 
 harbin et al   have   shown that on ultrasonography or ct examinations the cirrhotic livers can be differentiated from noncirrhotic livers by using c / rl with a sensitivity of 84% , a specificity of 100% , and an accuracy of 94% . in their study , the c / rl was calculated by using the main portal vein bifurcation as the landmark of the caudate lobe . 
 however , awaya et al   have proposed a modified c / rl for the quantitative evaluation of cirrhotic liver .   based on the fact that the caudate lobe receives blood supply from the posterior segmental branch in 51% of the cases   and 
 the intrahepatic course of the caudate branches is short and less influenced by fibrosis , the authors have considered that the landmark of the caudate lobe should be the bifurcation of the right portal vein . comparing the roc curve of the c / rl described by harbin with awaya s modified ratio for diagnosing cirrhosis 
 there was a significant higher az value for modified c / rl ( 0.797 ; p=0.040 ) than for harbin s ratio ( az=0.737 ) ( 9 ) . 
 in our study we used the c / rl modified by awaya and we obtained an az value of 0.848 for the diagnosis of cirrhosis . 
 the different az values may be the result of the fact that the patients included in awaya s study were reported having only mild or moderate cirrhosis in 65% of cases . 
 when we calculated the sensitivity , the specificity , and the accuracy of c / rl having a cut - off point of 0.50 we obtained a sensitivity of 72% , specificity of 87% , and accuracy of 78% . 
 our results are different from those reported by harbin . since the az value of the c / rl used in our study has shown higher values comparing with harbin s results , we would have been expect that the sensitivity , the specificity , and the accuracy would have been higher . 
 however , in their study   the authors have used different landmarks for c / rl and different criteria for including cirrhotic patients and control subjects . 
 harbin did not specify the underlying cause of cirrhosis and patients with diffuse liver diseases were also included in the control group . 
 the evaluation of the c / rl requires performing contrast - enhanced cross sectional imaging in order to identify the portal vein bifurcations . 
 therefore , the method gained limited use in clinical practice for assessment of cirrhotic patients . the right posterior hepatic notch and the expanded gallbladder fossa are two simple qualitative visual findings for cirrhosis [ 1 , 2 ] . 
 ito et al   described the rpn as a sharp indentation on the posteroinferior liver surface different from the renal impression . 
 the presence of the rpn in cirrhosis is the result of the atrophy of the right lobe and hypertrophy of the caudate lobe . 
 thus , both signs , c / rl and rpn , are dependent on the same lobar changes of the liver 
 . this may be the reason why the diagnostic sensitivity , specificity , and accuracy together with odds ratio of each sign are similar in our study . 
 when comparing the signs , we did not find any statistical significance difference between them for the diagnosis of cirrhosis . 
 moreover , there was no difference between these signs in the diagnosis of different classes of cirrhosis . 
 the sensitivity , specificity , and accuracy of rpn were reported by ito to be 72% , 98% , and 82% , respectively . 
 our results show lower values ( sensitivity 67% , specificity 87% , and accuracy 75% ) . 
 the rpn is a morphological sign more frequently seen in patients with alcoholic cirrhosis compared with patients with viral cirrhosis since the volume index of the caudate lobe is significant greater in alcoholic cirrhosis than in viral cirrhosis . in our study population 
 there was included only a small percentage of patients with alcoholic cirrhosis ( 5% ) . in comparison , 
 in the study of ito , 13% of the patients included in their study were with alcoholic cirrhosis . 
 this difference in the patient population together with the fact that the rpn is statistically more frequent in the alcoholic type of cirrhosis might explain the differences in the diagnosis performances between the two studies . 
 in contrast to c / rl and rpn , the expanded gallbladder fossa is a sign that reflects different segmental liver changes in a cirrhotic liver . 
 although , the hypertrophy of the caudate lobe and atrophy of the right lobe contribute to the presence of egf , another two segmental changes have an important role in the visualization of the sign : the atrophy of the medial segment of the left hepatic lobe and the cephalocaudal enlargement of the lateral segment of the left hepatic lobe . 
 the atrophy of the segment 4 in cirrhotic liver has been demonstrated by lafortune   who showed that regardless the cause or the severity of cirrhosis , the mean diameter of the segment 4 measured on ultrasonography is significant decreased in cirrhosis compared with normal liver . 
 torres et al   demonstrated on a volumetric evaluation of the cirrhotic liver a decrease in volume by 10.9% of the medial segment of the left lobe when compared with normals . on the other hand , 
 the enlargement of the lateral segment of the left hepatic lobe is seen only in compensated viral induced cirrhosis whereas in decompensated cirrhosis there is an atrophy of this segment . it has been also demonstrated that the left lobe does not increase in volume with the progression of the cirrhosis in contrast to the right lobe which shows a significant decrease in volume . 
 there is also a marked variability in the volumetric measurements of the lateral segment of the left lobe in normal livers . 
 these variations of the left lobe volume can be responsible for the low sensitivity ( 49% ) and low odds ratio ( or=10.18 ) of egf . 
 the low sensitivity of this sign in our study has been previously reported [ 1 , 2 ] . however , the specificity of egf in this study is high ( 91% ) and comes to confirm the results of other authors . when comparing egf with c / rl and rpn , there was a statistical significance difference between the last two signs and egf in diagnosing cirrhosis ( p<0.010 ) . 
 these results suggest that the egf is a useful sign in evaluation of cirrhosis when is used in conjunction with other morphological findings reported in previous studies [ 2 , 5 , 9 , 10 , 19 ] . 
 our study results demonstrated a significant statistical difference between c / rl and egf ( p=0.002 ) and between rpn and egf ( p=0.037 ) for child a class of cirrhosis . 
 no differences were found between signs for child b and child c classes of cirrhosis ( p>0.05 ) . 
 these data might express that in compensated and less severe clinical cirrhosis the morphological changes are mainly represented by the hypertrophy of caudate lobe and atrophy of the right lobe . 
 we did not evaluate the diagnosis performance of combined c / rl , rpn , and egf . 
 however , we tried to assess the individual role of each sign in the diagnosis of cirrhosis . 
 consideration of two findings or of all three signs of cirrhosis would improve the diagnosis accuracy . 
 another limitation is represented by the fact that we did not correlate the morphological signs with the underlying cause of cirrhosis . 
 further studies are needed to determine if these signs might help to differentiate the cirrhosis induced by different causes . 
 a potential criticism of this study may also be the exclusion of the patients with chronic liver diseases including fibrosis . 
 a relevant evaluation would involve an assessment of the morphological findings in cirrhotic patients in comparison with the findings in patients with chronic liver diseases . finally , the c / rl , the rpn , and the egf can not be only calculated and visualized on mr liver examinations . 
 . however , mr examinations is at this moment extensively used in the evaluation of cirrhosis since this method offer a more accurate and complete evaluation of the cirrhotic liver . 
 in conclusion , 
 the caudate to right lobe ratio and the right posterior hepatic notch have similar performance regarding the diagnosis of cirrhosis . 
 the expanded gall - bladder fossa is a sign with low sensitivity and high specificity and represents an important additional finding of cirrhosis .","background & aims the purpose of the study is to evaluate the accuracy of the c / rl , rpn , and egf in diagnosing cirrhosis . 
 methods the study population included 95 cirrhotic patients in the cirrhosis group ( 56 men , 39 women , age range 14 - 76;mean age 52.3 ) and 57 subjects in the control group ( 26 men , 31 women , age range 18 - 83;mean age 51 ) . 
 all mr examinations were performed by using the same protocol . 
 two radiologists independently assessed data sets in two different reading sessions . 
 the sensitivity , specificity , and accuracy and the relative risk of the signs in diagnosing cirrhosis were calculated . 
 the diagnosis accuracy of the c / rl sign was calculated using the roc curve . 
 the statistical significance of any difference of each sign between different classes of cirrhosis was also calculated . 
 results the interobserver agreement between the readers was excellent (  0.81;95% ci:0.92 , 1.0 ) . 
 there was a significant statistical difference of the diagnostic value of c / rl , rpn , and egf between cirrhotic patients and control group ( p<0.001 ) . 
 the sensitivity , specificity , and accuracy of c / rl were 72% , 87% , and 78% ; 67% , 87% , and 75% for rpn ; and 49% , 91% , and 65% for egf . 
 c / rl ( or=18.95 ) and rpn ( or=14.74 ) showed a higher risk for cirrhosis compared to egf ( or=14.74 ) . 
 there was a statistical significance difference between c / rl and egf ( p=0.002 ) and between rpn and egf for child a class of cirrhosis ( p-0.037 ) . 
 conclusion the c / rl and rpn have similar performance regarding the diagnosis of cirrhosis having a higher diagnostic performance compared to egf in cirrhosis .",pubmed
"participants from this study were included in a prospective longitudinal study designed to evaluate optic nerve structure and visual function in glaucoma ( the diagnostic innovations in glaucoma study ) conducted at the hamilton glaucoma center at the department of ophthalmology , university of california , san diego ( ucsd ) . 
 the institutional review board approved the study methodology , which adhered to the tenets of the declaration of helsinki and to the health insurance portability and accountability act . at each visit during follow - up , patients underwent a comprehensive ophthalmologic examination including review of medical history , best - corrected visual acuity , slit - lamp biomicroscopy , intraocular pressure , gonioscopy , dilated ophthalmoscopic examination , stereoscopic optic disc photograph ( kowa nonmyd wx3d ; kowa optimed , inc . , torrance , ca , usa ) , sd - oct testing ( spectralis ; heidelberg engineering , dossenheim , germany ) , and sap using the swedish interactive threshold algorithm ( sita standard 24 - 2 ; carl zeiss meditec , inc . , 
 subjects were excluded if they had any ocular or systemic disease that could affect the optic nerve or the visual field . 
 all patients had a diagnosis of glaucoma at baseline , based on the presence of repeatable visual field defects on sap and/or glaucomatous optic neuropathy on optic disc stereophotographs . 
 repeatable visual field defects on sap were defined as at least three consecutive abnormal sap results with pattern standard deviation ( psd ) with p < 0.05 , and/or glaucoma hemifield test results outside normal limits . 
 glaucomatous optic neuropathy was evaluated by masked assessment of stereophotographs and defined based on the presence of neuroretinal rim thinning , excavation , notching , or characteristic rnfl defects . 
 healthy subjects were recruited from the general population through advertisements or from the staff and employees at the ucsd . 
 healthy subjects had intraocular pressures less than 22 mm hg with no history of increased intraocular pressure and at least two reliable normal visual fields in both eyes , which were defined as a pattern sd within 95% confidence limits and a glaucoma hemifield test result within normal limits . for inclusion in the analysis , each glaucoma patient or healthy subject was required to have at least five visits ( with five sap tests and five oct tests ) over a follow - up duration of at least 2 years . the spectralis sd - oct ( software version 5.4.7.0 ) 
 all images were reviewed by the ucsd imaging data evaluation and analysis center to ensure that the scan was centered , the signal strength was more than 15 db , that there were no artifacts , and that there were no rnfl segmentation algorithm errors . 
 the oct parameter used in the study was the global rnfl thickness corresponding to the average of all rnfl thickness measurements acquired from a 3.45-mm circle centered on the optic disc consisting of 1536 a - scan points . 
 visual fields were performed using sap sita 24 - 2 and evaluated by the ucsd visual field assessment center ( visfact ) . 
 visual fields were excluded if they had more than 33% fixation losses or more than 15% false - positive errors . 
 visual fields were excluded in the presence of the following artifacts : eyelid , rim artifacts , fatigue effects , inappropriate fixation , or evidence that the visual field results were caused by a disease other than glaucoma or inattention . 
 visual fields exhibiting a learning effect ( i.e. , initial tests showing consistent improvement on visual field indices ) were also excluded . the mean sensitivity ( ms ) in decibels of each eye was calculated by converting each threshold point into a linear scale ( apostilbs ) and then averaging all 52 threshold points . 
 global parameters of structural and functional damage were selected for evaluation in this study to provide a fair comparison between the different tests . as comparable localized parameters 
 would be difficult to establish for sap and oct , we restrained our analyses to global structural loss as measured by the oct rnfl average thickness and global functional loss as measured by sap ms . 
 we used ms instead of mean deviation ( md ) , as md is an age - corrected parameter , whereas global rnfl thickness and ms are not . a challenge in evaluating the relative ability of different tests in detecting glaucoma progression resides in equating their specificities , so that sensitivities can be meaningfully compared . 
 when evaluating progression based on slopes of change , the specificity can be set by the type i error . 
 a statistically significant slope at p < 0.05 implies that in only 5% of the cases 
 the slope would be declared statistically significantly different from zero in cases where no change truly existed . however 
 , such approach may be insufficient to establish specificity due to possible changes over time that can occur from aging , instead of glaucoma progression . therefore , to take into account possible age effects , we established confidence limits for the rates of change of oct average rnfl thickness and sap ms using longitudinal data available from healthy eyes . 
 slopes of change were obtained using linear mixed models and cutoffs corresponding to the fifth percentile of rates of change in the healthy group were obtained for each parameter . 
 it is expected that 95% of healthy eyes will have rates of change slower ( i.e. , more positive ) than the cutoff value . 
 after cutoffs were obtained from the healthy group , individual slopes were calculated for each glaucoma eye and for each test using ordinary least squares regression . 
 progression was then determined to have occurred if the slope was statistically significant at p < 0.05 and also faster ( i.e. , more negative ) than the fifth percentile cutoff calculated from the healthy group . 
 this approach equated the specificities of the tests by requiring statistically significant slopes at the same type i error and also by taking into account age - related changes by establishing confidence limits from the healthy group . for each glaucoma eye 
 , slopes of change for each test were calculated using progressively cumulative visits over time , with a minimum of five visits . 
 for example , for an eye that had a total of 10 visits during follow - up , slopes of change were calculated from data including only the first 5 visits , as well as from the data including the first 6 visits , first 7 visits , and so on , up to 10 visits . 
 therefore , for an eye with 10 visits during follow - up , six slopes were calculated for each test . 
 progression ( yes / no ) was then determined based on the criteria set on the previous paragraph for each one of these slopes . 
 after progression ( yes / no ) was determined at each visit , a logistic regression model was used to evaluate the relative odds of progressing by oct and sap in the population . for the logistic regression , eyes that progressed exclusively by oct 
 were assigned the value 1 , whereas eyes that progressed exclusively by sap were assigned the value 0 . as we were interested in evaluating the factors associated with progressing by one method versus the other , 
 eyes that progressed by both methods or by none were excluded . as each eye could have had multiple slopes for each test depending on the number of visits during follow - up , each eye had a combination of 1s and 0s during follow - up , depending on whether progression was detected solely by oct or by sap at each point during follow - up . a generalized estimating equation ( gee ) model was used to deal with correlated observations from each eye . 
 the gee logistic model evaluated the effect of the number of tests during follow - up and disease severity at baseline on the relative odds of progressing by oct versus sap . 
 baseline disease severity was classified according to sap md in early ( > 6 db ) , moderate ( < 6 and > 12 
 statistical analyses were performed using commercially available software stata , version 13 ( statacorp lp , college station , tx , usa ) . the  level ( type i error ) 
 the spectralis sd - oct ( software version 5.4.7.0 ) was used to measure peripapillary rnfl thickness in the present study . 
 all images were reviewed by the ucsd imaging data evaluation and analysis center to ensure that the scan was centered , the signal strength was more than 15 db , that there were no artifacts , and that there were no rnfl segmentation algorithm errors . 
 the oct parameter used in the study was the global rnfl thickness corresponding to the average of all rnfl thickness measurements acquired from a 3.45-mm circle centered on the optic disc consisting of 1536 a - scan points . 
 visual fields were performed using sap sita 24 - 2 and evaluated by the ucsd visual field assessment center ( visfact ) . 
 visual fields were excluded if they had more than 33% fixation losses or more than 15% false - positive errors . 
 visual fields were excluded in the presence of the following artifacts : eyelid , rim artifacts , fatigue effects , inappropriate fixation , or evidence that the visual field results were caused by a disease other than glaucoma or inattention . 
 visual fields exhibiting a learning effect ( i.e. , initial tests showing consistent improvement on visual field indices ) were also excluded . the mean sensitivity ( ms ) in decibels of each eye was calculated by converting each threshold point into a linear scale ( apostilbs ) and then averaging all 52 threshold points . 
 global parameters of structural and functional damage were selected for evaluation in this study to provide a fair comparison between the different tests . as comparable localized parameters would be difficult to establish for sap and oct , we restrained our analyses to global structural loss as measured by the oct rnfl average thickness and global functional loss as measured by sap ms . 
 we used ms instead of mean deviation ( md ) , as md is an age - corrected parameter , whereas global rnfl thickness and ms are not . a challenge in evaluating the relative ability of different tests in detecting glaucoma progression resides in equating their specificities , so that sensitivities can be meaningfully compared . 
 when evaluating progression based on slopes of change , the specificity can be set by the type i error . 
 a statistically significant slope at p < 0.05 implies that in only 5% of the cases the slope would be declared statistically significantly different from zero in cases where no change truly existed 
 . however , such approach may be insufficient to establish specificity due to possible changes over time that can occur from aging , instead of glaucoma progression . 
 therefore , to take into account possible age effects , we established confidence limits for the rates of change of oct average rnfl thickness and sap ms using longitudinal data available from healthy eyes . 
 slopes of change were obtained using linear mixed models and cutoffs corresponding to the fifth percentile of rates of change in the healthy group were obtained for each parameter . 
 it is expected that 95% of healthy eyes will have rates of change slower ( i.e. , more positive ) than the cutoff value . 
 after cutoffs were obtained from the healthy group , individual slopes were calculated for each glaucoma eye and for each test using ordinary least squares regression . 
 progression was then determined to have occurred if the slope was statistically significant at p < 0.05 and also faster ( i.e. , more negative ) than the fifth percentile cutoff calculated from the healthy group . this approach equated the specificities of the tests by requiring statistically significant slopes at the same type i error and also by taking into account age - related changes by establishing confidence limits from the healthy group . for each glaucoma eye 
 , slopes of change for each test were calculated using progressively cumulative visits over time , with a minimum of five visits . 
 for example , for an eye that had a total of 10 visits during follow - up , slopes of change were calculated from data including only the first 5 visits , as well as from the data including the first 6 visits , first 7 visits , and so on , up to 10 visits . 
 therefore , for an eye with 10 visits during follow - up , six slopes were calculated for each test . 
 progression ( yes / no ) was then determined based on the criteria set on the previous paragraph for each one of these slopes . 
 after progression ( yes / no ) was determined at each visit , a logistic regression model was used to evaluate the relative odds of progressing by oct and sap in the population . for the logistic regression , eyes that progressed exclusively by oct were assigned the value 1 , whereas eyes that progressed exclusively by sap were assigned the value 0 . as we were interested in evaluating the factors associated with progressing by one method versus the other , 
 could have had multiple slopes for each test depending on the number of visits during follow - up , each eye had a combination of 1s and 0s during follow - up , depending on whether progression was detected solely by oct or by sap at each point during follow - up . a generalized estimating equation ( gee ) model was used to deal with correlated observations from each eye . 
 the gee logistic model evaluated the effect of the number of tests during follow - up and disease severity at baseline on the relative odds of progressing by oct versus sap . 
 baseline disease severity was classified according to sap md in early ( > 6 db ) , moderate ( < 6 and > 12 
 db ) , and advanced ( < 12 db ) . statistical analyses were performed using commercially available software stata , version 13 ( statacorp lp , college station , tx , usa ) . the  level ( type i error ) 
 the study included 462 eyes of 305 glaucoma patients and 62 eyes of 49 healthy controls . 
 table 1 shows the baseline clinical and demographic characteristics of the subjects included in the study . 
 glaucoma patients had a median of 8 visits ( range , 518 ) during an average follow - up of 3.6  0.9 years . 
 for healthy subjects , the median number of visits was 7 ( range , 510 ) , over an average follow - up period of 3.8  0.9 years . 
 mean age at baseline was 70.0  8.2 years in the glaucoma group and 69.1  8.5 years in the healthy group ( p = 0.470 ) . 
 average sap md was 4.8  5.9 and 0.0  1.2 db , and global rnfl thickness at baseline was 78.4  17.6 and 94.9  11.0 m , in glaucoma and healthy subjects , respectively . 
 baseline clinical and demographic characteristics of healthy subjects and glaucoma patients included in the study as the number of visits for glaucoma eyes ranged from 5 to 18 visits , the number of slopes ranged from 1 to 14 for each eye . 
 there were in total 1910 slopes for all the glaucomatous eyes included in the study . 
 the fifth percentile cutoff values for rates of change estimated from the healthy group were 0.35 db / y for sap ms and 1.48 m / y for global rnfl thickness . 
 progression was determined for each eye and each test if the slope was faster than these cutoff values and also if the slope was statistically significant at p < 0.05 . from the 1910 slopes , 
 progression was detected solely by oct in 363 slopes ( 19.0% ) , solely by sap in 183 slopes ( 9.5% ) , and by both oct and sap in 80 slopes ( 4.1% ) . 
 progression was not detected in 1284 slopes ( 67.2% ) . as we were interested in factors explaining the relative odds of progressing by one test versus the other 
 , subsequent analyses were conducted with the 546 slopes that showed progression exclusively by oct or exclusively by sap . 
 table 2 shows results of progression by oct versus sap according to the number of visits and baseline disease severity . 
 the raw data suggested that oct was able to detect more eyes as progressing in comparison to sap , except for eyes with moderate and severe disease and when a large number of tests were available ( fig . 
 total number of visits with progression detected by sap and sd - oct , according to baseline disease severity and the number of visits during follow - up estimated probabilities of detecting progression by sd - oct versus sap , according to different levels of disease severity at baseline and number of tests acquired during follow - up . 
 this was confirmed by the results of the gee logistic model , as shown in table 3 . 
 each 1 db higher md was associated with a 5% increase in the odds of detecting progression by oct versus sap ( odds ratio [ or ] = 1.05 per 1 db ; 95% confidence interval [ ci ] : 1.011.09 ; p = 0.005 ) . 
 that is , eyes with less severe disease at baseline had higher chance of being detected as progressing by oct but not by sap , whereas an increase in disease severity at baseline increased the chance that the eye would be detected as progressing by sap but not oct . a higher number of visits during follow - up was associated with a decrease in the relative odds of progressing by oct versus sap ( or = 0.86 per visit ; 95% ci : 0.810.93 ; p 
 although an increase in the number of visits would be expected to increase the chance of detecting progression by both oct and sap , it is important to keep in mind that the model evaluated the odds of progressing by one test versus the other . 
 the results show that for eyes with few visits during follow - up , oct had a higher chance of detecting progression compared with sap . 
 importantly , there was also a significant interaction between baseline disease severity and number of visits in explaining the relative odds of progressing by oct versus sap . for eyes with early disease and relatively small number of visits , 
 the odds of detecting progression by oct were in general much larger for oct compared with sap ( fig . 
 conversely , for eyes with advanced disease and relatively large number of visits available during follow - up , the odds of detecting progression by sap were greater than by oct . 
 figures 2 and 3 show cases of eyes included in the study illustrating differences in the ability to detect progression by oct and sap . 
 logistic gee model evaluating factors explaining the relative odds of progressing by oct versus sap example of an eye of a glaucoma patient with glaucomatous optic neuropathy but normal visual field at baseline ( mild disease ) . 
 significant progression was seen on sd - oct rnfl thickness measurements over time , with a rate of change of 2.28 m / y ( p = 0.001 ) . 
 progression was not seen on sap mean sensitivity measurements over time , with a slope of 0.25 db / y ( p = 0.188 ) . 
 example of an eye of a glaucoma patient with moderate disease at baseline ( baseline md deviation of 10.96 db ) . 
 significant progression was seen on sap with a slope of change in mean sensitivity of 0.51 db / y ( p = 0.009 ) . however , no significant change was seen on sd - oct measurements , with a slope of change of 0.02 m / y ( p = 0.961 ) . 
 the present study demonstrated that the relative odds of identifying progression by structural or functional tests varies according to the number of tests performed during the follow - up and also according to the disease severity at baseline . 
 these findings may have significant implications for the evaluation of glaucoma progression in clinical practice by allowing clinicians to tailor the frequency of sap and oct tests during follow - up , taking into account the relative odds of detecting progression at different levels of disease severity . 
 several studies have previously shown substantial disagreement when evaluating detection of glaucoma progression by structural and functional tests . 
 progression was detected simultaneously by sap and sd - oct in only 80 ( 13% ) of them . in the vast majority of cases , 
 progression was either shown by sd - oct or by sap , but not by both . 
 therefore , we built a model evaluating the relative odds of progression by sd - oct versus sap in an attempt to understand factors that could explain why some eyes are detected as changing by one test but not by the other . 
 the model showed that in eyes with less severe disease at baseline the odds of detecting progression by sd - oct were generally higher than the odds of detecting change by sap . 
 each 1 db higher baseline md was associated with a 5% increase in the odds of progression being detected by sd - oct versus sap . 
 this can be clearly seen on figure 1 . when slopes were calculated from eight visits , the probability of detecting progression by sd - oct when the baseline md was 1 db , for example , was 70% compared to only 30% for sap . 
 equal probabilities ( i.e. , 50% each ) for detecting progression for sap and sd - oct would occur for a baseline md of 10 db . for eyes with baseline 
 md worse than 10 db , the probability of detecting progression by sap would be larger than by sd - oct considering slopes calculated from eight visits . 
 for example , for a baseline md of 20 db , the estimated probability of detecting progression by sap was 70% versus only 30% for sd - oct . 
 these results are in agreement with previous suggestions in the literature that structural assessment by sd - oct would perform better for detection of disease progression in early stages of damage , whereas sap would perform better for more advanced cases . however , to the best of our knowledge , no previous study has provided a quantification of the impact of disease severity on the relative odds of detecting change by these two methods . 
 the relationship between ability to detect change and disease severity may be explained by a number of factors . 
 previous studies investigating the structure and function relationship in glaucoma have shown that when measurements are expressed in their original scales ( i.e. , db for sap and m for rnfl ) , a nonlinear relationship is seen . from this nonlinear relationship 
 , it can be extracted that for eyes at relatively early stages of damage , substantial change in rnfl measurements might be seen when only relatively small changes in visual function are seen in decibels . 
 in contrast , for eyes at advanced stages of disease , even relatively small neural losses will produce large changes in the decibel scale . 
 it is important to note that the nonlinear relationship between structure and function seems to be mostly the result of the logarithmic scaling of visual field data , as transformation of functional data to a linear scale ( or structural data to a decibel scale ) seems to produce a closely linear relationship between functional and structural measurements . 
 however , such post hoc transformations will not influence the ability of a test to detect change as long as the original measurements are still acquired in a decibel scale ( i.e. , the staircase procedures for threshold acquisition still acquired on decibel steps ) . 
 another factor influencing the relative ability of structural and functional tests in detecting progression is the presence of floor effects . for sd - oct average rnfl thickness measurements 
 , a floor effect seems to occur when measurements are close to 50 m , although there are variations according to specific instruments . 
 when the floor is reached , even further neural losses will go relatively undetected by rnfl thickness measurements . 
 the presence of such floor has been shown by several previous investigations and seems to be related to the presence of nonneural or glial tissue , as well as to the dynamic range of the instrument . 
 this would seem to contradict our findings of a relatively superior ability of sap in detecting progression as disease severity gets worse . 
 however , it is important to note that such relationship between disease severity and sap variability is valid for measurements expressed in the decibel scale . 
 even though variability in decibels increases with worsening visual field damage , due to the logarithmic scaling , a 1-db change in later stages of the disease corresponds to a much smaller change in terms of neural loss or structural damage than a 1-db change in early disease . 
 therefore , even relatively small losses of neural tissue in later stages of the disease may produce relatively large changes in visual sensitivity as measured in decibels , overcoming at least in part the limitation of increased variability and making detection of change in decibels relatively easier . 
 as expected , the number of tests ( visits ) acquired during follow - up also significantly influenced the ability to detect change . 
 it is expected that a greater number of visits will make detection of change easier for both structural and functional tests . 
 however , we also found a significant interaction between baseline disease severity and number of tests in explaining the relative odds of detecting change by sd - oct versus sap . 
 this is illustrated by figure 1 and also by the analysis of the raw data presented in table 2 . 
 when only a small number of tests were available during follow - up ( less than eight ) , detection of progression was more common for sd - oct than sap for all stages of the disease , even in eyes with moderate and advanced damage . 
 conversely , when a large number of tests were available ( > 12 tests ) , the odds of detecting change were higher for sap compared with sd - oct for eyes with moderate and advanced disease at baseline . 
 our findings may be important in providing clinicians with information that can be used to tailor the frequency and type of test according to the stage of disease 
 . however , such test customization strategy might be difficult to implement in clinical practice , as there are no clear cutoffs of when to perform one test versus the other . 
 additionally , even though under certain circumstances the relative odds of detecting change may be higher for test a compared with test b , this does not necessarily imply that all cases of progression will be detected by test a. therefore , algorithms that provide a combination of structural and functional data allowing effective monitoring throughout the disease continuum might offer a better solution to this issue . 
 this was done to allow equating the specificities of the tests , an essential requirement for comparing their abilities to detect change . 
 as there is no current gold standard for detecting change in glaucoma , our comparison was based on the relative proportion of glaucoma eyes that were detected as having statistically significant slopes of progression considering rates of change that would have only a 5% chance of occurring in a healthy population . 
 this was done to provide not only a statistically but also a clinically relevant criteria . 
 future studies should , however , investigate the relative ability to detect change by using other methods such as event - based detection of localized change or other techniques . as another limitation of our study 
 although this assumption is likely to correspond to how these tests are currently used in clinical practice for relatively short - term management decisions , it is likely that the true course of the disease is not linear in the long run . 
 recent investigations have also suggested nonlinear models for investigation of rates of visual field change that might provide an improvement over current simple linear models . 
 our results show that the ability to detect glaucoma progression by sap and sd - oct is significantly influenced by the stage of disease . 
 our results may provide useful information for guiding clinicians on the relative utility of these tests for detecting change throughout the disease continuum . 
 furthermore , they may also serve to guide the construction of indices or strategies combining structural and functional information for detection of change over time .","purposethe purpose of this study was to evaluate the effect of disease severity and number of tests acquired during follow - up on the relative odds of identifying progression by structural or functional tests in glaucoma.methodsthis was an observational cohort study involving 462 eyes of 305 patients with glaucoma and 62 eyes of 49 healthy subjects . 
 glaucoma patients and healthy subjects were followed for an average of 3.6  0.9 and 3.8  0.9 years , with a median ( interquantile range ) of 8 ( 69 ) and 7 ( 68 ) visits , respectively . at each visit 
 , subjects underwent visual field assessment with standard automated perimetry ( sap ) and retinal nerve fiber layer ( rnfl ) evaluation by spectral - domain optical coherence tomography ( sd - oct ) . 
 slopes of change in sap mean sensitivity and oct rnfl thickness over time were estimated by linear regression using progressively cumulative visits over time . 
 cutoff values for age - related expected rates of change for each test were obtained from the healthy group . 
 progression by sd - oct and/or sap was determined if the slope of change was statistically significant and also lower ( faster ) than the fifth percentile cutoff calculated from the healthy group . 
 a generalized estimating equation logistic regression model was used to evaluate the relative odds of progressing by oct versus sap in glaucoma eyes.resultseyes with less severe disease at baseline had a higher chance of being detected as progressing by sd - oct but not by sap , whereas an increase in disease severity at baseline increased the chance that the eye would be detected as progressing by sap but not sd - oct . 
 each 1 db higher md was associated with a 5% increase in the odds of detecting progression by sd - oct versus sap ( odds ratio = 1.05 per 1 db ; 95% confidence interval : 1.011.09 ; p = 0.005).conclusionsthe ability to detect glaucoma progression by sap versus sd - oct is significantly influenced by the stage of disease . 
 our results may provide useful information for guiding clinicians on the relative utility of these tests for detecting change throughout the disease continuum .",pubmed
"as has been the case in most other industries , the promise of new technologies and innovations has been cited as the way to address the healthcare industry s current challenges ; in particular , to circumvent the so - called  iron triangle 
 , simultaneously improving access , cost , and quality of care . at the same time , research based institutions across the globe are experiencing significant stress from more competition for less funding . from fy 2003 to 2015 , for example , the nih lost 22% of its capacity to fund research due to budget cuts , sequestration , and inflationary losses . 
 this resulted in fewer grants and discoveries , along with talented scientists and investigators leaving research and/or the us . 
 as such , it is more important than ever to increase the efficiency and effectiveness with which investments in fundamental research and development translate into products , services , and procedures that improve the health and wellbeing of people around the world . 
 examples abound of the power of technology and innovation to enable disruptive step changes in performance while simultaneously slashing costs . 
 moore s law in semiconductors , which projects a doubling of cpu capacity every 18 to 24 months , typifies the exponential power of technology . 
 innovator and futurist ray kurzweil extended moore s law to show that when a specific technology platform approaches some kind of physical limit , a new one emerges to extend the exponential growth , bypassing perceived barriers . 
 eroom s law typifies the experience in the pharma industry : rather than show an improvement , the trend for new drug approvals by the us food and drug administration per inflation - adjusted us dollars spent on r&d is a decrease of 50% over 9 years  a negative rather than positive exponential growth . while no generally accepted 
 law  exists for healthcare as a whole , labor productivity in healthcare , for example , is in decline , with technology not improving productivity as it has in other industries . 
 successful innovation in healthcare requires navigating a long and challenging journey between an unmet need or discovery generated from basic research to a viable commercial product or service . 
 while there are many potential stumbling points along the way , the so - called valley of death that exists between academic research and commercialization of a new product is often cited as a key obstacle for health innovations . 
 the valley of death has been shown to occur most frequently in the presence of non - economic investments , such as government expenditures on early stage , basic research without attention to the likelihood of commercially motivated investment at later stages if successful . 
 technologies that emerge from academia which do not have a promising commercialization pathway and clearly articulated value proposition are simply not considered by financially - motivated investors and , as a result , languish in the lab . 
 financial investors are not typically impressed with the great technical results that investigators focus on developing and are rewarded for publishing  they expect them . 
 they understand the reality is that the vast majority of new ventures fail not because of flawed science (  technical risk  ) , but because the market does not perceive a need or will not pay for the product or service (  market risk  ) . 
 many academic investigators and research teams are simply not prepared or motivated to think beyond their work in the lab to anticipate or address commercial issues such as market risk . 
 some are even discouraged from considering commercial issues while conducting research due to lingering concerns about aligning too closely with industry . 
 a commonly held misconception by many investigators is that once they are successful publishing results in a high impact journal , the commercial value will be so self - evident that companies will flock to get commercial rights . as a result , many leave commercialization related issues to be addressed only after a technology is shown to work ( i.e. proof - of - concept demonstrated ) , expecting them to be  transferred  to a company that will bring a product or service into practice . as a result , academic innovators pursuing translational research still often approach it with the mindset of doing science . 
 they do not develop an appreciation for which of the many paths available to them is most likely to lead to patient impact and hence financial return for investors . today 
 s challenging funding situation , with historically low pay - lines , creates even more pressure for investigators , particularly new ones , to focus on novelty and quickly move on to new areas once publishable results have been generated . 
 this often leaves institutional technology transfer offices ( ttos ) with the very challenging task of seeking licensees to technologies that have been shown to work , but with no clear commercial value and without much , if any , technical support . 
 simultaneously , financially driven investors are becoming less willing to take risks , particularly risks that they can not control or may only pay - off in the longer term . 
 they are looking to invest in opportunities for which they are well positioned to manage the risks , such as implementation related issues . 
 they do not want to take risks in areas for which they have no control , such as market acceptance , reimbursement and regulatory acceptance . as a result of these increasing pressures from both sides , 
 the only teams surviving the commercialization journey through today s more treacherous valley of death are ones with the most robust preparation , demonstrably de - risking both the technology and the market to succeed in attracting investors . 
 universities and academic medical centers are facing increasing competition to not only win scarcer research funding , but also to attract the best investigators and students . to compete successfully at both 
 , they need to do more in preparing and supporting those investigators interested in translating research into practice : excellence in science is still required , but is no longer sufficient . 
 investigators that wish to expand beyond basic research need new skills and resources that can help them focus on creating and developing technologies that have a better chance of surviving the commercialization journey long enough to engage investors and get products to the market . 
 one approach being taken at some institutions is to add a business development function to an institution s tto , expanding its role to include 
 while this approach can be very effective in advancing some projects , it does not address the fundamental issue of improving the commercial readiness or attractiveness of technologies emerging from labs . 
 in addition , balancing the roles of guardian and promoter of ip in a single cost center with increasingly constrained budgets can add an obstacle to commercialization which discourages all but the few that are selected for support . another approach taken by institutions , including stanford biodesign and the coulter foundation as well as by funders , including the nih and the nsf , are creating programs to assist academic investigators advance technologies toward commercialization while they are conducting their research . 
 they are providing educational opportunities to help investigators understand , anticipate and address commercialization challenges . 
 efforts range from didactic educational programs to accelerators which provide hands - on skills development and bridge / product development funding as well as support in the form of mentoring and  pitch 
 competitions , the goal being to help teams achieve a commercial exit : the point at which financially motivated investors such as angels , vcs and strategic players invest . 
 figure 1 schematically represents a range of these approaches taken by organizations to improve the effectiveness of the translational research they support . 
 figure 1.the gap filled by hcps in the portfolio approaches used by academic programs to improve the impact of translational research they support . the gap filled by hcps in the portfolio 
 the horizontal axis represents the typical focus of the effort . at one end of the spectrum 
 the focus is on building the skills of individuals , such as university based degree programs . 
 the other end of the spectrum is focused on helping advance projects , such as accelerator programs . 
 the vertical axis represents the typical intensity of the efforts . at one end of the spectrum 
 are activities that take a few hours and cost very little to deliver , such as webinars . at the other end 
 are programs that take years and cost in the hundreds of thousands of dollars , such as university based degree programs . 
 an emerging option for academic investigators is the development of healthcare commercialization programs ( hcps ) . 
 these programs attempt to strike a balance by cost - effectively advancing a project into commercialization , while simultaneously building the skills of team members so that they can learn while contributing to an entrepreneurial activity . 
 two recent examples are a modified version of the i - corps program , adapted in collaboration with the nih   and another being the coulter foundation c3i program . in 2013 , 
 the national heart , lung and blood institute ( nhlbi ) took a fresh approach to improving the effectiveness of the translation of academic based research into implementation . 
 it established three centers to accelerate innovations from the bench to the clinic : the nih centers for accelerated innovations ( ncais ) . 
 the ncais are to help advance projects into patient care by providing product development funds and project facilitation , while also providing skills development to empower academic investigators with a greater understanding of the commercial path of their technologies . 
 the ncais were provided additional funds by the nsf in the spring and summer of 2015 to explore how the i - corps program could help advance ncai supported projects into practice . 
 the university of california ncai   applied its pre - existing i - corps program and approach for teams from across the university of california network , with a focus on teams from the san francisco area . 
 the boston ncai , b - bic   ( through its collaborator the consortia to improve medicine with innovation and technology : cimit ) and cleveland s ncai ( the nih center for accelerated innovation at cleveland clinic ; ncai - cc ) took a different approach . 
 they choose to blend several methodologies into a program intended to address the unique challenges facing healthcare innovations . 
 they each built on the fundamentals of the i - corps program while integrating commercialization experiences gained from introducing therapeutic and healthtech products . 
 cimit built on its 18 + year history and experiences facilitating more than 600 projects with its unique approach to facilitating teams as well as its work with the coulter foundation and academic based programs at its member institutions . 
 cleveland clinic incorporated specific workshops focused on therapeutics , including regulatory and intellectual property with support of the nhlbi staff . a brief overview of each and some results are outlined below : cimit s focus is on healthtech innovations , the overlap of medicine and engineering , including devices , diagnostics , e / digital health and big data . 
 its initial pilot program had eight weekly sessions and was comprised of a cohort of 9 teams . 
 each team had a clinical lead , technical lead and entrepreneurial lead , which were supported by an executive from the cimit accelerator team . 
 the overall results were very promising , with four teams successfully attracting commercial funding within 6 months of completing the course , receiving more than $ 11 m in commercial investment . 
 three are now pursuing licensing opportunities instead of trying to create a newco , and two projects are being rethought after learning of the lack of a market need . 
 programmatically , these  quick kills  are viewed as being a success along with the teams that are able to advance to commercialization as it saves time and resources so that teams can work on more impactful problems . the teams all reported significant progress in a compressed period of time , and similar results 
 an example of a self - reported team assessment is:our experience at the cimit program and all the support from the faculty has been instrumental to our progress . 
 less than a year ago , i was an md - phd student with no business experience and no intentions of commercializing technology . 
 in fact , before the course , we did nt even know who our customer was or what our product was , let alone our business model . 
 cimit taught us to make hypotheses about our business and test them rigorously by speaking to all the stakeholders in the ecosystem . 
 not only did we convince ourselves of who our customer was and what we should be building , all the data we gathered also convinced investors .   our experience at the cimit program and all the support from the faculty has been instrumental to our progress . 
 less than a year ago , i was an md - phd student with no business experience and no intentions of commercializing technology . 
 in fact , before the course , we did nt even know who our customer was or what our product was , let alone our business model . 
 cimit taught us to make hypotheses about our business and test them rigorously by speaking to all the stakeholders in the ecosystem . 
 not only did we convince ourselves of who our customer was and what we should be building , all the data we gathered also convinced investors .  the ncai - cc hcp program was seven weekly sessions , specifically designed for therapeutics . 
 it provided specialization workshops on regulatory and intellectual property consideration with nhlbi staff and focused on the development of a target product profile , an accepted tool for pharmaceutical development projects . 
 the cohort included five teams , each with a principal investigator , entrepreneurial lead and a faculty member , one of the ncai product development directors . 
 the teams all began at different baselines but advanced their understanding of their commercialization process . 
 they defined the types and quality of data that would be required to attract a pharmaceutical company s interest in commercial development . 
 one team identified a path that includes formation of a new company and application for sbir support . 
 examples of the self - reported assessments include:taught to think differently about our technology  with a business / practical perspective never learned in graduate school or as a postdochelped prepare me for industry 
 i became better suited to write business reports such as a strategic plan focused for upper managementit was not until we talked to clinicians , patients and regulatory experts that we were able to capture a holistic view of how our technology fit in the therapeutic ecosystem . 
 taught to think differently about our technology  with a business / practical perspective never learned in graduate school or as a postdoc 
 i became better suited to write business reports such as a strategic plan focused for upper management   it was not until we talked to clinicians , patients and regulatory experts that we were able to capture a holistic view of how our technology fit in the therapeutic ecosystem . 
 the cimit and ncai - cc faculty coordinated activities in developing , delivering and critiquing their programs . despite the differences between the programs due to the different clinical foci , the faculty developed consensus 
 while there is strong agreement that there is no single  right way  to run an hcp , the following points are intended to help guide others wishing to develop an hcp . 
 the lessons reinforce the applicability of some of the fundamentals of the i - corps program as well as ways that an hcp should differ from i - corps to address the unique needs and challenges of healthcare innovators and innovations . 
 the fundamental premise of the i - corps program was found to apply very well to healthcare : get team members out of the lab to talk with stakeholders , customers in particular , and apply the scientific approach of developing , testing and validating hypotheses about the business . 
 there are many facets of the i - corps program that were found to apply ; some key examples include : 
 a weekly goal of around 10 exploratory interviews with a range of stakeholders needs to be stressed , and may be the most challenging as well as valuable thing for participants to learn . 
 most successful investigators are good advocates and teachers , re - learning to listen to feedback is often a challenge , particularly when based on partial information.the flipped - classroom structure with off - line assignments and weekly two - hour interactive session focused on practical applications of the materials and on teams presenting their work for robust feedback in a mix of physical and web - based meetings is time - efficient and allows participants to continue with their  day jobs.programs should run at least eight to ten weeks to allow time for a sufficient number of interviews along with the potential of at least one  pivot  - a change in direction based on validated feedback from the market . a weekly goal of around 10 exploratory interviews with a range of stakeholders 
 needs to be stressed , and may be the most challenging as well as valuable thing for participants to learn . 
 most successful investigators are good advocates and teachers , re - learning to listen to feedback is often a challenge , particularly when based on partial information . 
 the flipped - classroom structure with off - line assignments and weekly two - hour interactive session focused on practical applications of the materials and on teams presenting their work for robust feedback in a mix of physical and web - based meetings is time - efficient and allows participants to continue with their  day jobs  . 
 programs should run at least eight to ten weeks to allow time for a sufficient number of interviews along with the potential of at least one  pivot  - a change in direction based on validated feedback from the market . 
 it is not surprising that an effective hcp would differ from the i - corps program since it was developed with a different focus . 
 i - corps was designed to support principal investigator led academic teams with sbir awards in a broad range of industries . while many commercialization challenges transcend industries , there are some unique challenges facing healthcare innovators that require simultaneous attention in an hcp . 
 some key examples include : 
 1.buying dynamics : the user of the products / services ( user ) and those responsible for making a buying decision ( economic buyer ) in healthcare are usually not the same people , can be very hard to identify and may change from one institution to the next . in addition , users and economic buyers frequently have competing or misaligned objectives . 
 this is in contrast , for example , to selling to consumers who make the purchasing decision and use the products.2.reimbursement complexity : reimbursement for products / services in healthcare is a complex and highly regulated process . 
 this is in contrast to most industries in which the price a company can charge is established by a free market.3.regulatory pathway : while regulations exist in every industry , the impact that early and what may appear to be subtle decisions can have a major impact on the level of testing and validation that a medical product or service must undergo before being approved for sale or use 
 . the decision can mean the difference of years before introduction and 10 s of millions of dollars of investment and therefore commercial viability . 
 the option to start with a minimally viable product ( mvp ) and iterate with customer experiences to improve it is not the same as it is in industries with products that have less potential for harm , and are therefore less regulated.4.culture : healthcare professionals , be they doctors , nurses , therapists , technicians , etc . must invest many years and a great deal of money into education in order to practice . 
 the option for them to leave a position to focus on a start - up comes at a very high professional and personal risk and therefore is often not practical . 
 however , their constant engagement in a development effort and focus on addressing important unmet clinical needs is critical to success . 
 their engagement must be managed within their other time constraints , which are generally only increasing , and done in a way consistent with their expectations and culture.5.funding requirements : while there is a wide range of funding required to develop a new product across industries , healthcare innovations are typically at the higher cost end . 
 while the low - range may be $ 100k for an app and a few $ million for a consumer device , it now costs about $ 2 billion to bring a new drug to the market . in 2010 
 , medical devices typically cost between $ 31 million for those that require a 510(k ) and $ 94 million for higher - risk devices requiring the pma regulatory pathway ( excluding reimbursement and sales / marketing activities ) . as such , more resources , both quantity and magnitude 
 investors with smaller dollars are therefore less inclined to invest due to the potentially negative impact that later stage investments can have on the value of their investment . 
 buying dynamics : the user of the products / services ( user ) and those responsible for making a buying decision ( economic buyer ) in healthcare are usually not the same people , can be very hard to identify and may change from one institution to the next . 
 this is in contrast , for example , to selling to consumers who make the purchasing decision and use the products . 
 reimbursement complexity : reimbursement for products / services in healthcare is a complex and highly regulated process . 
 this is in contrast to most industries in which the price a company can charge is established by a free market . 
 regulatory pathway : while regulations exist in every industry , the impact that early and what may appear to be subtle decisions can have a major impact on the level of testing and validation that a medical product or service must undergo before being approved for sale or use . 
 the decision can mean the difference of years before introduction and 10 s of millions of dollars of investment and therefore commercial viability . 
 the option to start with a minimally viable product ( mvp ) and iterate with customer experiences to improve it is not the same as it is in industries with products that have less potential for harm , and are therefore less regulated . 
 culture : healthcare professionals , be they doctors , nurses , therapists , technicians , etc . must invest many years and a great deal of money into education in order to practice . 
 the option for them to leave a position to focus on a start - up comes at a very high professional and personal risk and therefore is often not practical . 
 however , their constant engagement in a development effort and focus on addressing important unmet clinical needs is critical to success . 
 their engagement must be managed within their other time constraints , which are generally only increasing , and done in a way consistent with their expectations and culture . 
 funding requirements : while there is a wide range of funding required to develop a new product across industries , healthcare innovations are typically at the higher cost end . 
 while the low - range may be $ 100k for an app and a few $ million for a consumer device , it now costs about $ 2 billion to bring a new drug to the market . in 2010 
 , medical devices typically cost between $ 31 million for those that require a 510(k ) and $ 94 million for higher - risk devices requiring the pma regulatory pathway ( excluding reimbursement and sales / marketing activities ) . as such , more resources , both quantity and magnitude 
 investors with smaller dollars are therefore less inclined to invest due to the potentially negative impact that later stage investments can have on the value of their investment . 
 below are some key distinguishing features and attributes the authors suggest to build on the i - corps fundamentals to address these healthcare specific issues in constructing and conducting an effective hcp : 
 1.team composition : teams need to include the perspectives and proactive engagement from the clinical as well as the technical and business environments . 
 while all team members need to share work and responsibilities , the person with the business / entrepreneurial focus needs to take the lead in coordinating the team s efforts as well as organize the outreach and implementation of stakeholder interviews . 
 clinicians are critical team members , and being time efficient is crucial to enable their participation . 
 this approach differs from i - corp that has fixed team member roles and only includes a technical and entrepreneurial lead along with an industry mentor supplied by the team . 
 successful implementation requires that each team member understands and is prepared to put in the time required , a total of about 40 hours / team / week . 
 senior faculty members should be discouraged from taking a team member slot if they intend to just oversee the work.2.executive faculty : given the inter - related complexities associated with healthcare innovations and need to balance multiple trade - off s simultaneously to effectively de - risk projects , the authors urge that the hcp faculty presenting materials be healthcare industry experts  and use healthcare examples . in particular , rather than having team - supplied mentors , the hcp should be responsible for supplying qualified , experienced industry veterans to provide teams with consistent feedback and guidance . to be cost effective , the same individuals can deliver most of the program s customized content as well as support teams . 
 it is important that in addition to the work they each do with a specific team that they work as group in providing regular synthesized feedback to teams . while increasing the cost , having experienced executives with a diversity of backgrounds ( technical , regulatory , operational , marketing , manufacturing , etc . ) 
 working together helps teams understand trade - offs and make better decisions to efficiently and cost effectively de - risk projects . 
 a key to successful implementation is finding and recruiting these individuals , which is a challenge . 
 the authors suggest tapping into existing entrepreneurial networks  locally or nationally  to get access to people with the diversity of skills needed.3.content : the content used in hcps should be designed to help teams address the specific challenges facing healthcare innovations . 
 it should cover the complex buying dynamics , reimbursement complexities , practice workflow implications , regulatory consequences and constraints in addition to traditional entrepreneurial topics . 
 the author s experience is that there is enough difference between pharma / biotech and healthtech , both in terms of content and the expertise of faculty and industry experts , to justify separate programs . 
 for example , the coulter foundation and stanford biodesign have well developed , publically available healthtech content for reference , so there is little need to create new content . 
 we suggest curating a repository using the participants feedback that can be accessed by team members as a reference library , not only during but also after the program.4.style : like i - corps , teams need frank , objective feedback . but unlike the rough treatment that is part of the i - corps pedagogy , faculty must also develop strong rapport with the team members . 
 they should be seen as being part of the team and not as outside assessors . 
 keeping objective metrics ( like the number of interviews ) visible keeps pressure on teams and engenders some inter - team competition . 
 team - team interactions are encouraged and generate some of the key learning opportunities by seeing the challenges and mistakes of other teams , as well as successful strategies they employ to overcome them.5.ecosystem leverage : in addition to the above ways an hcp should be conducted , the context in which an hcp is used can help address the funding challenges . 
 the primary goal of the program is for each team to establish if they can deliver against a validated value proposition with a convincing  pitch  that articulates the narrative of a viable commercialization pathway  or conclude that a  quick kill 
 it is critical that teams start their journey into the valley of death with a convincing pitch , but even better if they have some resources as they start their journey . 
 so , rather than conduct an hcp as a stand - alone activity at the end of a funding cycle ( such as the i - corps program after sbir funding ) , the authors strongly encourage hcps to be conducted as an integral part of an ecosystem or program , like the ncai program , while its other resources are available to assist teams advance . 
 this approach can significantly improve funding efficiency by applying the benefits of an hcp early , for example as a 
 this helps focus available resources on the teams with the highest likelihood of translational success and focus teams on tasks that are most needed to advance projects to commercially relevant , as well as technically and clinically important milestones . 
 team composition : teams need to include the perspectives and proactive engagement from the clinical as well as the technical and business environments . 
 while all team members need to share work and responsibilities , the person with the business / entrepreneurial focus needs to take the lead in coordinating the team s efforts as well as organize the outreach and implementation of stakeholder interviews . 
 clinicians are critical team members , and being time efficient is crucial to enable their participation . 
 this approach differs from i - corp that has fixed team member roles and only includes a technical and entrepreneurial lead along with an industry mentor supplied by the team . 
 successful implementation requires that each team member understands and is prepared to put in the time required , a total of about 40 hours / team / week . 
 senior faculty members should be discouraged from taking a team member slot if they intend to just oversee the work . 
 executive faculty : given the inter - related complexities associated with healthcare innovations and need to balance multiple trade - off s simultaneously to effectively de - risk projects , the authors urge that the hcp faculty presenting materials be healthcare industry experts  and use healthcare examples . 
 in particular , rather than having team - supplied mentors , the hcp should be responsible for supplying qualified , experienced industry veterans to provide teams with consistent feedback and guidance . to be cost effective , the same individuals can deliver most of the program s customized content as well as support teams . 
 it is important that in addition to the work they each do with a specific team that they work as group in providing regular synthesized feedback to teams . while increasing the cost , having experienced executives with a diversity of backgrounds ( technical , regulatory , operational , marketing , manufacturing , etc . ) 
 working together helps teams understand trade - offs and make better decisions to efficiently and cost effectively de - risk projects . 
 a key to successful implementation is finding and recruiting these individuals , which is a challenge . 
 the authors suggest tapping into existing entrepreneurial networks  locally or nationally  to get access to people with the diversity of skills needed . 
 content : the content used in hcps should be designed to help teams address the specific challenges facing healthcare innovations . 
 it should cover the complex buying dynamics , reimbursement complexities , practice workflow implications , regulatory consequences and constraints in addition to traditional entrepreneurial topics . 
 the author s experience is that there is enough difference between pharma / biotech and healthtech , both in terms of content and the expertise of faculty and industry experts , to justify separate programs . 
 for example , the coulter foundation and stanford biodesign have well developed , publically available healthtech content for reference , so there is little need to create new content . 
 we suggest curating a repository using the participants feedback that can be accessed by team members as a reference library , not only during but also after the program . 
 unlike the rough treatment that is part of the i - corps pedagogy , faculty must also develop strong rapport with the team members . 
 they should be seen as being part of the team and not as outside assessors . 
 keeping objective metrics ( like the number of interviews ) visible keeps pressure on teams and engenders some inter - team competition . 
 team - team interactions are encouraged and generate some of the key learning opportunities by seeing the challenges and mistakes of other teams , as well as successful strategies they employ to overcome them . 
 ecosystem leverage : in addition to the above ways an hcp should be conducted , the context in which an hcp is used can help address the funding challenges . the primary goal of the program is for each team to establish if they can deliver against a validated value proposition with a convincing  pitch  that articulates the narrative of a viable commercialization pathway  or conclude that a  quick kill 
 it is critical that teams start their journey into the valley of death with a convincing pitch , but even better if they have some resources as they start their journey . 
 so , rather than conduct an hcp as a stand - alone activity at the end of a funding cycle ( such as the i - corps program after sbir funding ) , the authors strongly encourage hcps to be conducted as an integral part of an ecosystem or program , like the ncai program , while its other resources are available to assist teams advance . 
 this approach can significantly improve funding efficiency by applying the benefits of an hcp early , for example as a  phase 0  award . 
 this helps focus available resources on the teams with the highest likelihood of translational success and focus teams on tasks that are most needed to advance projects to commercially relevant , as well as technically and clinically important milestones . 
 hcps offer institutions and funders an additional option to improve the efficiency with which the healthcare projects they support reach practice and improve people s health . 
 it offers a cost effective way for them to increase the likelihood of selecting and advancing projects to commercialization and patient impact while also helping build innovation capacity . 
 they teach academic based investigators important skills in a learn - by - doing mode to enable them to contribute to entrepreneurial activities without attempting to turn them into entrepreneurs . while no single approach for hcps is being advocated , the lessons learned synthesized from a diversity of programs can act as a guide for those interested in establishing a hcp to maximize the impact of their available resources .","academic investigators are generating a plethora of insights and technologies that have the potential to significantly improve patient care . however , to address the imperative to improve the quality , cost and access to care with ever more constrained funding , the efficiency and the consistency with which they are translated into cost effective products and/or services need to improve . 
 healthcare commercialization programs ( hcps ) are described and proposed as an option that institutions can add to their portfolio to improve translational research . in helping teams 
 translate specific healthcare innovations into practice , hcps expand the skillset of investigators and enhance an institution s innovation capacity . 
 lessons learned are shared from configuring and delivering hcps , which build on the fundamentals of the national science foundation s innovation corps program , to address the unique challenges in supporting healthcare innovations and innovators .",pubmed
"bladder cancer is the second most common cancer in the urogenital tract . in about 85% of cases at the time of diagnosis of cancer 
 is limited only to the urinary bladder in the remaining 15% of the observed expansion beyond the urinary bladder . 
 the first confirmed carcinogenic factors were the aromatic amines : o - toluidine , 2-naftylamine , 4-aminobiphenyl , and many other chemicals contained in tobacco smoke . in recent years 
 currently there are about 1.1 billion smokers . in poland , there are about 9.5 million people smoking , including 43% of men and 22% of omen . 
 passive smoking is also becoming a widespread social phenomenon , mainly at home ( 47% men & 52% women ) and work ( 55% men & 27% women ) . in poland 
 , smoking is considered a shameful habit so patients asked about their smoking history do not readily admit to active smoking or they say they quit smoking just before evaluation . 
 most patients know that smoking is harmful since they are ashamed to admit to active smoking ( embarrassed for their ignorance ) 
 . they do not realize that it is possible to assess the reliability of the collected information about the history of smoking . 
 we do not consider smoking danger warnings placed on cigarette packs and media - advocated smoking cessation as helpful to the no - smoking cause . 
 the international agency for research on cancer predicts that by 2020 the number of deaths related to smoking will rise to 10 million per year , of which 70% will be in developing countries . at present approximately 20% of cancer deaths 
 it is , however , satisfying that recently administrative rulings managed to limit the number of public places where smoking is permitted . 
 research was carried out , which allegedly confirmed the positive results of this reduced smoking . 
 the aim of this study was to assess the reliability of a subjects smoking history , and assess compliance with the recommendations of smoking cessation on the basis of a questionnaire and the measurement of cotinine , a marker of exposure . 
 the study is a part of the long - term study conducted in our department funded by the national research committee , grant no . 
 the evaluation of patients treated in the department of general , oncologic and pediatric urology of collegium medicum nicolaus copernicus university in bydgoszcz in the period from june 2009 to december 2010 who had confirmed primary or recurrent bladder cancer by histopathological examination was undertaken . 
 every patient who agreed to participate in the study signed an informed consent form and completed a questionnaire on smoking history and tobacco smoke exposure . 
 it was asked whether the patient smokes , if so , how many cigarettes per a day and for how many years . 
 smokers were asked when they stopped smoking , how many cigarettes were smoked per day , and for how long . 
 a model questionnaire on the history of smoking : 
 1 . 
 non - smoker and non - exposed to the tobacco smokenever smokedstopped smoking  < 1 year ago2 - 5 years ago>5 years ago 
 2 . 
 smokersthe number of cigarettes smoked , packages ( 1 pack = 20 pieces)since i smoke ? 
 3 . 
 exposed to tobacco smoke  ' passive smokingplace of exposure to tobacco smokehomeworkanother placethe residence time in tobacco smoke ( hours)number of smokers 1 . 
 exposed to tobacco smoke  ' passive smoking each patient had their urine cotinine levels determined immediately after admission to the hospital to reduce the number of false - negative results arising from the discontinuation of smoking during hospitalization . 
 cotinine ( name of the synthetic ( s)-1- methyl-5-(3-pyridinyl)-2-pirolydyna ) is an objective indicator of exposure to tobacco smoke and is found in the urine , even several hours after smoking ; with a half - life of 20 - 40 hours . in the body , it can be detected at very low concentrations in the limit 0.57 nmol / l = 0.1 ng / ml ( 6 ) . 
 the analysis of cotinine , the main metabolite of nicotine , were determined by high performance liquid chromatography ( hplc / dad ) . 
 application to the markings cotinine hplc / dad allowed the side to ensure adequate sensitivity of determination ( ng / l ) also identify the compound , determined by analysis of mass spectra . downloaded from those covered in the study sample of urine ( about 50 ml ) for analysis 
 were stored at minus 20 c. cotinine extraction from urine was carried out in alkaline ( ph = 11.1 mol naoh ) . 
 after evaporation of organic solvent in a nitrogen atmosphere the dry residue was dissolved in methanol and analyzed by hplc / dad with the following conditions : liquid chromatograph f - my agilent technologies ( binary pump & autosampler ) column eclipse xdb c8 zorba ( 150x4 , 6 mm , 5 l ) at 25 c , injection volume 20 ml . 
 the mobile phase was methanol / ammonium buffer ph = 3.5 with a flow of 0.8 ml / min . 
 detection of cotinine level in urine was performed in 96 patients , of which 35 patients ( 36% ) obtained results indicating active smoking ( cotinine levels in the urine of > 200 ng / ml , values range from 257 to 7929 ng / ml ) and in 4 patients ( 4% ) had cotinine levels equivalent to  passive smoking 
 ( level between 0 - 200 ng / ml , range of values from 40 to 187 ng / ml ) while in the remaining 57 patients ( 59% ) had no cotinine in urine ( table 1 ) . 
 subjects groups on basis cotinine level we analyzed the questionnaire completed by patients and compared it with test results of cotinine in the urine . among the 96 patients,18 admitted to smoking ( at about 19% ) , 
 in fact , in 35 ( 36% ) patients were confirmed by cotinine levels indicating active smoking , in 4 patients , exposure to tobacco smoke at home . among smokers , 
 the average smoking time is approximately 33 years ( range from 20 to 45 years ) . 
 the number of cigarettes smoked was on average 15.6 pieces per day ( range of values from 15 to 20 pieces a day ) within 18 months of observation in 3 patients in the first test was confirmed in the urine cotinine ( values from 2709 to 4760 ng/ ml ) in a subsequent study found no cotinine in 2 patients , with a group of non - smokers in another examination confirmed that indicates the level of active smoking ( values from 839 to 1110 ng/ ml ) was not confirmed in the questionnaire . 
 it is widely know from numerous publications that bladder cancer and smoking are strongly linked ; however , no significant correlation between the type of tobacco smoking , nicotine content of the smoked cigarettes , and higher risk of bladder cancer was confirmed . 
 all cigarette smoking , both  strong  ( with a higher nicotine content ) and  weak  ( with reduced nicotine content ) , with or without a filter , has the same negative effect on our body without clinically important differences . 
 there are no further reports assessing how smoking affects the course of bladder cancer , the number of recurrences , and the progression of the disease . 
 no such analysis is probably due to the limited reliability of the smoking questionnaire and due to expensive and time - consuming methods of monitoring cotinine in urine in all patients , which can only be afforded by a few university centers . 
 the destructiveness of tobacco should always be presented to all smoking patients in an attempt to persuade the smokers to stop smoking . 
 this study found that not all patients admit to smoking , and not all follow the recommendations to quit smoking . in the present study patients did not know that the information contained in it would be verified objectively before filling in the questionnaire . 
 therefore , 18% of patients admitted to active smoking and 4% of patients were unaware of the adverse effects of tobacco smoke exposure . 
 three smoking patients quit smoking by the 18-month follow - up while two from the group of non - smokers had confirmed active smoking in subsequent examinations , although the questionnaire strongly denied that those patients smoke . using a marker of exposure to tobacco smoke 
 each patient was determined in urine cotinine levels immediately after admission to hospital to reduce the number of false - negative results resulting from smoking cessation during hospitalization , despite the fact that the period of persistence of cotinine in the urine is so long that it enables detection , even after several hours . 
 detection of cotinine in urine proved to be a convenient and easy way that was acceptable to the patient and chosen by the authors of the study to eliminate the stress factor associated with the collection of a blood sample . 
 application to the markings cotinine hplc / dad allows side to ensure adequate sensitivity of determination ( ng / l ) also denoted the identification of compound . 
 statement and determine the levels of cotinine in the urine of patients who do not smoke tobacco will allow the realization of risks arising from the presence in tobacco smoke to those who reside there but involuntarily and unknowingly risk individuals [ 11 , 12 ] . in our study , people exposed to tobacco smoke ,  passive smoking  was not aware that being in smoky areas are directly exposed to the harmful effects of tobacco smoke . 
 satisfactory is the fact that in recent years through administrative rulings have managed to limit the number of public places where smoking is permitted . 
 still , smoking in enclosed areas , especially in households causes harmful substances that remain after combustion of nicotine on the surfaces of various objects for many hours and adversely affect people who are in such areas , in particular , children . on the question how smoking affects of bladder cancer evaluation 
 will answer the study conducted in our clinic during the long observation of patients with bladder cancer . 
 detection of cotinine level allows for credible assessment of medical history related to tobacco smoking.questions and answers indicated that the credibility of the questionnaire is limited to the disclosure and does not accurately relate smoking by the patients.assessment of active and passive smoking should be carried out based on evaluation of the questionnaire and measurement of the nicotine metabolite in the urine.monitoring of cotinine level allows for an objective assessment and to effectively convey the recommendations for quitting tobacco smoking . 
 detection of cotinine level allows for credible assessment of medical history related to tobacco smoking . 
 questions and answers indicated that the credibility of the questionnaire is limited to the disclosure and does not accurately relate smoking by the patients . 
 assessment of active and passive smoking should be carried out based on evaluation of the questionnaire and measurement of the nicotine metabolite in the urine . 
 monitoring of cotinine level allows for an objective assessment and to effectively convey the recommendations for quitting tobacco smoking .","an analysis of the reliability of a questionnaire on smoking in 96 patients with transitional cell carcinoma ( tcc ) of the bladder . 
 the credibility of the questionnaire was evaluated based on the detection of cotinine , an objective marker of tobacco smoke exposure , in urine . 
 it was confirmed that approximately 18% of smokers did not admit to smoking , did not comply with recommendations to stop smoking , and about 4% of non - smokers were exposed to tobacco smoke unknowingly .",pubmed
"anal incontinence is a bothersome ailment associated with many health complaints and discomfort in daily life : hygienic problems , limitations in occupational and social life , sexual dysfunction , reduced quality of life , and altered self - esteem . 
 prevalence and severity of anal incontinence are measured by patient self - reporting and no objective assessment methods exist . 
 obstetric anal sphincter injury ( oasis ) is one of the main causes for female ai reported in nonpregnant women . 
 additionally , multiple vaginal deliveries can increase the risk of ai regardless of anal sphincter injury [ 2 , 3 ] . 
 age , obesity and medical conditions such as diabetic neuropathy and gastrointestinal disorders also increase the risk of anal incontinence [ 2 , 4 , 5 ] . 
 prevalence of anal incontinence among women differs largely ( 228% ) in previous studies and differs between different study populations [ 46 ] . 
 postpartum studies show a high prevalence of ai in women having suffered from oasis , 3859% [ 68 ] . 
 women attending gynecological outpatient clinics have higher prevalence of ai ( 1628% ) compared with the general female population ( 4.4% ) [ 2 , 5 ] . 
 women with pelvic floor disorders have higher prevalence of ai than women without pelvic floor disorders . 
 community - based studies show differences in prevalence of ai between age groups , with increasing prevalence by increasing age [ 4 , 5 , 9 ] . 
 most frequent ai is found among nursing home residents ( 5060% ) , among the oldest women with frequent additional complaints and comorbidity . 
 few previous studies have assessed the prevalence of anal incontinence in a female population of fertile age , and few studies have included nulliparous women [ 1114 ] . 
 the aim of this study was therefore to assess the prevalence and risk factors for anal incontinence in an unselected female population across parity groups in second trimester of pregnancy . 
 this study is part of a comprehensive perineum research study , which was approved by the regional committee for medical research ethics in south - eastern norway ( ref . 
 this study was conducted as a survey of pregnant women attending free of charge routine ultrasound examination at second trimester , from september 2009 to august 2010 , in a large university hospital in oslo , norway . 
 the pregnant women attending the ultrasound screening in our hospital represent a nonselected population from the entire oslo area . all pregnant women in norway 
 are offered a free of charge second trimester routine ultrasound examination in gestational week 1820 , and 98% attend . in our hospital , this routine ultrasound is performed by specially educated midwives at the fetal medicine unit . 
 the hospital receives admission notes from the local general practitioners when the woman is pregnant in the first trimester . 
 the invitation to participate in our study , the questionnaire , and the informed consent were included as a part of the invitation to the routine ultrasound appointment . 
 midwives performing the routine ultrasound examination reminded the women of the study and collected the questionnaire and the signed informed consent . from the 7256 women who were posted a questionnaire , 
 973 women were not found in our postpartum labor ward database : they did not achieve 18 weeks pregnancy ( pregnancy loss ) , they did not deliver in our hospital , or they had moved out of oslo area or norway , resulting in 6283 women eligible for study participation . 
 four women returned two questionnaires ( twice during the same pregnancy ) , and one woman returned the questionnaire shortly after the index delivery . 
 thus , five filled - out forms were excluded from the analyses and the study group consisted of 2846 ( of 6283 invited ) women , resulting in a response rate of 45% . 
 the questionnaire consisted of 105 questions concerning anal and urinary incontinence , general health condition , drug use , and worries concerning pregnancy and delivery . 
 the major part of the questions was chosen from validated questionnaires such as due and ottesen , st . 
 mark 's , nugg , hunt , and cambridge worry scale ( cws ) [ 19 , 20 ] . 
 additionally , we collected demographic data , obstetrical history , educational level , household income , and country of origin of the participant . 
 anal incontinence was identified by self - reported leakage of gas , loose , or solid stools , lack of ability to defer defecation for 15 minutes ( fecal urgency ) , use of pads or plugs , and alteration of lifestyle described in st . 
 mark 's score from 0 to 2 were analyzed as a control group ( no or infrequent ai ) . 
 urinary incontinence was defined as self - reported symptoms of stress or urge urinary incontinence . 
 thus , women with cesarean delivery only ( never having delivered vaginally before ) were categorized as  vaginal primiparous . 
 continuous data were categorized and the independent variables are presented as frequencies . univariate analysis was performed to identify significant risk factors for anal incontinence . 
 the results from this regression analysis are presented as adjusted odds ratios ( aors ) for ai with 95% ci . for each model 
 the assumptions underlying a valid logistic regression analysis were checked and found to be adequately met . 
 mark 's score 3 or more ) in the entire study group was 8.4% ( 238/2846 ) . 
 most of the women ( 80.3% ) reported complete anal continence ( 2268/2846 ) with st . 
 mark 's score 0 , and 11.3% ( 322/2846 ) women reported infrequent ai with st . 
 mark 's score 1 or 2 . inability to control flatus was the most frequent complaint , reported by 18.0% ( 513/2846 ) . of these , 
 fecal incontinence was reported by 6.0% ( 171/2846 ) and fecal urgency by 3.2% ( 90/2846 ) of the women . 
 urinary incontinence ( ui ) was significantly associated with reported anal incontinence among all parity groups and 32.4% of the women with ai also reported ui ( p < 0.001 ) . 
 prevalence of ui was threefold among parous women compared to nulliparous women and increased slightly with increasing vaginal parity ( p < 0.001 ) ( data not shown ) . 
 the majority of the 2846 women had answered the questionnaire when they were in the second trimester ( 84% ) , 12.2% in the first trimester , and 1.2% in the third trimester . 
 mean height was 168  cm and mean weight was 66.7  kg . mean bmi was 23.6 , range 16.042.4 . 
 smoking was infrequent ; only 2% ( 57/2851 ) women reported that they smoked during this pregnancy ( table 1 ) . 
 there was a significant difference in prevalence of ai among women with different obstetric histories . 
 mark 's score of 3 or above ) increased with increasing vaginal parity ( data not shown ) . of the nulliparous women , 7.8% ( 139/1792 ) reported anal incontinence . in the univariate analysis , non - western background , low household income , 
 being unmarried or single , low educational level , age 35 or more , answering the questionnaire in the first trimester ( as opposed to second trimester ) , dermatological disease , ulcerative stomach disease , hypertension , rheumatoid arthritis , and muscle - skeletal complaints were significantly associated with anal incontinence among the nulliparous women ( tables 1 and 2 ) . in the multivariate analysis , low educational level , dermatological disease , and rheumatoid arthritis remained as significant factors for ai ( table 2 ) . after excluding the 140 women with previous cesarean delivery only , the subgroup of vaginal parous women consisted of 914 women . 
 overall anal incontinence among parous women was 9.8% ( 90/914 ) . in the group of women with one previous vaginal delivery , 8.5% ( 61/714 ) reported ai , whereas the group of women with more than one previous vaginal delivery , as many as 14.5% ( 29/200 ) , reported ai ( p = 0.004 ) . 
 of the parous women , 15.9% had previously delivered at least one macrosomic ( > 4000  g ) infant ( 145/914 ) , 156 reported previous delivery with vacuum extraction , and 24 women reported a previous forceps delivery . an obstetric history with instrumental delivery or a macrosomic infant 
 previous delivery with oasis was reported by 41 women ( 4.5% ) and was strongly associated with ai .   in the univariate analysis , 
 previous delivery with oasis , non - western background , low household income , being unmarried or single , low educational level , age 35 or more , bmi 25 or more , dermatological disease , and use of pain killers were significantly associated with anal incontinence among the parous women ( tables 1 and 2 ) . in the multivariate analysis , 
 previous delivery complicated with oasis and dermatological disease remained as significant risk factors for ai ( table 2 ) . 
 the risk of ai was threefold among women with previous oasis compared to women without ( 24.4% and 8.1% , resp . ) . 
 the higher risk of ai associated with previous oasis remained threefold in the more severe forms of ai , if defining ai as self - reported st . 
 mark 's score of 5 or above ( 12.2% and 3.8% ) or if 7 or above ( 7.3% and 2.3% ) instead of 3 or above ( table 3 ) . 
 the subgroup of parous women with previous cesarean only ( n = 140 ) and no vaginal deliveries was also analyzed separately . in this subgroup 
 the prevalence of ai was the lowest ( 6.4% , 9/140 ) compared with all other parity groups but was too small to further analysis of risk factors . when the analyses were repeated with this subgroup of women added to the subgroup of nulliparous women , the conclusions remained unaltered ( data not shown ) . 
 women who reported having a dermatological disease reported also more anal incontinence than women without this disease ( table 2 ) . there was a significant association between dermatological disease and several other complaints : allergy , migraine and headache , constipation , and psychological problems . 
 women who reported dermatological problems also more frequently reported use of vitamins , allergy medication , and stomach and bowel regulators . 
 these women also reported more worries about the cambridge worry scale than the women without dermatological problems . 
 this population - based study showed that previous obstetric anal sphincter injury was the strongest risk factor for self - reported ai among pregnant parous women . among the nulliparous women , a low educational level and comorbidity 
 the group of women with previous deliveries with cesarean section only had the lowest prevalence of ai , indicating that pregnancy per se may not represent a major risk factor for ai . 
 these findings support the notion that the process of vaginal delivery may be more damaging to the anal continence mechanisms than pregnancy per se . 
 we found a surprisingly high frequency of self - reported ai among nulliparous women ( 7.8% ) . 
 low socioeconomic status ( low income , low education ) is a well - known reason for lower health status and increased morbidity , and previous studies show that self - rated health predicts morbidity well [ 2123 ] ; therefore , there is now a reason to doubt the correctness of the self - reported ai . 
 socioeconomic differences have been found in occurrence of almost all conditions and illnesses [ 22 , 23 ] . 
 this might explain part of the results for the group of nulliparous pregnant women in our study , where low educational level remained as significant risk factor for ai in the multivariate analysis . 
 a previous oasis was the strongest risk factor for self - reported anal incontinence in all analyses in parous women , with and without adjusting for other factors , and in all categories of severity of anal incontinence . in our study , women with previous oasis reported a lower prevalence of ai than women in previous studies on nonpregnant women [ 68 , 24 ] . 
 all the participants in our study were pregnant , and the low prevalence of ai among women with previous oasis might indicate that fewer women with severe complaints of ai embark on a new pregnancy . the risk of ai increased with increasing number of vaginal deliveries , a result similar to previous studies . 
 interestingly , previous delivery with a macrosomic infant ( > 4000  g ) was not associated with self - reported ai during pregnancy in our study , and was not a previous delivery with vacuum extraction or forceps . 
 this is in contrast to some previous studies , where previous forceps delivery and macrosomy are reported as risk factors for ai [ 8 , 26 , 27 ] . in our study of pregnant women , 
 maternal age was not a significant risk factor for ai in the subgroup of parous women , probably because our study group was young , the oldest participant was 45 years old , and age related increased risk of anal incontinence is probably more important in older age groups [ 2 , 5 , 27 ] . 
 women with overweight ( bmi 2529.9 ) and obesity ( bmi > 30 ) were more likely to suffer from anal incontinence than women with normal bmi ( < 25 ) in our study , but in the multivariate analysis this effect disappeared , due to the strong effect of previous oasis . 
 the large effect of oasis exceeded all other factors ( except dermatological illness ) . 
 many previous studies of ai describe only the frequency of the different components of ai . 
 we chose to describe the prevalence of ai as a score , to be able to perform multivariable analyses of the assessed variables in our study . 
 mark 's score 3 as cutoff for ai was to be able to compare the results from this study to our previous study   and also to our future study , a followup of the participating women after delivery . as a limit of 3 for defining ai 
 may be questioned for clinical relevance , we repeated all statistical analyses with different cutoffs ( 4 , 5 , and 7 ) for st . 
 the main conclusions remained unaltered , oasis was the most important predictor for ai ( for all these cutoffs for st . 
 mark 's score ) among parous women , and low socioeconomic status and comorbidity were the most important indicators for ai among nulliparous women . 
 variables with a p value over 0.05 were also included in the primary analyses to ensure that no risk factors were missed among our registered variables , but no such factors were revealed . similarly to our previous study of nonpregnant women , urinary incontinence was reported by 19.2% of the participants . 
 prevalence of urinary incontinence was threefold among women who reported ai compared to women without ai , in both nulliparous and parous subgroups of women ( p < 0.001 ) . 
 this might indicate that some women are in higher overall risk for incontinence , possibly associated with tissue type , or the pathophysiological mechanism may be the same for both diseases . 
 strength of this study is that the pregnant population was unselected and consisted of all parity groups , including nulliparous women . 
 the majority of studies on female anal incontinence have assessed nonpregnant women 624 months after delivery . 
 another strength of this study is that we also assessed comorbidity and medication use in addition to obstetrical history . to our knowledge , 
 no previous studies have assessed anal incontinence and comorbidity among pregnant women . among nulliparous women 
 further research is needed to explore whether this is a consistent finding across population groups and to explore which mechanisms could underlie such an association . 
 a weakness in our study is that the response rate among the invited women was less than 50% , and this can cause self - selection bias among the study participants . 
 similar selection bias was observed in the norwegian moba study , where higher educated women more likely agreed to participate . 
 however , the effect of such selection bias was found low in the moba study , and we have no reason to believe that our response rate of 45% negatively affected our study either . 
 low prevalence of comorbidity and medication use may indicate that the participants did not have lower health status than nonparticipants or the general population in oslo . 
 bias of women with a previous oasis and complaints of ai having been more eager to participate in the study is unlikely , since the prevalence of ai in the subgroup of women with previous oasis was lower ( 24.4% ) than that in the previously reported studies ( from norway ) [ 6 , 7 ] . 
 we compared the study population 's basic clinical data with an anonymous electronic database covering all patients delivering at the same time period as the participants in this study . 
 the distribution of nulliparous women in our study population was higher than in the overall delivery population in our hospital ( 63% and 52% , resp . ) . 
 we did not find any differences in mean age between responders and nonresponders , but the distribution of women with non - western background was higher among the nonresponders ( data not shown ) , as expected , as the questionnaire and patient information were in norwegian . 
 all data in this study was based on self - reporting from the participants , and thus information of their obstetric history can include errors . 
 it is likely that women remember correctly the number of previous deliveries , delivery mode , and infant birth weight , but not all women are aware of having suffered from oasis   when they leave the hospital after delivery . lacking information of oasis 
 might strengthen our conclusions of oasis being a strong risk factor for ai : if women unaware of previous oasis reported ai and were analyzed as having no previous oasis , the risk of ai after oasis is even higher than calculated in this study . 
 on the other hand , if more women who were unaware of having oasis reported no ai , our results would show too strong effect of oasis as a risk factor to ai . 
 we found an association between self - reported dermatological disease and self - reported ai for all parity groups , which has not been reported before . 
 we can only speculate reasons for this association ; women that have ai may also be more sensitive to dermatological bother than others , perhaps associated with the fecal incontinence with affection of perianal skin . possibly , there could be a common tissue specific risk for both ai and dermatological conditions . 
 women who reported dermatological problems also reported more worries ; another explanation could be that these women were in general more sensitive to different symptoms and signs . in a further study 
 more detailed questions about the type of dermatological disease would be of interest when assessing comorbidity in relation to symptoms of ai . 
 we have performed a large number of analyses due to a large number of detailed information about obstetric history and maternal characteristics . 
 the study population is relatively young , and thus , frequency of comorbidity and medication use was very low among the participants , which can give results by chance . 
 this did not alter the conclusions ; low educational level among nulliparous and oasis among parous women were the most important factors associated to ai . 
 we conclude that among parous women , previous oasis is the most important risk factor for anal incontinence and other obstetrical events only had a minor effect on development of ai . as oasis is a modifiable risk factor , and frequency 
 may rapidly be altered after introduction of obstetrical perineal support programs [ 3033 ] , prevention of obstetrical sphincter injury is likely the most important factor for reducing bothersome anal incontinence in fertile women .","the aim of this study was to assess the prevalence and risk factors of anal incontinence in an unselected pregnant population at second trimester . 
 a survey of pregnant women attending a routine ultrasound examination was conducted in a university hospital in oslo , norway . a questionnaire consisting of 105 items concerning anal incontinence ( including st . 
 mark 's score ) , urinary incontinence , medication use , and comorbidity was posted to women when invited to the ultrasound examination . 
 results . 
 prevalence of self - reported anal incontinence ( st . mark 's score  3 ) was the lowest in the group of women with a previous cesarean section only ( 6.4% ) and the highest among women with a previous delivery complicated by obstetric anal sphincter injury ( 24.4% ) . among nulliparous women the prevalence of anal incontinence was 7.7% and was associated to low educational level and comorbidity . 
 prevalence of anal incontinence increased with increasing parity . 
 urinary incontinence was associated with anal incontinence in all parity groups . 
 conclusions . 
 anal incontinence was most frequent among women with a history of obstetric anal sphincter injury . 
 other obstetrical events had a minor effect on prevalence of anal incontinence among parous women . 
 prevention of obstetrical sphincter injury is likely the most important factor for reducing bothersome anal incontinence among fertile women .",pubmed
"the incidence of hypotension in non - obstetric patients ranges between 5% and 66% , depending on the definition of hypotension limit , timing of measurements , and differences between data acquisition methods and patient characteristics . 
 previous cohort studies reported that hypotension due to spinal anesthesia was approximately twice as common in hypertensive patients [ 13 ] . 
 it has been shown that intraoperative hypotension is associated with myocardial ischemia and increased stroke risk . 
 in addition , because hypertension is an independent risk factor for coronary heart disease , congestive heart failure , and cerebrovascular diseases , it is considered that the negative effects of hypotension will occur more easily in this patient group . 
 the number of hypertensive patients in the adult population in turkey is estimated to be approximately 15 million ( 31.8% ) , and almost one - third is unaware of their condition . 
 although hypertension is a problem frequently encountered by anesthesiologists , and despite advances in the definition and management of hypertension , there are only a few randomized controlled studies that have investigated the hemodynamic effects of spinal anesthesia in these patients . in this study we aimed to compare normotensive patients and hypertensive patients under control by medical treatment , with respect to the hemodynamic effects of spinal anesthesia performed with hyperbaric bupivacaine . 
 the study was approved by the ethics committee of haseki training and research hospital , and all patients gave written and verbal consent prior to the study . 
 sixty patients ages 1865 with asa scores i  ii and who were scheduled for elective surgeries of lower abdomen , lower limbs , and perineum under spinal anesthesia were included into the study . 
 blood pressures and heart rates were measured under standard conditions and the basal values were recorded . 
 patients with a history of hypertension and who regularly used antihypertensive medications constituted group h ( n=30 ) , and patients with basal blood pressure values below 140/90 mm hg and no history of use of drugs effective on the cardiovascular system constituted group n ( n=30 ) . 
 hypertension was inadequately controlled but blood pressure was below 180/100 mm hg in all patients of group h. antihypertensive drugs were given on the morning of surgery . 
 patients in whom lumbar puncture failed in the first attempt , who had blood loss necessitating transfusion , or had any contraindications against spinal anesthesia were excluded from the study . 
 an intravenous catheter was placed in the premedication room , then the ecg , non - invasive blood pressure ( nibp ) and peripheral oxygen saturation ( spo2 ) were monitored ( drger infinity delta , drger medical systems , inc . 
 the patients were premedicated with 11.5 mg midazolam and received 10 ml / kg isotonic nacl according to the ideal body mass calculated with formula ( [ height100 ( height150)/4 ] for male and [ height100 ( height150)/4]0.9 for female ) in 20 minutes , then they were taken into the operating room . spinal anesthesia injected with a 22-gauge quincke spinal needle from the l34 intervertebral space with the patient in sitting position . as the local anesthetic , 3.5 ml of 0.5% hyperbaric bupivacaine ( marcaine 0.5% , spinal heavy 0.5% ( 5 
 mg / ml ) astrazeneca ) was applied to the subarachnoid space for 15 seconds . 
 the patients were then immediately brought into supine position with the head elevated to 30 degrees . 
 sensory block was evaluated with the pinprick test , and motor block was evaluated with the modified bromage scale ( 0=no motor block , 1=inability to raise extended leg , but able to move knees and feet , 2=inability to raise extended leg and move knee , but able to move feet , 3=inability to flex ankle joint ) . 
 the systolic ( sbp ) , diastolic ( dbp ) , and mean ( mbp ) blood pressures , and heart rate ( hr ) were measured and recorded by an assistant at the following time points : basal ( the average of 3 consecutive measurements in the premedication room ) , after fluid loading , and 1 , 3,5 , 10 , 20 , 30 , 40 , 50 , and 60 minutes after spinal anesthesia . 
 a decrease of mbp of more than 30% from the baseline was accepted as hypotension and was treated with intravenous ephedrine 5 mg . 
 a decrease in hr to below 50 beats / minute was accepted as bradycardia and was treated with intravenous atropine 0.5 mg . 
 patients who received ephedrine and atropine , and the time of treatment were recorded . during surgery 
 , all patients inspired 100% o2 at a flow rate of 2 l / minute using a face mask , and isotonic nacl was infused at 8 ml / kg / hour . 
 data were evaluated with the spss 17.0 statistics software for windows ( spss inc . , 
 categoric variables between the groups ( sex , asa , the frequency of hypotension and bradycardia ) were compared with the chi - square test . 
 numeric variables ( sex , body weight , height , and hemodynamic parameters ) were evaluated with the kolmogorov - smirnov test with respect to normal distribution . 
 repeated measurements analysis of variance ( rmanova ) was used for intragroup comparison of the hemodynamic data , and bonferroni test was used for post hoc multiple comparison . 
 the data are shown as mean s.d . and p<0.05 was accepted as statistically significant . 
 there was no significant difference between the groups with respect to age , sex , weight , height , maximal height of sensory block , incidences of hypotension and bradycardia , or the amount of fluids infused ( p>0.05 ) . 
 however , in group h the number of patients with asa ii physiologic scores was statistically higher compared to group n ( p<0.001 ) ( table 1 ) . 
 antihypertensive medications used by the patients are also shown in table 1 . in the hypertensive patient group , 
 the sbp , dbp , and mbp values were significantly higher than the normotensive patient group at all measurement times ( p<0.05 ) . 
 comparison within the groups did not reveal any significant differences in either group compared to the basal values ( p>0.05 ; figures 13 ) . 
 we also found that hr values did not show a significant change compared to the basal measurements ( p>0.05 ; figure 4 ) . 
 six patients in group h and 7 patients in group n received atropine due to bradycardia . 
 we observed that bradycardia resolved with a single dose of 0.5 mg intravenous atropine . in patients who developed hypotension , a single dose of 5 mg ephedrine treatment was sufficient ( table 1 ) . 
 in this study we showed that there was no difference between the incidence of hypotension in hypertensive versus normotensive patients who underwent spinal anesthesia with hyperbaric bupivacaine . 
 however , the number of patients who developed hypotension was greater in the hypertensive group ( 6/30 ) compared to the normotensive group ( 1/30 ) . in contrast to our results , racle et al 
 .   reported that hypotension due to spinal anesthesia with 0.5% isobaric bupivacaine was more frequent in hypertensive patients than in normotensives . in their study , the incidence rate of hypotension was 33% ( 10 out of 30 patients ) in the hypertensive group and 10% ( 3 out of 30 patients ) in the normotensive group , which were higher than our results . on the other hand , 2 other studies showed , as in our study , that the incidence rates of hypotension in hypertensive and normotensive patient groups were not different . 
 however , the rates of hypotension were 55.5% and 67% in hypertensive patients , and 43.8% and 73% in normotensive patients , which were higher than our findings . 
 the patients in the studies of racle   and nishikawa   were 1 or 2 decades older than ours ( mean age was above 75 ) . 
 hypotension due to spinal anesthesia is caused by decreased systemic vascular resistance and venous return to the heart , caused by sympathetic system blockage . 
 there is an increase in the basal sympathetic system activity and plasma norepinephrine levels associated with aging . 
 therefore , sympathetic blockage after spinal anesthesia may cause a greater decrease in the systemic vascular resistance in elderly patients . 
 hypertensive patients also have increased sympathetic activity and norepinephrine levels , as well as decreased parasympathetic activity . 
 persistent sympathetic stimulation , independent of hypertension itself , causes loss of elasticity in the arterial wall and induces structural changes that in turn result in increases in peripheral vascular resistance . 
 it has been shown that there is a close relationship between basal sympathetic activity and decrements in blood pressure that occur after sympathetic blockage . 
 the reduction in blood pressure after spinal anesthesia is correlated with the degree of preoperative blood pressure . 
 fukuda   and nishikawa   found that when preoperative blood pressure was normalized with antihypertensive treatment , there was no difference between the incidence of hypotension following spinal anesthesia in hypertensive versus normotensive patients . 
 our results support these findings  the reason for this is the improvement in vascular structural changes and the achievement of a decrease in basal sympathetic activity with the use of effective antihypertensive treatment . 
 prehydration under the guidance of heart rate variability , an indicator of sympathovagal balance , was seen to decrease the risk of hypotension after spinal anesthesia . 
 venous capacitance is decreased in hypertensive patients ; therefore it is thought that a sympathetic block after spinal anesthesia will cause a further decrease in the central volume and venous return to the heart . 
 the fact that antihypertensive treatment provides an improvement in venous capacitance as much as peripheral vascular resistance explains why we did not find any significant decrease in the incidence of hypotension between normotensive and hypotensive patients . in this study , although we showed that there was no difference between the 2 groups with respect to the frequency of hypotension , the sbp , dbp , and mbp were higher in hypertensive patients compared to normotensive patients at all measurement times  this is related to the higher basal blood pressure values in the hypertensive group compared to the normotensive group . 
 our study did not measure the basal sympathetic activities of the patients , which is this study s most significant limiting factor . 
 although an improvement in this increase and blood pressure regulation is achieved with appropriate antihypertensive treatment , lack of measurement of the basal sympathetic activities of the patients was the most significant limiting factor . 
 in conclusion , we found that there was no significant difference between normotensive and hypertensive patients in the incidence of hypotension caused by spinal anesthesia with 0.5% hyperbaric bupivacaine .","backgroundhypotension is the most common problem with spinal anesthesia . 
 this prospective study aimed to compare normotensive and hypertensive patients with respect to the hemodynamic effects of spinal anesthesia performed with hyperbaric bupivacaine.material/methodssixty patients who were scheduled to undergo various elective operations under spinal anesthesia were included into the study . 
 the patients were separated into 2 groups : hypertensive patients constituted group h ( n=30 ) and normotensive patients constituted group n ( n=30 ) . after fluid loading , spinal anesthesia was performed with 3.5 ml 0.5% hyperbaric bupivacaine . 
 demographic characteristics and incidence of hypotension and bradycardia were compared . 
 systolic ( sbp ) , diastolic ( dbp ) , and mean blood pressures ( mbp ) and heart rate ( hr ) were also compared before and after spinal anesthesia.resultsthere was no significant difference between the groups with respect to demographic characteristics , maximal height of sensory block , incidences of hypotension and bradycardia , and the amount of fluids infused ( p>0.05 ) . in the hypertensive patient group , 
 the sbp , dbp , and mbp values were significantly higher than in the normotensive patient group at all measurement times ( p<0.05 ) . 
 comparison within the groups did not reveal any significant differences in either group compared to the basal values ( p>0.05 ) . 
 there were no significant differences in hr between or within groups ( p>0.05).conclusionsthere was no significant difference between normotensive and hypertensive patients in the incidences of hypotension caused by spinal anesthesia with 0.5% hyperbaric bupivacaine .",pubmed
"chronic pain and depression are among the most common health problems reported by patients attending primary care [ 13 ] . diagnosing pain and its coexistence with depression and getting an overview of the pain patient 's situation can be challenging and can have great consequences for the treatment outcome . 
 numerous studies have found pain associated with a significant limitation in daily activities , physical activities and with poor self - rated health [ 3 , 4 ] . 
 pain is also associated with increased prevalence of depressive disorders [ 5 , 6 ] . 
 studies from primary care have demonstrated that 2240% of all contacts to primary care are due to pain . 
 persistent pain is often comorbid with depression both in general pain conditions [ 4 , 8 ] and in disease - specific conditions . 
 though health care providers are advised to pay special attention to pain symptoms in patients with depression , studies have demonstrated that primary care physicians tend to associate pain with depression to a significant lesser extent than other somatic symptoms , although pain is the most prevalent somatic symptom in patients with depression . 
 this tendency to underestimate the intensity of burden has also been proven for the symptom pain with significant differences between the patient 's and the healthcare provider 's grading of the intensity of the pain [ 11 , 12 ] . 
 patients with depression seek care more often for somatic symptoms than for psychological symptoms , why care providers must have an increased awareness for this coexistence . 
 there is evidence that optimizing antidepressant therapy and pain self - management in primary care patients may result in improvement of both depression and pain . 
 coexistence of depression and pain has also shown to be a major risk factor for opioid misuse both by using opioids for stress and sleep problems instead of for pain alone , and by using more opioids than prescribed . 
 it is therefore important that healthcare provider and the patient have an ongoing awareness and understanding of this connection between depression and pain and preferably share understanding of the current state of the condition . 
 dolotest ( figure 1 ) has been developed to provide such a shared understanding of the patients situation . 
 it is a validated , pain- and health - related quality of life ( hrqol ) assessment tool integrating measurement of the intensity of pain with assessment of the intensity of the impact of the pain including mood . 
 the aim of this study was to determine to which degree dolotest is useful as a screening tool for depression in primary care for adult ( all persons 18 years or older ) seeking primary care , and to find the sensitivity and specificity for dolotest used for screening for depression . 
 the study is a diagnostic study among the population of patients visiting primary care physician no matter the reason for the visit . 
 laegehuset noerretorv  in grindsted , denmark in the period from october 1st 2008 to december 31st , 2008 . 
 all patients being 18 years old or older visiting the clinic and not complying with the exclusion criteria were asked to fill in the questionnaires : dolotest   and  major depression inventory 
 the study included a study population of 715 patients equal to a response rate of 68,5% . 
 when arriving at the clinic , a clinic nurse screened for inclusion / exclusion criteria . 
 patients who could enter the study were handed the questionnaires together with a letter of introduction and the standardized dolotest patient introduction . 
 patients were also asked about age and gender and whether their visit was due to a pain problem or not . 
 patients were excluded if they meet exclusion criteria : cognitive dysfunction , visual impairment , and patients whose physical condition made filling the test impossible or prior participation in the study . 
 dolotest is a validated pain- and health related quality of life ( hrqol ) assessment tool integrating measurement of the intensity of pain with assessment of the intensity of the impact of the pain on eight 100  mm visual analogue scales ( vas ) . 
 the design of dolotest provides a presentation of the test result as a visual profile ( dolotest - profile ) , with  no problems  toward the centre of each vas - line and  worst possible  toward the periphery . 
 the larger the dolotest - profile the worse the hrqol , which is immediately understandable for both the patient and the healthcare provider . 
 the dolotest - profile enables the patient to take part in the evaluation of the test result and improve communication about the situation including problems related to depression like mood , sleep problems , tiredness functional problems , and allows the patient and the healthcare provider to evaluate response to treatment together . 
 the test results are also available as a dolotest - score , the sum of all scored domains , ranging from 0 to 800 . 
 mdi is a validated self - assessment questionnaire developed to cover the diagnostic criteria in who 's icd-10 classification of major depressive disorders . 
 mdi was chosen because it follows the icd-10 criteria and is easy to use as a self - assessment questionnaire . 
 the mdi can be used both as a measuring instrument and as a diagnostic instrument with algorithms leading to the icd-10 categories of depression . 
 when used as a diagnostic instrument like in this study , the mdi items are dichotomized to indicate the presence or absence of each of the symptoms . 
 data comes from 715 consecutive patients coming to primary care , no matter the reason for their visit . 
 statistical analysis was made using mann - whitney test to compare groups and chi - square test to compare frequencies using analyse - it software v. 2.12 . 
 the study was according to danish law ethical approved by  the danish data protection agency  , copenhagen , denmark , and all patients participated voluntary and were provided with both oral and written information . 
 a total of 1044 patients were asked to participate . of those , 302 ( 31,5% ) 
 were not included either because they did not want to participate or due to the exclusion criteria . 
 of the 715 patients , 458 ( 64.1% ) were women and 257 ( 35.9% ) men . 
 there were no statistically significant difference between the gender distribution in the study group and the excluded group ( p = 0.59 ) . 
 the average age in the study population was 45.7 years and in the excluded group 55.4 years ( p < 0.0001 ) . 
 table 1 shows that 246 patients ( 34.4% ) stated their visit was due to a pain problem ( study group ) and 469 patients ( 65.6% ) came for other reasons than pain ( control group ) . 
 the study and control groups were sociodemographic identical with no statistical significant difference between the gender and age distributions , 33.6% of the woman and 35.8% of the men came due to pain ( p = 0.56 ) . 
 table 2 shows that in all domains there are statistically significant difference ( p < 0.0001 ) between the study group and for the control group . 
 low spirits  and  pain  were the only domains in study group with statistically significant differences between men and women , women having the highest score . in the total study population , 
 the women in average scored statistically significantly higher than men in all domains except for  pain  and  problems with more strenuous activity  .  in the control group 
 , women scored statistically significantly higher in al domains except  pain  , where the women also scored higher than men , however not statistically significant . 
 the dolotest - score was statistically significantly higher for women in the whole study population and also for the control group . in the study population 
 the women 's dolotest - score had a tendency to be higher than the men 's . 
 table 1 shows the prevalence of patients with depression for the study and control groups . 
 115 ( 16.1% ) of all the patients met criteria for depression using the mdi - scale . 
 table 2 presents the prevalence of depression together with the average mdi - rating score for the whole study population as well as specified for the study group and for the control group . in the whole study population , 
 32 men ( 12.8% ) and 83 women ( 18.1% ) met the criteria for depression using the mdi scale , and the corresponding prevalence for control group was 11 men ( 6.7% ) and 38 women ( 12.5% ) with statistically significant difference between genders in both groups ( p = 0.05 ) . in the study population , 
 21 men ( 22.8% ) and 45 women ( 29.2% ) met criteria for depression ( p = 0.27 ) . 
 the prevalence of patients with depression in study group was statistically significantly higher than in the control group ( p = 0.0002 ) for men and p < 0.0001 for women .   for patients meeting criteria for depression and patients not meeting the depression criteria 
 , there was a difference for all dolotest domains ( p < 0.0001 ) ( table 3 ) . 
 the domains were scored equally by men and women meeting criteria for depression due to the mdi scale ; however , for patients not meeting criteria for depression , women scored statistically significantly higher than men on five of the domains ( problems with light physical activity , reduced energy and strength , low spirits , reduced social life , and sleeping problems ) ( table 3 ) . 
 the numbers in table 3 are visualized by presented as average dolotest profiles in figure 1 . a cut - off value for the domain 
 to what extent do you experience low spirits  for depression was found using roc curve analysis . 
 it is desirable to find a cut - off with both a high sensitivity and a high specificity , and from a clinical point of view defining a cutoff with few false positive and high predictive value of positive test is important to help making an accurate clinical decision . selecting a cutoff point of  65 
 ( figure 2 ) will provide a sensitivity of 0.78 ( 95% ci : 0.700.85 ) and a specificity of 0.95 ( 95% ci : 0.930.96 ) the predictive value of positive test being 0.74 for detecting that a patient meet criteria for depression . 
 dolotest has in this study proved useful as a screening tool for patients ' experienced pain and the impact of pain on the patient 's hrqol as well as a screening tool for patients meeting criteria for depression and thereby for the coexistence of pain and depression . a cut - off value of  65  on the  low spirits 
 domain provides both high sensitivity ( 0.78 ) and high specificity ( 0.95 ) and a predicted value on positive test at 0.74 . 
 this study is the first to present health related quality of life ( hrqol ) data as dolotest profiles and dolotest scores for all patients seeking a primary care both for patients whose visit are due to a pain problem and for patients whose visit has other reasons . 
 furthermore , the study contributes to the growing knowledge about coexistence of pain and depression , finding the prevalence of depression to be statistically significantly higher for both genders in the group visiting primary care physician because of a pain problem than for other problems combined . 
 pain symptoms have been found to be associated with reduced psychological and physical health as well as with limitations in daily activities and with social isolation [ 19 , 20 ] and are found to be a major reason for seeking health care [ 19 , 20 ] . in this study , 34.4% of the patients stated that their visit to physician was due to pain , which is in accordance with findings in other studies . 
 our study shows that 16.1% of all patients met criteria for depression , and 26.8% whose visit was due to pain , met depression criteria . 
 these findings are in good correlation with the literature , though this study captures the pain intensity during the last week , for example , not necessarily persistent pain . 
 the findings in this study are clinically important since the visual dolotest profiles can facilitate both awareness and often otherwise difficult communication about depression and pain , a coexistence leading to increased risk of reduced function , reduced work capacity , increased risk of opioid misuse , reduced hrqol , and other related problems . 
 it has been shown that suffering from both pain and depression at the same time besides the low hrqol is associated with dramatically increased health care costs [ 22 , 23 ] compared to depression alone , and that treating depression alone does not give sufficient improvement on somatic symptoms like pain [ 21 , 24 ] . 
 based on this knowledge , health care providers are encouraged to be aware of pain symptoms in patients with depression as well as depressive symptoms in patients with pain [ 21 , 25 ] . 
 furthermore , it is stated that targeted screening for the cooccurrence of pain and depression is warranted . 
 the respect trial   found that effective treatment of either depression or pain is dependent on the treatment of the other when they are co - existing . 
 the study concludes  it makes no longer sense to treat one condition without considering the other  . 
 it is therefore of high importance to provide healthcare professionals with tools to screen and identify these patients at risk for suboptimal treatment if not found . 
 in other studies using visual analogue scale ( vas ) as screening tool for depression , it has shown to be a reliable method   and vas has thereby showed to be a valuable tool for assessment of mood . a study looking at using one question from the subjective health complaints ( shc ) inventory found this useful with sensitivity 79% , specificity 81% and predictive value of positive test to be 67% . in the present study 
 , dolotest has proved as a tool screening both pain and depression , since the dolotest domain  low spirits  is associated with depression . 
 a cut off value of  65  has been found optimal for the dolotest domain  low spirits  with a sensitivity of 78% and specificity of 95% to detect persons meeting criteria for depression , and with a predicted value on positive test at 0.74 . 
 pain is not included in neither the icd-10 nor the dsm - iv criteria for depression , and it is also noteworthy that pain is not included in the most used tools for depression diagnostics like hospital anxiety and depression scale ( hads ) and beck depression inventory ( bdi )   or in the major depression inventory   ( mdi ) used in the present study , which increase the risk for not finding the coexistence . both persistent pain and somatization 
 have been found to be frequently associated with depression , yet few studies on somatization are controlled for depression . 
 data is regarded representative for denmark as the present study was conducted in a clinic serving both urban and rural areas . in denmark , all citizens have free and equal access to primary care , as the primary care is financed through the danish taxation system . 
 as expected the age distribution was statistically significantly higher in the excluded group due to the exclusion criteria cognitive and visual impairment . 
 the scoring on both the dolotest and mdi was made by the patients themselves and not controlled . 
 same physician made all diagnoses of meeting criteria for depression based on the mdi scorings ; however , a depression diagnosis was not provided through interview or other further examination . 
 dolotest used as a screening tool for depression criteria had a sensitivity of 78% and specificity of 95% , and a predicted value on positive test at 0.74 . 
 patients meeting criteria for depression had statistically higher pain score and reduced health related quality of life than patients not meeting criteria for depression . 
 dolotest has in this study proved an easy - to - use screening tool in clinical settings for pain and the impact of pain as well as a screening tool for patient meeting criteria for depression . 
 this ease the process of identifying patients with the important coexistence enabling easy identification of this large group of patients needing individualized treatment for more than the pain alone in order to get best possible health related quality of life . treating both pain and depression when co - existing is very important since it otherwise is found to be associated with risk of reduced function , reduced work capacity , increased risk of opioid misuse , and increased healthcare costs .","background . coexistence of pain and depression has significant impact on the patient 's quality of life and treatment outcome . 
 dolotest is a pain and hrqol assessment tool developed to provide shared understanding between the clinician and the patient of the condition by a visual profile . aim . to find the sensitivity and specificity of dolotest as a screening tool for depression for patients in primary care . 
 methods . 
 all patients coming to a primary care clinic were asked to fill in a dolotest and a major depression inventory . 
 results . 715 ( 68.5% ) of 1044 
 patients entered the study . 
 34.4% came due to pain . 
 16.1% met depression criteria , and 26.8% of patients coming due to pain met criteria for depression . 
 65.6% of the men and 54.2% of the women meeting the criteria for depression came due to pain . 
 depressed patients had statistically significant higher scores on all dolotest domains . selecting the cutoff value for the domain  low spirits  to be  65  ( 0100 ) for depression gave a sensitivity of 78% ( 7085% ) and a specificity of 95% ( 9396% ) for meeting depression criteria . 
 conclusion . 
 dolotest can with a high sensitivity and specificity identify persons meeting criteria for depression and is an easy - to - use screening tool to identify patients with the coexistence of pain and depression .",pubmed
"human papillomavirus ( hpv ) is a non - enveloped deoxyribonucleic acid ( dna ) virus belonging to the family papillomaviridae . 
 this family includes more than 130 genotypes,1 many of which infect the mucosal areas of the human upper digestive tract and the anogenital region through sexual contact,2,3 leading to increased risk of development of cancer.1 these genotypes are grouped into  high - risk  and  low - risk  according to the degree of risk of development of cancer after infection . 
 infection with the high - risk serotypes of hpv can lead to cervical cancer and are associated with other mucosal anogenital , and head and neck cancers.4,5 infection with the low - risk serotypes is known to cause benign or low - grade cervical tissue changes and genital warts ( condyloma acuminata ) on the cervix , vagina , vulva , and anus in women and on the penis , scrotum , and anus in men.6,7 genital hpv infection is one of the most common sexually transmitted infections in sexually active adolescents and young women.8,9 it has been estimated that at least 50% of sexually active adults have had a genital hpv infection2,9 and that globally 75% of individuals ( males and females ) will experience an hpv infection at least once in their lifetime , with the highest rates of infection occurring in those under the age of 25 years.10 in a recent meta - analysis , a global hpv prevalence of 11.7% was reported . 
 the hpv prevalence in north america and europe was estimated at 11.5% and 14.2% , respectively , while the prevalence in africa was estimated at 21.1% , with sub - saharan africa topping the list at 24%.11,12 in nigeria , the prevalence of hpv is high in all female age groups , and highest in women aged 1523 years.12,13 studies have indicated that high - risk hpv genital infections in young females are transient and have little long - term significance.2,14,15 however , when the infection persists , as in 5%10% of infected women , there is a high risk of developing a precancerous lesion of the cervix , which can progress to invasive cervical cancer 1520 years later.4,1622 persistent infection following acquisition of a high - risk hpv is generally defined by continued detection of cervical dna of the same hpv type.23 cervical cancer is an important health problem worldwide , being the second most common cancer among women , and ranking first in many developing countries.9,17 half a million women develop cervical cancer annually and more than half die from the disease.9 in 2008 , more than 270,000 women died of cervical cancer worldwide , with nearly 85% of these deaths occurring in developing countries.13,24 cervical cancer is the second most common cancer in women aged 1544 years in nigeria and the incidence rate is 27/100,000.13,24 current estimates indicate that every year 14,089 women are diagnosed with cervical cancer and 8,240 die from the disease in nigeria.13 a prevalence of 26.3% for hpv in the general population has been reported in southern nigeria.25 the incidence of hpv in women with cervical cancer is reported to be 24.8%,12,24 while hpv prevalence in the general population ( among women with normal cytology ) is 23.7%.13 risk factors associated with hpv infection include heterosexuality , promiscuity , smoking,26,27 high parity , early sexual debut,3 infection with other sexually transmitted diseases,26 prolonged use of contraceptives , dietary factors , and genetic disorders such as whim ( warts , hypogammaglobulinemia , immunodeficiency , myelokathexis ) syndrome.28,29 infection with hpv , diagnosed by detection of antibodies to hpv in the serum or detection of hpv dna , is the primary risk factor contributing to development of cervical intraepithelial neoplasia and invasive cervix carcinoma . 
 detection of anti - hpv has been shown to reflect the overall hpv infection rate in a population more effectively than detection of hpv dna.3 this study was conducted in an area where the seroprevalence of hpv has not as yet been reported . 
 the study population comprised 350 women of reproductive age ( 1549 years ) attending the reproductive health clinic at ahmadu bello university teaching hospital , shika , zaria , nigeria 
 . this clinic offers counseling and contraceptive services to delayers , spacers , and limiters . 
 post abortion care , endometrial biopsy , papanicolaou smear , cervical punch biopsy , and colposcopy are other services offered . after obtaining ethical approval from the hospital s ethics committee , 
 the women were recruited and counseled , and those who gave their consent were enrolled in the study . 
 women who were pregnant , those who refused to give consent , and those not of reproductive age were not enrolled . 
 a 3 ml blood sample was collected by venipuncture from each of the 350 women with the assistance of a laboratory scientist under the supervision of a physician . 
 the blood samples were transported immediately to the laboratory in the department of microbiology , faculty of science , ahmadu bello university , zaria , which is located 5 km from the hospital . 
 the blood samples were allowed to clot for 30 minutes and then centrifuged at 1,000 g for 10 minutes to separate the serum . 
 the serum samples were stored at 20c until analysis . an enzyme - linked immunosorbent assay ( wkea med supplies corporation , changchun , people s republic of china ) was used to screen for hpv immunoglobulin g ( igg ) antibodies in the serum samples . 
 all reagents were brought to room temperature and mixed thoroughly by gentle swirling before use . 
 the cutoff value was calculated using the manufacturer s specifications . the average optical density value of the negative control wells plus 0.15 was taken as the cutoff value . 
 a negative hpv result was interpreted as any sample with an optical density value less than the calculated cutoff value , and samples with an optical density greater than the calculated cutoff value were reported as positive for igg to hpv . 
 the results and data from the questionnaires were analyzed , reduced to percentages , and presented as tables and figures . 
 the statistical analysis was done using statistical package for the social sciences version 17 software ( spss inc . , 
 associations between variables were identified by pearson s chi - square analysis and relationships between hpv and risk factors were identified by spearman s rank correlation . 
 the study population comprised 350 women of reproductive age ( 1549 years ) attending the reproductive health clinic at ahmadu bello university teaching hospital , shika , zaria , nigeria 
 . this clinic offers counseling and contraceptive services to delayers , spacers , and limiters . 
 post abortion care , endometrial biopsy , papanicolaou smear , cervical punch biopsy , and colposcopy are other services offered . after obtaining ethical approval from the hospital s ethics committee , 
 the women were recruited and counseled , and those who gave their consent were enrolled in the study . 
 women who were pregnant , those who refused to give consent , and those not of reproductive age were not enrolled . 
 a 3 ml blood sample was collected by venipuncture from each of the 350 women with the assistance of a laboratory scientist under the supervision of a physician . 
 the blood samples were transported immediately to the laboratory in the department of microbiology , faculty of science , ahmadu bello university , zaria , which is located 5 km from the hospital . 
 the blood samples were allowed to clot for 30 minutes and then centrifuged at 1,000 g for 10 minutes to separate the serum . 
 an enzyme - linked immunosorbent assay ( wkea med supplies corporation , changchun , people s republic of china ) was used to screen for hpv immunoglobulin g ( igg ) antibodies in the serum samples . 
 all reagents were brought to room temperature and mixed thoroughly by gentle swirling before use . 
 the cutoff value was calculated using the manufacturer s specifications . the average optical density value of the negative control wells plus 0.15 was taken as the cutoff value . 
 a negative hpv result was interpreted as any sample with an optical density value less than the calculated cutoff value , and samples with an optical density greater than the calculated cutoff value were reported as positive for igg to hpv . 
 the results and data from the questionnaires were analyzed , reduced to percentages , and presented as tables and figures . 
 the statistical analysis was done using statistical package for the social sciences version 17 software ( spss inc . , 
 associations between variables were identified by pearson s chi - square analysis and relationships between hpv and risk factors were identified by spearman s rank correlation . 
 the sociodemographic data for the women obtained from the questionnaires were analyzed and the results showed that women aged 4549 years were most frequently seen ( 32.6% , 114/350 ) in the clinic while those aged 1519 years were the least often seen ( 4.3% , 15/350 , table 1 ) . 
 further analysis showed that 38.0% ( 133/350 ) of the women were from tribes other than the three major nigerian ethnic groups . 
 further , 39.7% ( 139/350 ) were civil servants , 65.1% ( 228/350 ) were married , 56% ( 199/350 ) were in monogamous marriages , and 4.3% ( 15/350 ) admitted to smoking ( table 2 ) . 
 the reproductive characteristics and sexual behavior of the women are shown in tables 3 and 4 , respectively . 
 the data show that 54.9% ( 192/350 ) of the women had been married for less than 25 years and 56.3% ( 197/350 ) had had less than seven successful pregnancies ( parity ) , ie , reaching at least 28 weeks gestation . in total , 69.1% ( 242/350 ) of the women had children , with 57.1% ( 200/350 ) having 16 children . 
 further analysis of the data showed that 60.3% ( 211/350 ) of the women had never used any form of contraception , and a sizeable proportion ( 3.1% , 11/350 ) had used other forms or non - modern contraception ( table 3 ) . 
 the most frequent age of sexual debut was 19 years ( 26.9% , 94/350 ) while 8.9% ( 31/350 ) had their first child aged 1518 years ( table 4 ) . 
 human papillomavirus igg antibodies were detected in serum samples from 42.9% ( 150/350 ) of women . 
 antibodies to hpv initially increased with age , decreased in women aged 3039 years , and then increased again from age 40 years , peaking in women aged 4549 years ( 50.0% , 57/114 ) . 
 women aged 3539 years had the lowest prevalence ( 31.7% , 13/41 , table 1 ) . 
 the hpv infection rate did not differ significantly according to patient age ( =6.192 , df = 6 , p=0.402 ) . 
 women belonging to tribes other than the three major nigerian ethnic groups had the highest hpv prevalence ( 44.0% , 66/119 ) , while igbo women had the lowest prevalence ( 4.7% , 7/25 ) . 
 there was a marginal association between hpv infection rate and tribal origin ( =7.595 , df =3 , p=0.05 ) . 
 hpv antibodies were detected significantly ( =3.938 , df = 1 , p=0.03 ) more often in christian women ( 47.1% , 99/203 ) than in muslim women ( 36.4% , 51/140 ) . retired women had the highest prevalence ( 75.0% , 9/12 ) and unemployed women had the lowest prevalence ( 35.1% , 20/57 ) ; however , this finding was not statistically significant ( =7.820 , df = 4 , p=0.098 ) . 
 the seroprevalence of hpv was highest among widows ( 53.1% , 17/32 ) and lowest among divorcees ( 35.7% , 5/14 ) , but the difference was not statistically significant ( =1.739 , df =3 , p=0.773 ) . 
 the seroprevalence of hpv igg antibodies was similar between monogamous ( 43.2% , 86/199 ) and polygamous women ( 42.7% , 32/75 ; =0.007 , df = 1 , p=0.935 ; odds ratio 1.023 ; 95% confidence interval 0.5981.749 ) . 
 hpv infection was significantly associated with educational status ( =6.594 , df = 4 , p=0.043 ) . 
 antibodies were detected with the highest prevalence among women with secondary education ( 49.2% , 29/59 ) while those with quranic education had the lowest prevalence ( 21.7% , 5/23 , figure 1 ) . 
 antibodies to hpv were detected with a slightly higher frequency ( 43.1% , 140/325 ) in women who were not smoking compared with those who admitted to smoking ( 40.0% , 10/25 ; =0.090 , df=1 , p=0.765 ) . 
 analysis of the results according to years of marriage showed that the highest frequency of antibodies to hpv was among women who had been married for 1620 years ( 55.3% , 21/38 ) and the lowest frequency in those who had been married for 2630 years ( 28.6% , 6/21 ; =8.734 , df = 7 , p=0.262 ) . 
 no statistically significant relationship was found between seroprevalence of hpv and parity ( =3.700 , df = 4 , p=0.448 ) , but the prevalence was highest amongst women who had 79 pregnancies ( 46.7% , 21/45 ) and lowest in those of parity > 9 ( 25.0% , 3/12 ) . women with children had a higher prevalence of antibodies to hpv ( 44.6% , 108/242 ) than those without children ( 38.9% , 42/108 ) . although the difference observed was not statistically significant ( =1.004 , df = 1 , p=0.316 ) , women with children were 1.3 times more likely to be infected than women without children ( odds ratio 1.267 ; 95% confidence interval 0.7982.011 ) . 
 women who had 79 children had the highest seroprevalence ( 46.9% , 15/32 ) while those with more than nine children had the lowest seroprevalence ( 16% , 1/6 ; =2.851 , df = 4 , p=0.583 ) . 
 the seroprevalence of igg antibodies to hpv was not significantly associated with use of contraceptives ( =1.169 , df = 4 , p=0.883 ) ; women who were using other types of contraception ( eg , not modern or local contraceptives ) had the highest prevalence ( 54.5% , 6/11 ) while those using oral contraception had the lowest prevalence ( 39.4% , 13/33 ) . 
 antibodies to hpv were detected most frequently among women who had their sexual debut aged 2023 years ( 51.1% , 46/90 ) and least often in women who had their sexual debut aged 2426 years ( 37.9% , 25/66 ) . 
 however , this difference was not statistically significant ( =3.114 , df = 5 , p=0.682 ) . 
 the frequency of detection of igg antibodies to hpv according to age at birth of first child was highest among women older than 30 years ( 68.8% , 11/16 ) and lowest among women aged 1518 years ( 35.5% , 11/31 ; =8.163 , df = 5 , p=0.147 ) . 
 the prevalence of hpv was also investigated according to vaginal symptoms reported by the women during the study . 
 women with normal vaginal discharge had the highest frequency of hpv antibodies ( 45.1% , 110/244 ) while women with abnormal discharge ( 37.7% , 40/106 ) had the lowest frequency ( =1.628 ; df = 1 ; p=0.202 ) . 
 further , women with genital rash at the time of the study had the highest seroprevalence ( 61.9% , 13/21 ) , with the lowest seroprevalence seen in women with vaginal itching ( 33.3% , 26/78 ) . 
 the difference in prevalence observed according to vaginal symptoms was almost statistically significant ( =7.461 , df =3 , p=0.059 ) . 
 half of the women ( 50.0% , 9/18 ) with a family history of cervical cancer had igg antibodies to hpv compared with those without a family history of cervical cancer ( 42.5% , 141/332 ; =0.395 , df = 1 , p=0.348 ) . 
 antibodies to hpv tended ( =0.806 ; df = 1 ; p=0.274 ) to be detected with a higher prevalence in women without a history or signs of whim as a genetic disorder ( 43.3% , 146/337 ) than in those with such a history ( 30.8% , 4/13 , table 5 ) . 
 the sociodemographic data for the women obtained from the questionnaires were analyzed and the results showed that women aged 4549 years were most frequently seen ( 32.6% , 114/350 ) in the clinic while those aged 1519 years were the least often seen ( 4.3% , 15/350 , table 1 ) . 
 further analysis showed that 38.0% ( 133/350 ) of the women were from tribes other than the three major nigerian ethnic groups . 
 further , 39.7% ( 139/350 ) were civil servants , 65.1% ( 228/350 ) were married , 56% ( 199/350 ) were in monogamous marriages , and 4.3% ( 15/350 ) admitted to smoking ( table 2 ) . 
 the reproductive characteristics and sexual behavior of the women are shown in tables 3 and 4 , respectively . 
 the data show that 54.9% ( 192/350 ) of the women had been married for less than 25 years and 56.3% ( 197/350 ) had had less than seven successful pregnancies ( parity ) , ie , reaching at least 28 weeks gestation . in total , 69.1% ( 242/350 ) of the women had children , with 57.1% ( 200/350 ) having 16 children . 
 further analysis of the data showed that 60.3% ( 211/350 ) of the women had never used any form of contraception , and a sizeable proportion ( 3.1% , 11/350 ) had used other forms or non - modern contraception ( table 3 ) . 
 the most frequent age of sexual debut was 19 years ( 26.9% , 94/350 ) while 8.9% ( 31/350 ) had their first child aged 1518 years ( table 4 ) . 
 human papillomavirus igg antibodies were detected in serum samples from 42.9% ( 150/350 ) of women . 
 antibodies to hpv initially increased with age , decreased in women aged 3039 years , and then increased again from age 40 years , peaking in women aged 4549 years ( 50.0% , 57/114 ) . women aged 3539 years had the lowest prevalence ( 31.7% , 13/41 , table 1 ) . 
 the hpv infection rate did not differ significantly according to patient age ( =6.192 , df = 6 , p=0.402 ) . 
 women belonging to tribes other than the three major nigerian ethnic groups had the highest hpv prevalence ( 44.0% , 66/119 ) , while igbo women had the lowest prevalence ( 4.7% , 7/25 ) . 
 there was a marginal association between hpv infection rate and tribal origin ( =7.595 , df =3 , p=0.05 ) . 
 hpv antibodies were detected significantly ( =3.938 , df = 1 , p=0.03 ) more often in christian women ( 47.1% , 99/203 ) than in muslim women ( 36.4% , 51/140 ) . retired women had the highest prevalence ( 75.0% , 9/12 ) and unemployed women had the lowest prevalence ( 35.1% , 20/57 ) ; however , this finding was not statistically significant ( =7.820 , df = 4 , p=0.098 ) . 
 the seroprevalence of hpv was highest among widows ( 53.1% , 17/32 ) and lowest among divorcees ( 35.7% , 5/14 ) , but the difference was not statistically significant ( =1.739 , df =3 , p=0.773 ) . 
 the seroprevalence of hpv igg antibodies was similar between monogamous ( 43.2% , 86/199 ) and polygamous women ( 42.7% , 32/75 ; =0.007 , df = 1 , p=0.935 ; odds ratio 1.023 ; 95% confidence interval 0.5981.749 ) . 
 hpv infection was significantly associated with educational status ( =6.594 , df = 4 , p=0.043 ) . 
 antibodies were detected with the highest prevalence among women with secondary education ( 49.2% , 29/59 ) while those with quranic education had the lowest prevalence ( 21.7% , 5/23 , figure 1 ) . 
 antibodies to hpv were detected with a slightly higher frequency ( 43.1% , 140/325 ) in women who were not smoking compared with those who admitted to smoking ( 40.0% , 10/25 ; =0.090 , df=1 , p=0.765 ) . 
 analysis of the results according to years of marriage showed that the highest frequency of antibodies to hpv was among women who had been married for 1620 years ( 55.3% , 21/38 ) and the lowest frequency in those who had been married for 2630 years ( 28.6% , 6/21 ; =8.734 , df = 7 , p=0.262 ) . 
 no statistically significant relationship was found between seroprevalence of hpv and parity ( =3.700 , df = 4 , p=0.448 ) , but the prevalence was highest amongst women who had 79 pregnancies ( 46.7% , 21/45 ) and lowest in those of parity > 9 ( 25.0% , 3/12 ) . 
 women with children had a higher prevalence of antibodies to hpv ( 44.6% , 108/242 ) than those without children ( 38.9% , 42/108 ) . although the difference observed was not statistically significant ( =1.004 , df = 1 , p=0.316 ) , women with children were 1.3 times more likely to be infected than women without children ( odds ratio 1.267 ; 95% confidence interval 0.7982.011 ) . 
 women who had 79 children had the highest seroprevalence ( 46.9% , 15/32 ) while those with more than nine children had the lowest seroprevalence ( 16% , 1/6 ; =2.851 , df = 4 , p=0.583 ) . 
 the seroprevalence of igg antibodies to hpv was not significantly associated with use of contraceptives ( =1.169 , df = 4 , p=0.883 ) ; women who were using other types of contraception ( eg , not modern or local contraceptives ) had the highest prevalence ( 54.5% , 6/11 ) while those using oral contraception had the lowest prevalence ( 39.4% , 13/33 ) . 
 antibodies to hpv were detected most frequently among women who had their sexual debut aged 2023 years ( 51.1% , 46/90 ) and least often in women who had their sexual debut aged 2426 years ( 37.9% , 25/66 ) . 
 however , this difference was not statistically significant ( =3.114 , df = 5 , p=0.682 ) . the frequency of detection of igg antibodies to hpv according to age at birth of first child was highest among women older than 30 years ( 68.8% , 11/16 ) and lowest among women aged 1518 years ( 35.5% , 11/31 ; =8.163 , df = 5 , p=0.147 ) . 
 the prevalence of hpv was also investigated according to vaginal symptoms reported by the women during the study . 
 women with normal vaginal discharge had the highest frequency of hpv antibodies ( 45.1% , 110/244 ) while women with abnormal discharge ( 37.7% , 40/106 ) had the lowest frequency ( =1.628 ; df = 1 ; p=0.202 ) . 
 further , women with genital rash at the time of the study had the highest seroprevalence ( 61.9% , 13/21 ) , with the lowest seroprevalence seen in women with vaginal itching ( 33.3% , 26/78 ) . 
 the difference in prevalence observed according to vaginal symptoms was almost statistically significant ( =7.461 , df =3 , p=0.059 ) . 
 half of the women ( 50.0% , 9/18 ) with a family history of cervical cancer had igg antibodies to hpv compared with those without a family history of cervical cancer ( 42.5% , 141/332 ; =0.395 , df = 1 , p=0.348 ) . 
 antibodies to hpv tended ( =0.806 ; df = 1 ; p=0.274 ) to be detected with a higher prevalence in women without a history or signs of whim as a genetic disorder ( 43.3% , 146/337 ) than in those with such a history ( 30.8% , 4/13 , table 5 ) . 
 in this study , hpv igg antibodies were detected in serum samples from 150 of 350 women studied , giving a seroprevalence of 42.9% . 
 similar to the 40% reported in southern mozambique.31 the higher seroprevalence in the present study compared with that in a previous report from nigeria may be due to a difference in the study population . another reason could be the high sensitivity of the enzyme - linked immunosorbent assay that permits detection of the hpv antibody in samples with low hpv antibody which would probably otherwise have been scored as negative for hpv . 
 it has been shown that detection of antibodies to hpv can evaluate the overall hpv infection rate in a population more effectively than hpv dna.3 in view of this , and because ahmadu bello university teaching hospital is a referral center where patients from zaria and its environs are seen , and 40% of the women attending the clinic there have been infected with hpv , this percentage represents the actual seroprevalence of hpv in the state . 
 this high seroprevalence could be due to the lifestyle of the local population in the study area , where women are constantly exposed to the virus , by means of early sexual debut , early marriage , multiple sexual partners due to polygamy , and high divorce rates . 
 acquisition of hpv infection has been shown to be strongly related to sexual behavior , and the prevalence of hpv increases with increasing number of sexual partners and early sexual debut.3,30 about one third of the women attending the reproductive health clinic were aged 4549 years , also the group in which hpv was most prevalent . 
 the detection of hpv in older women in this study is consistent with a previous report,32 and could be due to the sexual mode of transmission of the virus which allows reinfection and persistence of the virus for years.33,34 these women might have acquired the virus at an earlier age , considering that the virus has been shown to persist in a significant percentage of women.20,22 antibodies to hpv were also detected in younger women in this study , probably because of early acquisition of infection as a result of early indulgence in sexual activity and early marriage . 
 women in the study area often marry as young as age 15 years , and it has been reported that the age of sexual debut in nigeria is 910 years.35 however , detection of antibodies to hpv , which signifies infection , does not mean eventual development of cervical cancer . 
 this is because most hpv infections in younger women are transient or asymptomatic , often spontaneously regress,36 and have little long - term significance . 
 moreover , the incidence of cervical cancer in women younger than 30 years is very low , and 70% of cases of hpv infection resolve in one year and 90% in 2 years.22,37 however , persistent infection with one or more high - risk types of hpv is an important etiologic factor in the development of cervical intraepithelial neoplasia and progression to cervical cancer.20,22,3840 in addition , virologic , environmental , immunologic , and genetic factors have also been implicated in the development of cervical cancer.41 the prevalence of hpv according to occupational status of the woman was not statistically significant in this study . 
 this means that all women , regardless of occupation were at similar risk of being infected . 
 single and married women in this study had a similar seroprevalence of hpv , indicating a similar rate of sexual activity . 
 the seroprevalence of hpv was higher in widows than in other studies , which reported a higher prevalence among married women.31,39 yet other studies have reported the highest prevalence among single women.33,41 the prevalence of antibodies to hpv was similar between women who married into monogamous homes and those who married into polygamous homes . 
 this study presents a paradoxical picture that is in contrast with the widely held belief that sexual activity in individuals with multiple partners increases the risk of hpv infection . 
 it has been shown that educational level is a socioeconomic factor with an effect on risk of hpv infection . 
 more educated women had the highest infection rate in our study , with the highest prevalence seen in women with secondary school education . 
 this result is consistent with previous research,32,39 but in contrast with a report by marrazzo et al42 who showed that the hpv infection rate decreased with increasing level of education . 
 this observation could be due to early indulgence in sexual activity and lack of awareness of its consequences . 
 however , in our study , hpv was not significantly associated with smoking and was detected with a higher prevalence among nonsmokers . 
 this observation contrasts that of schlecht et al.39 an earlier study reported that daily cigarette smoking had a deleterious effect and contributed to development of low - grade squamous intraepithelial lesions.43 several epidemiologic studies have identified a role of cigarette smoking in invasive cervical cancer.26,27,44 nicotine and other cigarette metabolites have been found in cervical mucus.45 it is also suspected that the relationship between cigarette smoking and low - grade squamous intraepithelial lesions reflects a link between smoking and immune dysregulation,46 and it has been suggested that smoking may induce an impaired antibody response in young women infected with hpv16/18.27 studies have shown that use of oral contraceptives is a risk factor for acquiring hpv , and prolonged use of oral contraceptives is associated with development of squamous intraepithelial lesions.29,47 in this study , even though , hpv antibodies were detected with the highest prevalence among women who used non - modern contraceptives compared with those using modern contraceptives , the difference in prevalence was however , not statistically significant . 
 this result contrasts with previous reports.33,39 other studies have recorded a higher prevalence of hpv infection in women who do not use any form of contraception.32,42 however , a recent study conducted in kano , nigeria , did not find a significant association between use of oral contraceptives and hpv infection.30 number of years of marriage had no significant effect on infection with hpv in this study , implying that hpv can be acquired at any time during marriage . 
 women with high parity had the least prevalence , as previously reported.39 low parity was recently reported to be a significant risk factor for acquisition of hpv in northern nigeria.30 in contrast , okolo et al,25 in southern nigeria , observed that the prevalence of hpv increases with increasing parity . 
 this increase in prevalence of hpv infection with increasing parity has been attributed to increased sexual activity.3,34 in our study , women who had children had a higher prevalence of antibodies to hpv than women with no children . although this difference in prevalence did not reach statistical significance , women who had children were 1.3 times more likely to be infected with hpv than those who did not . 
 this observation is consistent with a report by firnhaber et al.48 it was also observed that antibodies to hpv were detected more often in women who had had multiple pregnancies than in women who had never been pregnant . 
 a similar observation was made by trottier et al,34 who also agreed with the view that parity is a risk factor for acquisition of hpv infection . according to the american cancer society,49 women who have had three or more full - term pregnancies have an increased risk of developing cervical cancer due to having unprotected intercourse to become pregnant , 
 further , studies have pointed to hormonal changes during pregnancy as a possible factor making women more susceptible to hpv infection or development of cancer.49 women in our study who had their sexual debut aged 2023 years had the highest prevalence of hpv antibodies . 
 this agrees with previous reports of an increased hpv among women who indulge in sexual activity at an early age.25,30,32,41 acquisition of hpv infection is strongly associated with sexual behavior . 
 the prevalence of hpv increases with number of sexual partners and earlier sexual debut.3 antibodies to hpv were detected more often in women who had had their first child at age older than 30 years . in 2012 , 
 the american cancer society reported that women who were younger than 17 years when they had their first pregnancy were almost twice as likely to develop cervical cancer later in life than women who did not become pregnant until they were aged 25 years or older.49 abnormal discharge was found in 37.7% of our women , although this finding was not statistically significant . 
 abnormal discharge is the commonest sign observed when there is infection in the reproductive organs.50 discharge is always seen when neoplasia is present and in the advanced stages of hpv infection.51 the majority of the women attending our reproductive health clinic had normal vaginal discharge . 
 abnormal vaginal symptoms included itching , rash , and ulcer , with vaginal itching being the most common ( p=0.059 ) . 
 half of the women with a family history of cervical cancer had antibodies to hpv . 
 cervical cancer may run in some families . if cervical cancer is known in a family to be genetic , the chances of developing the disease are 23 times higher than if no one in the family has it.49 some researchers suspect that in some cases this familial tendency is caused by an inherited condition that makes some women less able to fight off hpv infection than others.49 four of the 13 women who had a history of or some signs of whim as a genetic disorder had antibodies to hpv , but the antibodies were not detected with a statistically significance difference . 
 it has however been reported that the main clinical problem of patients with whim , is unusual susceptibility to infection with hpv51,52 and the most common signs are repeated bacterial infections , neutropenia and extensive infection with hpv leading to dermal and genital warts.52 
 hpv igg antibodies were detected in 42.9% of the women enrolled in this study , indicating that the women had been infected and that the virus is circulating with a high prevalence in kaduna state . 
 infection with hpv is a major risk factor contributing to the development of cervical intraepithelial neoplasia and invasive cervical carcinoma . 
 women aged 4549 years and those who had their sexual debut when aged 2023 years had the highest seroprevalence of hpv . 
 infection with hpv varied with the women s reproductive characteristics , sexual behavior , and sociodemographic factors but did not reach statistically significant levels except for educational status , tribe , and religion . 
 in view of the high prevalence of antibodies to hpv detected in this study , which denotes a high infection rate in the area , we recommend introduction of a subsidized hpv vaccine in our national immunization schedule in other to prevent the current scourge of cervical cancer in nigeria . 
 two prophylactic vaccines , ie , gardasil ( merck and co , inc . , whitehouse station , nj , usa ) and cervarix ( glaxosmithkline , london , uk ) , that are safe and effective against anogenital hpv,53 are currently available for the prevention of genital hpv infection.1 in view of the results of the present study and those of another recent study showing low knowledge of hpv and its vaccines among nigerian mothers with a high awareness for cervical cancer but little knowledge of its link to hpv,12 we recommend improving knowledge at the population level by education on the mode of transmission of the virus and the risks associated with the virus as a cause of cervical cancer . in addition , cervical cancer screening programs need to be put in place in nigeria , given that cervical cancer has been shown to disproportionately affect african american women , who are nearly twice more likely than european american women to die of the disease.22,54 it is important to screen women for anti - hpv , and if positive , detect the genotypes . 
 those found to be infected with the high - risk genotypes should have papanicolaou screening annually in order to prevent cervical cancer . 
 the majority of the women who took part in this study could not communicate in the english language and used an interpreter , so some of the data might not accurately reflect their sociodemographic , reproductive characteristics , or sexual behavior . 
 in addition , some were not willing to disclose all information concerning their reproductive characteristics and sexual behavior . 
 also , we were not able to determine the presence of hpv dna in hpv igg - positive serum .","backgroundhuman papillomavirus ( hpv ) is the cause of 90%95% of squamous cell cancers . 
 persistent infection with high - risk hpv can lead to development of precancerous lesions of the cervix in 5%10% of infected women , and can progress to invasive cervical cancer 1520 years later . 
 this study was conducted to determine the seroprevalence of hpv immunoglobulin g ( igg ) antibodies among women of reproductive age attending a reproductive health clinic at ahmadu bello university teaching hospital , zaria , nigeria.methodsthe study was descriptive , cross - sectional , and experimental , combining the use of a structured questionnaire and analysis of serum samples obtained from 350 consecutive consenting women . 
 the serum samples were analyzed for igg antibodies to hpv by enzyme - linked immunosorbent assay.resultswe found a seroprevalence of 42.9% ( 150/350 ) for igg antibodies to hpv in these women . 
 women aged 4549 years and those who had their sexual debut aged 2023 years had the highest hpv seroprevalence , ie , 50% ( 57/114 ) and 51.1% ( 46/90 ) , respectively . 
 presence of antibodies varied according to sociodemographic factors , but was significantly associated with educational status , tribe , and religion ( p<0.05 ) . 
 human papillomavirus infection was not significantly associated with the reproductive characteristics and sexual behavior of the women . 
 antibodies to hpv were detected in 50.0% ( 9/18 ) of women with a family history of cervical cancer and in 30.8% ( 4/13 ) of those with a history or signs of whim ( warts , hypogammaglobulinemia , immunodeficiency , myelokathexis ) syndrome as a genetic disorder ( p>0.05).conclusionfurther studies are needed to determine the hpv serotypes and evaluate the risk of natural development of hpv - related malignancies among women in the study area .",pubmed
"today s world witnesses rapid innovations and developments in the area of health care ( 1 ) , so that medical knowledge has expanded rapidly over the past fifty years ( 2 ) and the number of scientific papers published has doubled over 10 years ( 3 ) . on the other hand , studies indicate that in the patient s beside , 5 questions per every one hospitalized patient and about 2 questions per every one outpatient are raised for the physicians ( 4 ) . 
 knowledge transfer from research to routine clinical practice is one of the main obstacles preventing from benefiting medical sciences ( 5 ) . 
 some have assessed that more than 40% of the patients do nt receive care in accordance with the best scientific evidences and 20% of patients receive care which is not sufficient or harmful ( 6 ) . 
 generally there is a gap between what physicians do in their routine practice and what they must do ( 7 ) . 
 evidence based medicine ( ebm ) is a new paradigm and philosophy which attempts to direct clinical services and care toward effective , efficient services with minimal complications and errors . 
 in addition , ebm is influential in filling the knowledge gap in helping physicians to find the best evidence for patients care . 
 thus , it is a systematic approach for solving clinical problems which allows integration of available best evidence with clinical experience and preferences and values of the patients ( 8) . 
 this new paradigm may confirm or reject previous acceptable practice methods or may suggest new care methods which are more effective and efficient and less vulnerable ( 9 ) . 
 the main challenge today is transfer and translation of knowledge which help physicians to make the best clinical decisions based on the best evidence in the minimum time ( 10 ) . using the best scientific evidence , 
 sources can be distributed equitably and demands for  ineffective  treatment would be removed and effective services would be assured ( 11 ) . regarding the facts mentioned it seems that common sources are not effective enough for obtaining information on the current method . as it was noted , evidence based medicine is integration of the best available evidence with clinical experiences and patients preferences , and thus it is a solution for problem of lack of update information for physicians ( 12 ) . despite of these benefits , currently there is several factors affect the use of evidence - based medicine and evidence - based policy ( 13 , 14 ) . regarding the role and importance of paradigm of evidence - based practice and its remarkable impact on the effectiveness and efficiency of clinical services and healthcare , development of an integrated system seems necessary in order to manage dispersed data and ensure using evidences in clinical decision making . 
 hence , in this paper a model is presented in order to produce and implement evidence in the form of a system . in order to achieve higher goals such as promotion of clinical effectiveness , patient safety , effectiveness and improvement of health outcomes , 
 national evidence - based health care system insures the best evidence is generated , stored , used , shared and finally monitored and evaluated in order to provide effective and safe services in a systematic process and according to needs of different stakeholders . 
 national health care system is one of the main subsystems of the health system which is responsible for providing health service and health care . 
 in fact , subsystem of health service delivery is regarded as the most objective and most tangible function of the health system and it reflects impacts of other health system functions . 
 the ultimate goal of health care system in every country is maintaining and improving community health . 
 achievement of this goal depends on effectiveness of delivered services and consistency of the services with national and local priorities . on the other hand , among these functions 
 , stewardship is considered as the central function of the health system and the trusted and reliance point of health systems in achievement of their goal . 
 this function means determining and enhancing excusive rules and providing directions and strategies for all actors of the system and accepting accountability at highest level which ministry of health and medical education on behalf of the government is responsible for this task . 
 stewardship status and social accountability of the governments necessitates that national health care system to provide the best quality health care and service for the people . 
 clinical effectiveness. in order to achieve clinical effectiveness , the best practice should be realized in the country . 
 the requirement for the best practice is insuring reference to the best local evidence in different parts of the health care . 
 the system which provides reference to the best local evidence and guarantees evidence based decision making ( ebdm ) in practice is called evidence based health care system ( ebhcs ) . in other words , a system which is able to provide local knowledge required by evidence based clinical decision making ( ebcdm ) and guarantee and institutionalize its application among care providers , 
 national health care system is one of the main subsystems of the health system which is responsible for providing health service and health care . 
 in fact , subsystem of health service delivery is regarded as the most objective and most tangible function of the health system and it reflects impacts of other health system functions . 
 the ultimate goal of health care system in every country is maintaining and improving community health . 
 achievement of this goal depends on effectiveness of delivered services and consistency of the services with national and local priorities . on the other hand , among these functions 
 , stewardship is considered as the central function of the health system and the trusted and reliance point of health systems in achievement of their goal . 
 this function means determining and enhancing excusive rules and providing directions and strategies for all actors of the system and accepting accountability at highest level which ministry of health and medical education on behalf of the government is responsible for this task . 
 stewardship status and social accountability of the governments necessitates that national health care system to provide the best quality health care and service for the people . 
 clinical effectiveness. in order to achieve clinical effectiveness , the best practice should be realized in the country . 
 the requirement for the best practice is insuring reference to the best local evidence in different parts of the health care . 
 the system which provides reference to the best local evidence and guarantees evidence based decision making ( ebdm ) in practice is called evidence based health care system ( ebhcs ) . in other words , a system which is able to provide local knowledge required by evidence based clinical decision making ( ebcdm ) and guarantee and institutionalize its application among care providers , 
 this paper is a study of comparative type which has been written in three stages : 
 investigation of structure and process of evidence based practice in selected countries , investigation and analysis of current status in iran in this regard in terms of service delivery and medical educationinvestigation and recommendation of strategies and methods which make model implementation more feasible in the country . 
 investigation of structure and process of evidence based practice in selected countries , investigation and analysis of current status in iran in this regard in terms of service delivery and medical education investigation and recommendation of strategies and methods which make model implementation more feasible in the country . in order to investigate status of evidence based practice system , 
 following related literature review , some developed countries including england , usa , canada , australia , and scotland were selected which were leading countries in this regard . using search engines of google scholar , pubmed and implementation science , key terms of  evidence based medicine ,  evidence based practice and  evidence based national system were searched accompanied by the name of the countries . used sources included studies related to introduction and analysis of data bases and system associated to responsible agencies in respective countries . 
 evidence based practice were studied in the respective countries in terms of dimensions of organization and stewardship , knowledge products , distribution and support of evidence application and the way of performance monitoring . 
 results of these studies are given in table 2 . in order to investigate and analyze the status in the country , 
 12 of the scholars , instructors and experts related to the subject were selected using purposeful sampling and focus group discussion was used in order to analyze and classify . 
 strengths and weak points and barriers to evidence based practice in the same areas ( organization and stewardship , used knowledge products , distribution and support of evidence application , and performance monitoring ) as well as knowledge of the physicians on the evidence based practice and motives of the physicians for change in practice system were evaluated and analyzed . 
 finally , focus group discussion was used in order to investigate strategies which are applicable and feasible in iran , and strategies were studied in terms of the following aspects : 
 needed structureprocessesknowledge products and outputsmotivationneeded support regulationperformance monitoring knowledge products and outputs needed support regulation performance monitoring delphi technique was used so as to finalizing the country s status and results of the focus group discussion were sent to 46 experts and it was finalized following twice modification . 
 evidence based health care system in england , usa , canada , australia , new zealand , and scotland were studied in terms of the organization and stewardship , knowledge products , distribution and support of evidence application and performance monitoring . 
 according to studies , insuring an evidence based practice culture in the country requires a system called national evidence - based health care system which consists of three subsystems as follows : 
 national system of clinical knowledge management , national evidence - based practice systemintegrated national network of clinical effectiveness 
 . national system of clinical knowledge management , national evidence - based practice system integrated national network of clinical effectiveness . national clinical knowledge management system ( nckms ) includes activities , actions , processes and structures which promote , facilitate and guarantee clinical knowledge management in the country ( fig . 
 1 ) . this part of the national evidence based health care system attempts to provide the best evidence for  evidence based decision making through analysis , synthesis and localization of the fragmented raw data , if necessary . 
 when subject of insuring clinical knowledge management through frameworks of the national system of clinical knowledge management is raised , it means insuring monitoring and providing possibility for regular control of the new data which are published in scattered manner . 
 the outcome of processes and actions carried out in the national system of the clinical knowledge management regarding dominant social , economic , cultural circumstances and values of the country , leads to products which are called knowledge products . 
 these products which are considered as the main constituents of the system in units of the clinical knowledge management , are produced based on two approaches : reactive and proactive . 
 reactive approach is the approach in which reaction is given regarding current problems and difficulties in the health system of the country and by developing and distributing appropriate knowledge products it is attempted to solve the problems . proactive or prospective approach is the approach in which clinical knowledge management is done in parallel to knowledge push in the world . 
 in other words , monitoring of the new information is done with a proactive approach regarding national priorities in different clinical levels in order to match knowledge resulting from this information with national needs of the country . 
 clinical knowledge management units , multi - disciplinary teams of clinical knowledge management , and clinical knowledge managers are considered as the most important pillars of the national system of clinical knowledge management . 
 in addition , this system provides structure , frameworks , incentives and requirements so as to achieve its goals so that production of knowledge products needed in the country are facilitated , promoted and insured . 
 knowledge products produced in this system include pay knowledge , health technology reporting , clinical guidelines , and systematic reviews . in case 
 knowledge products possess considerable value added , they are considered in the national committee on clinical policy making and are imparted as a 
 clinical policy. in this case , its observation would be mandatory for all provides on public sector and non - governmental sector and licensing system at individual level and validating system at organizational level support such policies . 
 national evidence based practice system ( nebps ) includes actions , activities , processes and structures which promote , facilitate and guarantee usage of clinical policies or knowledge products produced at national level ( table 3 ) . 
 as it can be seen from table 3 , national evidence based practice system is composed of various components . in this system , 
 evidence based provides in evidence based health centers that their structure , processes and infrastructures facilitate , promote and guarantee evidence based decision making , are seeking for establishment of such a system that institutionalize delivery of the most effective services to patients through production and application the local knowledge products . 
 evidence based care providers make reference to knowledge products produced by national system of clinical knowledge management and local knowledge products produced by them in order to make decision . 
 providers in this system have a reactive approach to knowledge production since no clinical decision - making body would be able to acquire all the needed knowledge and predict all clinical questions needing answer . 
 thus , both evidence based care providers and clinical knowledge managers produce evidence , but there is essential difference in features and characteristics of the process of evidence production by these two groups ( table 4 ) . evidence demonstrated that less than 20% of the clinical actions and decisions of these providers are based on the best acceptable evidence ; while evidence based providers have the necessary skill for achieving lifelong learning and thus , they are constantly trying to enhance their personal practice and increase effectiveness and safety of their interventions . 
 hence , evidence based care providers are divided into three levels based on their performance in production and application of the knowledge products , that is , care providers of level one , two and three . in this system , in addition to classification of care providers , service provider centers are also classified based on their evidence based units performance . 
 this system is part of national integrated clinical effectiveness network ( nicen ) which facilitates evidence based practice in the country . in this system , 
 there such components as electronic evidence based performance portfolio , certificate and characteristics of the evidence based centers and departments , educational service on principles of evidence based decision making and evidence based practice . 
 national evidence based practice system ( nebps ) includes actions , activities , processes and structures which promote , facilitate and guarantee usage of clinical policies or knowledge products produced at national level ( table 3 ) . 
 as it can be seen from table 3 , national evidence based practice system is composed of various components . in this system , 
 evidence based provides in evidence based health centers that their structure , processes and infrastructures facilitate , promote and guarantee evidence based decision making , are seeking for establishment of such a system that institutionalize delivery of the most effective services to patients through production and application the local knowledge products . 
 evidence based care providers make reference to knowledge products produced by national system of clinical knowledge management and local knowledge products produced by them in order to make decision . 
 providers in this system have a reactive approach to knowledge production since no clinical decision - making body would be able to acquire all the needed knowledge and predict all clinical questions needing answer . 
 thus , both evidence based care providers and clinical knowledge managers produce evidence , but there is essential difference in features and characteristics of the process of evidence production by these two groups ( table 4 ) . 
 evidence demonstrated that less than 20% of the clinical actions and decisions of these providers are based on the best acceptable evidence ; while evidence based providers have the necessary skill for achieving lifelong learning and thus , they are constantly trying to enhance their personal practice and increase effectiveness and safety of their interventions . 
 traditional care providers have the least self - learning and therefore effort . hence , evidence based care providers are divided into three levels based on their performance in production and application of the knowledge products , that is , care providers of level one , two and three . in this system , in addition to classification of care providers , service provider centers are also classified based on their evidence based units performance . 
 this system is part of national integrated clinical effectiveness network ( nicen ) which facilitates evidence based practice in the country . in this system , 
 there such components as electronic evidence based performance portfolio , certificate and characteristics of the evidence based centers and departments , educational service on principles of evidence based decision making and evidence based practice . 
 preserving and promoting service quality and health care is one of the major tasks of the governments . 
 trustee s role and social responsibility of the ministry of health and medical education as a custodian of the country s health system bounds it to provide infrastructures , processes and frameworks so that national service delivery system offer services and health care with the highest quality(20 ) . 
 clinical effectiveness is one of quality aspects , requirement of which is access to the best evidence . 
 thus evidence base health care system ensures that the best evidence are produced , stored , disseminated , used and shared for providing effective and safe services through a systematic process according to needs of different stakeholders and finally it is monitored and evaluated . 
 it can not be achieved without active participation of ministry of health and medical education and other involved organizations in health policy making . 
 investigation of consideration process and emphasis on the health quality and care in uk and usa indicates that without imposing macro interventions at national level , quality improvement and especially clinical effectiveness promotion as the core of quality would not be possible(20 ) . 
 for example , report by institute of medicine ( iom ) in usa in 2001 mentioned several basic interventions for quality promotion all of which suggest involvement of health system stewardship in this process and following cases can be mentioned : prohibition of approval of drugs with name , packaging , color and appearance similar to it , change in payment system , search management and access to the best evidence , changes in the educational system , mandatory use of computerized physician order entry ( cpoe)- and use of reminders(21 ) . 
 lack of legal requirement to rely on the best available evidence , insufficient skills in the use of evidence in daily practice , evidence - based decision - making , inability to produce documentary evidence , lack of financial and non - financial incentives to produce evidence according to local conditions and similar experiences in countries like australia(22 ) in the nhmrc program(22 ) , uk in nice program(23 ) , scotland in sign program(24 ) , usa in ahrq program(25 ) , canada in cma program(26 ) and new zealand in nzgg program(27 ) demonstrate that only presence of national plan and macro interventions can guarantee evidence production and application . 
 implementation of the evidence based practice requires a system and this system needs a set of micro and macro strategies enabling facilitations , promotion and guaranteeing production and application of the clinical knowledge in the country . 
 thus , the government should adopt policies and interventions for changing behavior in three levels of promotional , facilitation and guarantying . 
 policies and interventions should influence on both at individual and organization level and certainly implementation of such interventions needs external and internal cooperation of the various stakeholders in the country . 
 ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .","background : regarding the role and importance of paradigm of evidence based practice and its remarkable impact on the effectiveness and efficiency of clinical services and healthcare , development of an integrated system seems necessary in order to manage dispersed data and ensure using evidence in clinical decision making , thus the aim of this study was designing a model for implementing national system of evidence based health care in iran.methodos:this paper is a study of comparative type which has been written in three stages : investigation of structure and process of evidence based practice in selected countries , investigation and analysis of current status in iran in this regard and recommendation of strategies which make model implementation feasible in the country . 
 such methods as review of literature , focus group discussion and delphi technique were used for investigation.results:according to studies , insuring an evidence based practice culture in the country requires a system called national evidence based health care system which consists of three subsystems including national system of clinical knowledge management , national evidence - based practice system and integrated national network of clinical effectiveness.conclusion:the ultimate goal of health care system in every country is maintaining and improving community health . 
 achievement of this goal depends on effectiveness of delivered services and consistency of the services with national and local priorities . in order to achieve clinical effectiveness , 
 the best practice should be realized in the country , implementation of which requires a set of macro and micro strategies enabling facilitation , promotion or guaranteeing clinical knowledge application in the country .",pubmed
"human metapneumovirus ( hmpv ) is a negative single - stranded rna virus of the family paramyxoviridae and closely related to the avian metapneumovirus ( ampv ) subgroup c [ 1 , 2 ] . 
 hmpv is an important aetiological agent of respiratory tract infection ( rti ) in infants , or senior and immunocompromised individuals . 
 this infection caused different symptoms ranging from influenza like syndromes ( i.e. , fever , cough , and rhinorrhea ) to severe lower respiratory tract infection . 
 previous studies have shown that many children exposed to this virus and also easily to be reinfected as common [ 35 ] . 
 therefore , hmpv is becoming as a major concern in child respiratory tract viral infection . 
 genome sequencing and comparative analysis provides us a useful approach to analyze the pathogenicity of organisms . 
 moreover , this analysis might also provide us an approach to understand its evolution history and cell - host interaction . as previously reported hmpv genome is approximately 13  kb in length , and the gene composition from 3 terminal to 5 terminal 
 is n - p - m - f - m2 - 1/m2 - 2-sh - g - l [ 6 , 7 ] . 
 comparative analysis suggests that its genomic organization is similar to human respiratory syncytial virus ( hrsv ) , which just lacks 2 nonstructural genes , ns1 and ns2 . 
 moreover , hmpv has been demonstrated the existence with two main genetic lineages termed as subtype a and b , which also containing within them the subgroups a1/a2 and b1/b2 , respectively . 
 the genetic diversity analysis shows the a2 sublineage exhibits the greatest diversity among all the sublineages of hmpv . 
 as we all know , there are differences in the frequency of occurrence on synonymous codons in coding dna , which termed as synonymous codon usage bias . 
 briefly , there are 64 different codons ( 61 codons encoding for amino acids plus 3 stop codons ) in each organism , but only 20 different translated amino acids . 
 these alternative codons for the same amino acids are termed as synonymous codons . in general 
 , codon usage variation may be the product of natural selection and/or mutation pressure for accurate and efficient translation in various organisms [ 810 ] . 
 synonymous codon usage bias on virus can provide us with a better understanding on the evolution profile , gene expression , and virus - host interaction [ 1114 ] . however , there is still lacking about codon usage pattern of hmpv genome and its major influence factors . 
 herein , we firstly performed the comparative analysis of synonymous codon usage in hmpv genomes and analyzed their influencing factors . 
 this study will provide a new insight to understand the pathogenicity and its evolution history of hmpv . 
 in this study , a total 17 complete hmpv genomes which representing two genotypes were retrieved from ncbi ( http://www.ncbi.nlm.nih.gov/ ) until december , 2011 . the serial number ( sn ) , genbank number , genotype , and other information 
 moreover , 10 ampv genomes sequences were retrieved from ncbi database as reference frame ( in supplemental table  1 supplementary material available online at doi:10.1155/2012/460837 ) . to investigate the characteristics of synonymous codon usage , relative synonymous codon usage ( rscu ) values of each complete coding region in 17 hmpv genomes and 
 the rscu value of each codon for their amino acid was calculated as previously described . 
 a codon with an rscu value of more than 1.0 has a positive codon usage bias , while a value of less than 1.0 has a negative codon usage bias . 
 when the codon with rscu values close to 1.0 , it means that this codon is chosen equally and randomly . 
 the codon usage data of human cell and bird cell were obtained from the codon usage database online ( http://www.kazusa.or.jp/codon/ ) . 
 the effective number of codons ( enc ) is used to measure deviation from expected random codon usage of hmpv and is independent of hypotheses involving natural selection . 
 if only one codon is used for each amino acid , this value would be 20 , while all of codons are used equally , it will be 61 . 
 moreover , the index of gc3s was used to calculate the fraction of the nucleotides g + c content at the synonymous third codon position ( excluding aug [ met ] , ugg [ trp ] , and the termination codons ) . 
 multivariate statistical analysis can be used to explore the relationships between variables and samples . in this study , 
 correspondence analysis was used to investigate the major trend in codon usage variation among genomes . in this study , 
 the complete coding region of all 17 hmpv genomes was represented as a 59 dimensional vector , and each dimension corresponds to the rscu value of one sense codon ( excluding met , trp , and the termination codons ) . 
 correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern . 
 in order to investigate the synonymous codon usage of hmpv , we calculated various rscu values of various codons from 17 different strains , including different genotypes . 
 as shown in table 3 , the preferred codons in hmpv are gca , aga , aau , gau , ugu , caa , gaa , gga , cau , cau , auu , uua , aaa , uuu , cca , uca , agu , aca , uau , guu . interestingly , all preferred codons in hmpv genomes are ended with a / u , while none of them is ended with g / c . 
 this result suggests that hmpv genome has a great synonymous codon usage bias , and this phenomenon might highly associate with the nucleotide composition in its genomes . 
 as shown in table 2 , the g + c content of hmpv genome is 36.91% , which shares similar extent with another rna virus . 
 there are over 68.57% codons are ended with a / u among hmpv genomes , and 40.87% codons are a3 end , and 27.7% codons are u3 end . 
 this high abundance of a / u nucleotides is consistent with all preferred codons , which are ended with a / u . 
 this phenomenon reflects that nucleotide composition is the main force to affect the codon usage bias in hmpv genome . 
 moreover , as shown in table 2 , the enc values among hmpv genomes show a range from 45.127 to 48.28 , and its average value of 45.785 and sd value of 0.8458 . 
 the stable enc values suggest that their genomic compositions are much conserved among hmpv genomes . 
 natural selection and mutation pressure have been considered to be two key factors which have effect on codon usage patterns of organisms . in order to investigate whether mutation pressure or natural selection as a determinative factor for codon usage mutation in hmpv , we calculated correlation relation between a% , u% , g% , c% , gc% and a3% , u3% , g3% , c3% , gc3% . 
 as shown in table 4 , there exhibits a very complex correlation map observed in nucleotide compositions . in detail 
 , u3% has a significant positive correlation with u% ( r = 0.7821 , p < 0.01 ) , while shared negative correlation with c% ( r = 0.7153 , p < 0.01 ) and gc% ( r = 0.7474 , p < 0.01 ) . 
 c3% has positive correlation with c% ( r = 0.8331 , p < 0.01 ) and gc% ( r = 0.7880 , p < 0.01 ) , and negative correlation with a% ( r = 0.6199 , p < 0.01 ) and g% ( r = 0.5846 , p < 0.05 ) . 
 gc3% has significant positive correlation with c% ( r = 0.7178 , p < 0.01 ) and gc% ( r = 0.8052 , p < 0.01 ) , but has negative correlation with u% ( r = 0.7342 , p < 0.01 ) . 
 interestingly , the gc3% shows positive correlation with c% , while shows negative correlation with g% . 
 we calculated the gc3 skew by using formula as cg3  skew = ( c3  g3)/(c3 + g3 ) . 
 the gc3 skew of hmpv range s from 0.371 to 0.592 , which reveals that gc composition involved in the codon usage bias . 
 these data suggest that the nucleotide constraint might play an important role in influencing synonymous codon usage bias . 
 furthermore , the correlation analysis of first two principle axes ( f1 and f2 ) in hmpv and its nucleotide contents were performed ( table 5 ) . 
 apparently , the first principle axis ( f1 ) has a significantly negative correlation with u3% , and negative correlation with gc3% . 
 this result suggests that nucleotide u3% and gc3% are the major factor influencing the synonymous codon usage pattern in hmpv genome . 
 moreover , we observed the second principle axis ( f2 ) shared a significant positive correlation with g3% , and negative correlation with c3% and gc3% . 
 therefore , compositional constraint is a major factor which is involved in shaping the pattern of synonymous codon usage bias in hmpv genome . to investigate the effect of different hpmv genotypes on synonymous codon usage 
 , we analyzed the codon usage bias of different genotypes with correspondence analysis . from the correspondence analysis , the first dimension variable f1 and 
 the second dimension variable f2 can reflect 43.27% and 33.38% of total mutation , respectively . 
 as the plot of correspondence analysis shown ( figure 1 ) , each genotype is mainly separated and clustered into two clades . 
 however , the sublineage of each genotype did not exhibit any significant difference among them . 
 but this might be due to the limited number of hpmv genomes available in current study . 
 therefore , the phylogenetic distant might effect on the variation of synonymous codon usage in hmpv , and this difference might reflect on their biological effect , such as viral replication , virulence , and so forth . from the enc - gc3% plot analysis ( figure 2 ) , 
 the plots of each hmpv genomes are all under the expected curve , none of them shows above the curve . 
 moreover , there are still some other factors that can effect on the codon usage bias of hmpv . in the current study 
 as shown in table 3 , the pattern of synonymous codon usage in hmpv shows a complementary profile , which shows in human cell . in detail , hmpv and human host cell shared only 1 preferred codon ( aga ) , which encoded for arginine , while there are 17 different preferred codons between them . as a reference frame , 
 ampvs were enrolled in this study , and it also shows a complementary pattern with its host , bird cell . 
 the comparative analysis among hmpv and human host cell , hmpv and ampv host bird cell , and hmpv and ampv were analyzed . to compare the complementary ability of hmpv with bird cell , 
 there are more overlays ( 6 preferred codons overlay ) than hmpv with its human host cell ( only 1 preferred codons overlay ) . 
 this result shows human cell has much more complementary pattern with hmpv than bird cells . 
 this might be more suitable for hmpv survive and persist infect in human host environment . 
 this result also suggests that host factor plays an important role in codon usage bias in hmpv . 
 this complementary trend will benefit for virus replication instead of competitive with its host and it might help us to understand the mechanism of hmpv persistent infection . 
 interestingly , hmpvs are shares with more than 15 preferred codons to ampv , which might be due to a close phylogenetic distance . 
 synonymous codon usage analysis can reveal much about virus genome . to understand the extent and causes of codon usage bias is essential for studying the viral evolution , particularly the interaction between viruses and host immune response . in this study 
 as we know , the variation and evolution of virus generally happened in the changes of nucleotide composition . therefore , the nucleotide composition bias is the main force to influence the synonymous codon usage patterns . in this study 
 first of all , in hmpv , all preferred codons are ended in a / u nucleotide , which occupied the majority of nucleotide composition in hmpv genome . 
 this phenomenon confirmed that nucleotide composition was the main force in shaping the pattern of codon usage . 
 secondly , enc was used to quantify the codon usage bias , which is one of the best overall estimators of absolute synonymous codon usage bias . in this study , we observed enc of these genomes fluctuated from 45.13 to 48.28 with a mean 45.78  0.85 . this enc value of hmpv is consistent with other previously reported rna virus in the same family paramyxoviridae , ranging from 43.8 to 55.1 , that is , measles virus 55.1 , mumps virus 54.3 , parainfluenza-3 virus 43.8 , and respiratory syncytial virus 44.3 [ 8 , 24 , 25 ] . 
 moreover , the enc of hmpv is more close to respiratory infection agents , rsv and parainfluenza-3 , which reveals that the similar extent of codon usage bias among viruses might have similar infection syndrome . 
 this observation helps us to address an interesting assumption that synonymous codon usage bias of virus might associate with its pathogenicity . 
 mutation pressure and natural selection are generally treated as the main factors that account for codon usage bias in different organisms . 
 enc - gc plot was considered as a part of the general strategy to investigate patterns of synonymous codon usage [ 9 , 18 , 26 ] . 
 herein , all the plots are laid below the expected curve , suggesting that codon usage bias in all these 17 hmpv genomes was principally influenced by mutation bias , which consistent with that mutation pressure rather than natural selection is the most important determinant of the codon usage in human rna virus [ 8 , 2731 ] . 
 this observation can be explained as the mutation rates in rna viruses much higher than those in dna viruses . 
 mutation pressure is the main force in shaping synonymous codon usage bias of rna virus . 
 however , based on correspondence analysis , we observed an interesting phenomenon that codon usage bias in hmpv showed distinct differences among different phylogenetic types . 
 it might suggest that codon usage bias plays an important role in hmpv evolution history . 
 this similar phenomenon also observed in several other viruses , this might reflect that phylogenetic difference is a common influencing factor in shaping codon usage bias [ 25 , 27 , 3034 ] . 
 therefore , we hypothesize that the difference of codon usage bias might influence its virulence in different genotypes .   in this study , we also observed that hmpv showed a complementary trend with human cells by comparing the codon usage . 
 this complementary will be benefit for the survive of virus , which can keep replication by using the nonpreferred codons in the host cell without competition , and this could be one of the mechanisms of virus persistent infection in the human environment . moreover , this pattern might also be caused by the longitude selection and evolution between human hosts with virus . 
 therefore , this characteristic is important for hmpv keeping balanced with their host on the codon usage side , and also for understanding the cell - host interaction and viral evolution . 
 in summary , we firstly reported the synonymous codon usage pattern in hmpv genomes and revealed that mutation pressure is the main force in shaping its codon usage bias . 
 phylogenetic difference and host factors are also discussed , and this information can provide better understanding on the molecular evolution and its pathogenicity of hmpv .","human metapneumovirus ( hmpv ) is an important agent of acute respiratory tract infection in children , while its pathogenicity and molecular evolution are lacking . herein , we firstly report the synonymous codon usage patterns of hmpv genome . 
 the relative synonymous codon usage ( rscu ) values , effective number of codon ( enc ) values , nucleotide contents , and correlation analysis were performed among 17 available whole genome of hmpv , including different genotypes . 
 all preferred codons in hmpv are ended with a / u nucleotide and exhibited a great association with its high proportion of these two nucleotides in their genomes . 
 mutation pressure rather than natural selection is the main influence factor that determines the bias of synonymous codon usage in hmpv 
 . the complementary pattern of codon usage bias between hmpv and human cell was observed , and this phenomenon suggests that host cells might be also act as an important factor to affect the codon usage bias . 
 moreover , the codon usage biases in each hmpv genotypes are separated into different clades , which suggest that phylogenetic distance might involve in codon usage bias formation as well . 
 these analyses of synonymous codon usage bias in hmpv provide more information for better understanding its evolution and pathogenicity .",pubmed
"as more and more genomes are automatically sequenced , comprehensive protein annotation is a needed step after gene identification . even in good annotated genomes ( human , mouse ) about 30% of all proteins are not functionally identified ( 13 ) , and thus often a similarity search will not be sufficient . here , we present a suite of protein tasks , protsweep , domainsweep and 2dsweep , which perform analysis from sequence similarity to small domains and structural elements . 
 this includes similarity searches against protein sequence databases and specialized motif collections , prediction of secondary structural elements , attributing each sequence to known super - families , protein localization prediction , physicochemical protein characteristics and domain functional assignation . 
 our strategy for assigning relevant functional roles is based on the joint use of both global ( homology similarity ) and local ( domain and motif ) sequence similarities ( 4 ) . 
 several query sequences can be uploaded by the usual  copy & paste procedure into the input box using fasta format . 
 if more than one sequence is to be queried , a multiple fasta file can be used . 
 then the user will be redirected to an application page , and the runbutton can start the task . 
 additionally , there is a link to an online help , indicated with a  ? 
 , with the following topics : short description , programs employed , algorithm , output , additional options and acknowledgments . 
 results can be received by selecting the tab  go to results page. the results are provided as html for visual inspection or can be downloaded as xml for storage in private databases . in case of error when clicking on the application name in the results manager page , a log - file is displayed where more human readable error messages can be found . 
 standard protein databases used by the tasks like uniprot / swissprot , uniprot / trembl and refseq are automatically updated whenever new versions become available . concerning ensembl , 
 the situation is more complex . due to possible inconsistencies between the different ensembl api versions , which are used in the tasks , 
 it is not possible to automatically update the ensembl data and this needs to be done by hand . 
 the different databases and the way they were used in these pipelines are described in table 1 . 
 table 1databases used in the different pipelines , and the programs and parameters used to search themserverdatabaseslinksprogramparametersprotsweepuniprot / swissprothttp://www.ebi.ac.uk / swissprotblastp - nofilter -exp = 10.0uniprot / tremblhttp://www.ncbi.nlm.nih.gov / refseq / blastp - nofilter -exp = 10.0refseqftp://ftp.ebi.ac.uk / pub / databases / trembl / sptrembl / blastp - nofilter -exp = 10.0ensemblhttp://www.ensembl.org / blastp - nofilter -exp = 10.02dsweepdssphttp://www.sander.ebi.ac.uk / dssp / blastp - exp = 0.001 -extension = 10 - nogappedalignnrpep ( non - redundant ncbi protein database)ftp://ftp.ncbi.nih.gov / blast / db / fastapsiblast - b20000 -a5 -j2 -e0.001uniprot / swissprot + uniprot / trembl + updateshttp://www.expasy.ch / sprot / sprot - top.html 
 http://www.ebi.ac.uk/swissprotmsfgenerator-exp = 0.001 -overlap = 75 -customrange = 5 custompercentage =  80,60,50,45,40,35domainsweepprositeftp://ftp.expasy.ch / databases / prositemotifs & pfscandefaultpfamhmmhttp://www.sanger.ac.uk / software / pfam / hmmscan - lib = pfam.hmm -dprintsftp.bioinf.man.ac.ukhmmscan - lib = prints.hmm -dsmarthttp://smart.embl - heidelberg.de / hmmscan - lib = smart.hmm -dtigrfamshttp://www.tigr.org / tigrfams / index.shtml / hmmscan - lib = tigrfams.hmm -dscophttp://scop.mrc - lmb.cam.ac.uk / scopscopscandefaultblocksftp://ftp.ncbi.nih.gov / repository / blocks / unixblockssearcher - cutoff = 0.01 dinterprohttp://www.ebi.ac.uk / interpro / srs queriesprodomhttp://www.toulouse.inra.fr / prodom.htmlprodomblastdefault databases used in the different pipelines , and the programs and parameters used to search them 
 standard protein databases used by the tasks like uniprot / swissprot , uniprot / trembl and refseq are automatically updated whenever new versions become available . concerning ensembl , the situation is more complex . due to possible inconsistencies between the different ensembl api versions , which are used in the tasks , 
 it is not possible to automatically update the ensembl data and this needs to be done by hand . 
 the different databases and the way they were used in these pipelines are described in table 1 . 
 table 1databases used in the different pipelines , and the programs and parameters used to search themserverdatabaseslinksprogramparametersprotsweepuniprot / swissprothttp://www.ebi.ac.uk / swissprotblastp - nofilter -exp = 10.0uniprot / tremblhttp://www.ncbi.nlm.nih.gov / refseq / blastp - nofilter -exp = 10.0refseqftp://ftp.ebi.ac.uk / pub / databases / trembl / sptrembl / blastp - nofilter -exp = 10.0ensemblhttp://www.ensembl.org / blastp - nofilter -exp = 10.02dsweepdssphttp://www.sander.ebi.ac.uk / dssp / blastp - exp = 0.001 -extension = 10 - nogappedalignnrpep ( non - redundant ncbi protein database)ftp://ftp.ncbi.nih.gov / blast / db / fastapsiblast - b20000 -a5 -j2 -e0.001uniprot / swissprot + uniprot / trembl + updateshttp://www.expasy.ch / sprot / sprot - top.html 
 http://www.ebi.ac.uk/swissprotmsfgenerator-exp = 0.001 -overlap = 75 -customrange = 5 custompercentage =  80,60,50,45,40,35domainsweepprositeftp://ftp.expasy.ch / databases / prositemotifs & pfscandefaultpfamhmmhttp://www.sanger.ac.uk / software / pfam / hmmscan - lib = pfam.hmm -dprintsftp.bioinf.man.ac.ukhmmscan - lib = prints.hmm -dsmarthttp://smart.embl - heidelberg.de / hmmscan - lib = smart.hmm -dtigrfamshttp://www.tigr.org / tigrfams / index.shtml / hmmscan - lib = tigrfams.hmm 
 -dscophttp://scop.mrc - lmb.cam.ac.uk / scopscopscandefaultblocksftp://ftp.ncbi.nih.gov / repository / blocks / unixblockssearcher - cutoff = 0.01 dinterprohttp://www.ebi.ac.uk / interpro / srs queriesprodomhttp://www.toulouse.inra.fr / prodom.htmlprodomblastdefault databases used in the different pipelines , and the programs and parameters used to search them 
 protsweep is an approach to the functional characterization of unknown proteins based on a cascade of similarity searches . 
 it is well known that protein databases do not completely overlap and differ in their annotation quality ( 5 ) . 
 this task takes into account the significant differences among databases ( supplementary table 1 ) to improve the quality of the protein characterization . 
 it selects the order in which the databases have to be searched and combines the annotation found depending on the results . 
 protsweep classifies proteins into the following categories : identical , homolog , similar , weakly similar and putative proteins . 
 the query protein starts the blast ( 6 ) cascade against swissprot ( 7 ) first ( figure 2 ) . 
 we do take into account three parameters to classify the blast hits : ( i ) percentage of identity , ( ii ) 
 spercent. the two last parameters are related to the length of the total alignment , being 
 qpercent the percentage of the query sequence length covered in the alignment with the database hit and 
 spercent the percentage of the hit ( subject ) sequence length covered by the alignment ( figure 2 ) . depending on the classification of the blast hits according to these parameters and the hit protein annotation 
 if the hit has 100%  qpercent and  spercent and more than 98% identity , it is considered an identical protein and the swissprot i d will be searched in ensembl ( 8) . 
 if it is successful , all information from both databases will be combined ( supplementary table 2 ) and stored in the xml output . 
 if the i d can not be found in ensembl then a blast search is performed with the query protein against ensembl . 
 the best ensembl hit is selected and compared against the swissprot hit using the smith 
 if the identity between sequences is greater than 98% , then the information from both sources and the blast alignment will be added to the final output , if the identity is less , only swissprot annotation and the alignments will be added to the xml . 
 if the  qpercent and  spercent is between 80% and 98% and the identity is between 85% and 98% , the hit is classified as homologous and follows the same strategy with ensembl as already described ( figure 2 ) . in case , 
 spercent are greater than 85% , then the blast cascade continues with sptrembl and refseqprot . in the case that no identical or homologous hits can be found in any of the databases , the best similar hit among the three databases 
 is selected and classified as similar , weakly similar or putative ( figure 2 ) . 
 if the protein is characterized , information concerning the coding gene , about the splicing variants and orthologous genes is also provided . 
 depending on the degree of homology , protein function , transcript of origin , genomic localization , and go annotation or partial similarities will also be shown . 
 hypothetical proteins will only be presented in the result when no other information about identical or homologous proteins can be found in any of the databases ( supplementary figure 1 ) . 
 the web output of protsweep ( supplementary figure 1 ) is divided in five sections : ( i ) general information , ( ii ) identified protein and transcripts , ( iii ) features and functions , ( iv ) genomic localisation and ( v ) homology to other organisms / genes . the information provided in each of these sections is provided in figure 2 and supplementary table 2 . 
 the user has immediate access to all complete application outputs and database entries via hyperlinks . at the bottom of the html output 
 there is a link to the explanatory legend as well as to the xml output containing all the generated information . 
 domainsweep identifies the domain architecture within a protein sequence and therefore aids in finding correct functional assignments for uncharacterized protein sequences ( figure 3 ) . 
 it employs different database search methods to scan a number of protein / domain family databases . among these models , in increasing complexity , 
 are : prodom ( 10 ) , automatically generated protein family consensus sequences , prosite ( 11 ) regular - expression patterns , blocks ( 12 ) , ungapped position - specific scoring matrices of sequence segments , prints ( 13 ) sequence motifs , prosite profiles ( 7 ) , gapped position - specific scoring matrices and hidden markov models like pfam ( 14 ) , smart ( 15 ) , tigrfams ( 16 ) and scop ( 17 ) . 
 each database covers a slightly different , but overlapping set of protein families / domains . 
 each model has its own diagnostic strengths and weaknesses and for each of these protein / domain family databases used we have established different thresholds . 
 for example , in the case of the database pfam - a , we compare the input sequence against the hidden markov model profile of each pfam protein family . in principle , it is possible to decide the significance of a match upon its e - value . 
 however , there are a few complications such as that there is no analytical results available for accurately determining e - values for gapped alignments , especially profile hmm alignments . 
 we use as threshold the trusted cut - off value ( tc ) existing for each pfam family . 
 this value is the lowest score for sequences included in the family ( e.g. in the full alignment ) . 
 therefore , we consider a hit very significant if scores better than the trusted cut - off and at the same time has a significant e - value . in the case of scop , 
 we use the scop filtering mechanism to look for consistency in the hmmscan output , and filtering out inconsistent hits . in the case of smart 
 we use only the e - value . for each of the protein / domain databases used 
 afterwards domainsweep takes all true positive hits of all individual database searches for further data interpretation . 
 domain hits are listed as  significant : 
 if two or more hits belong to the same interpro family . 
 the task compares all true positive hits of the different protein family databases grouping together those hits , which are members of the same interpro family / domain.if the motif shows the same order as described in prints or blocks . 
 both databases characterize a protein family with a group of highly conserved motifs / segments in a well - defined order . 
 the task compares the order of the identified true positive hits with the order described in the corresponding prints or blocks entry . only hits in correct order 
 the task compares all true positive hits of the different protein family databases grouping together those hits , which are members of the same interpro family / domain . if the motif shows the same order as described in prints or blocks . 
 both databases characterize a protein family with a group of highly conserved motifs / segments in a well - defined order . 
 the task compares the order of the identified true positive hits with the order described in the corresponding prints or blocks entry 
 it is clear that any automatically produced sequence analysis implies a reasonable compromise between sensitivity and selectivity , and that no ideal recognition threshold exists that would allow for perfect separation of true and false similarities . 
 our thresholds tend to be rather conservative and stringent and thus the possibility of extending false positives is very limited . 
 the output in the web consists of two groups of graphs , those corresponding to the significant and putative hits , and one table output containing all reported protein domains ( supplementary figure 2 ) . 
 putative hit a cartoon of the sequence with the domain corresponding to the match , the hit i d , description , begin , end and gene ontology ( go ) annotation . 
 the user has immediate access to all complete application outputs and database entries ( via hyperlinks ) by clicking on the corresponding part of the picture . at the bottom of each graph 
 the table output contains all hits , ids , descriptions and links to the original output . 
 the xml output containing all the generated information is available via hyperlink at the bottom of the task output . 
 2dsweep identifies the structural domains in the protein and therefore aids in finding structural elements . 
 it reports on predictions for alpha - helix , beta - strand , coiled - coil and helix - turn - helix motifs , transmembrane regions , signal sequences , hydrophobicity , antigenicity , protease cleavage sites and more . 
 when predicting the secondary structure of a protein , it is useful to exploit the features of several available prediction algorithms rather than to rely on a single program . unfortunately , combining prediction methods on a large scale is complicated by the fact that prediction programs have very different input requirements and output formats . 
 some of them perform much better when they have a multiple sequence alignment covering different degrees of similarity as input instead of a single sequence . 
 we have developed msfgenerator , a program , which creates a multiple sequence alignment for a single protein sequence according to user , defined rules ( supplementary data msf ) . 
 it performs a blast search against a non - redundant protein database following different strategies that will generate different kind of alignments ( supplementary data msf , figure 4 ) . 
 the output of msfgenerator is an alignment in msf format ( multiple sequence file ) . 
 the generated msf will be used as input for four different structure prediction programs : psipred ( 18 ) , jnet ( 19 ) , prof ( 20 ) , and dsc ( 21 ) . 
 psipred is a two - stage neural network that bases its prediction on position specific scoring matrices , jnet is a neural network method that works by utilizing an alignment as input , alongside psiblast ( 22 ) and hmm profiles . 
 dsc is based on decomposing secondary structure prediction into basic concepts and then uses simple and linear statistical methods to combine them . 
 since dsc is known to perform worse than the other prediction methods employed in 2dsweep , the usage of dsc is optional . 
 figure 4.2dsweep flowchart . as a second concept , 2dsweep searches for dssp ( definition of secondary structure of the protein , ( 23 ) annotation for the input protein . 
 found it extracts secondary structure elements ( if any ) from the structure definition of the dssp database . 
 if there is more than one element covering the same sequence region , 2dsweep uses a simple majority vote to determine the structure at each position . 
 the result of this procedure is shown together with the prediction of the different secondary structure prediction tools . 
 first , the distribution of small , charged and hydrophilic amino acids are shown and probable antigenic regions are indicated . furthermore , the task searches for transmembrane helices and intervening loop regions using four different methods : tmhmm ( 24 ) , das ( 25 ) , tmap ( 26 ) and tmpred . in eukaryotic protein sequences 
 finally , information is given about molecular weight , isoelectric point , the distribution of protease cleavage - sites , and the possible sub - cellular localization of the protein . 
 the web output of 2dsweep ( supplementary figure 3 ) is divided in five sections : ( i ) general information , ( ii ) secondary structure , ( iii ) features and ( iv ) cleavage sites . 
 the information provided in each of these sections is shown in figure 4 and supplementary table 3 . 
 the complete results can be viewed by clicking on the corresponding part of the picture . at the bottom of each graph 
 there is a link to the corresponding explanatory legend . as in the other tasks the xml output containing all the generated information is available via a hyperlink at the bottom of the task output 
 these servers have been implemented using the w3h task framework ( 27 ) , which allows the execution of compound jobs using work and data flow descriptions in a heterogeneous bioinformatics environment using meta - data information . 
 the system regulates the dataflow by specifying dependency rules between the used applications in the meta - data , which allows the design of high complexity bioinformatics tasks , and stores the results of the different applications together with the new results computed during the process . 
 the final output of the task is an xml file which contains all relevant information generated . 
 the xml information is transformed by means of w2h 's ( 28 ) post - processing mechanism into an html page for the task report using the extensible style - sheet language transformations ( xslt ; http://www.w3.org/tr/xslt for facilitating a final visual inspection of the results . 
 furthermore , the xml output can be also required and used for further analysis ( i.e. direct integration in user 's databases , additional pipeline analysis ) . 
 all public databases used by these servers are installed under the sequence retrieval system ( srs ) at the dkfz ( 29 ) . 
 the dkfz srs server contains more than 500 databases that are automatically updated whenever new releases become available ; this means that the webservers will be using the very last version of each database . 
 therefore , as new and improved algorithms and methodologies are developed , they are incorporated into the protein analysis process without having to redesign the entire task . 
 it is also possible to incorporate specific sets of databases as they become available , and to implement arbitrary configuration parameters . 
 the development of the three pipelines presented here , has been user - driven from the beginning . 
 their functionalities are continually being updated and extended in response to requests and suggestions emerging from our core users like lifedb ( 30,31 ) , where these servers are actively used in their protein analysis and annotation . 
 we are currently developing checks especially through the application of filtering strategies and algorithms that will take into account the relationships between domain structure and homology searches . at the moment 
 we are starting to develop a filtering system for the homology searches results taking into account the different quality of annotation in different protein databases with the idea to assign confidence levels and cross - checking results between tasks . 
 we are additionally working on the implementation of directed text mining using the keywords of the proteins description . 
 ","the wealth of transcript information that has been made publicly available in recent years has led to large pools of individual web sites offering access to bioinformatics software . however , finding out which services exist , what they can or can not do , how to use them and how to feed results from one service to the next one in the right format can be very time and resource consuming , especially for non-experts.automating this task , we present a suite of protein annotation pipelines ( tasks ) developed at the german cancer research centre ( dkfz ) oriented to protein annotation by homology ( protsweep ) , by domain analysis ( domainsweep ) , and by secondary structure elements ( 2dsweep ) . 
 the aim of these tasks is to perform an exhaustive structural and functional analysis employing a wide variety of methods in combination with the most updated public databases . 
 the three servers are available for academic users at the husar open server http://genius.embnet.dkfz-heidelberg.de/menu/biounit/open-husar/",pubmed
"delirium is a common and severe complication among elderly patients and is associated with increased morbidity and mortality , prolonged hospital stay , increased risk of post - discharge institutionalization and dementia . 
 identifying accurate biomarkers for delirium may shed light on the pathophysiology and may help to improve delirium recognition and care . 
 oxidative stress and disturbances in serotonergic and dopaminergic neurotransmission might all be involved in the pathophysiology of delirium and probably act together . within the central nervous system , tetrahydrobiopterin ( bh4 ) functions as an essential cofactor in enzymatic reactions responsible for the production of serotonin and dopamine . 
 in addition , bh4 is a cofactor for nitric oxide synthase ( nos ) that catalyzes the production of nitric oxide ( no ) and citrulline from arginine . 
 , when bh4 is partially deficient , some cellular sources of nos may generate superoxide ( o2 ) instead of no and citrulline . in patients with delirium , bh4 
 status has only been investigated after elective cardiac surgery . in order to assess bh4 status , we measured amino acid levels and subsequently calculated the phenylalanine / tyrosine ( phe / tyr ) ratio . 
 this ratio is an indicator for the bh4 status as it reflects the activity of the enzyme phe hydroxylase , an enzyme that uses bh4 as a cofactor . 
 furthermore , we determined the ratios of tryptophan ( trp ) , phe and tyr to the other large neutral amino acids ( lnaas ) . 
 trp is the precursor of serotonin , while phe and tyr are the precursors of dopamine . 
 the lnaas ( trp , phe , tyr , valine , isoleucine and leucine ) compete with each other for transport across the blood - brain barrier . 
 therefore , a decreased trp / lnaas ratio is suggestive for a decline in the amount of trp that enters the brain and consequently for reduced synthesis of serotonin . 
 moreover , we measured plasma levels of the dopamine metabolite homovanillic acid ( hva ) , approximately 30% of which is estimated to originate from dopamine neurons in the central nervous system and which is therefore thought to be a reliable indicator for central dopamine activity . 
 finally , we measured plasma levels of arginine and citrulline to investigate the production of no by nos . 
 the aim of the study was to investigate bh4 status , potential disturbances in serotonergic and dopaminergic neurotransmission and the production of no in patients with and without delirium . 
 the present study was performed within the delirium in the old ( dito ) study in which mean levels of neopterin , interleukin-6 and insulin - like growth factor-1 were compared between patients with and without delirium . in the dito study , a cross - sectional study , we included patients who were admitted to the wards of internal medicine and geriatrics of the erasmus university medical center and the ward of geriatrics of the havenziekenhuis , rotterdam , the netherlands . 
 exclusion criteria were a diagnosis of lewy body dementia , parkinson 's disease , neuroleptic malignant syndrome , tardive dyskinesia , ongoing treatment with antipsychotics or other psychiatric medications except haloperidol and benzodiazepines , aphasia , insufficient understanding of the dutch language and a mini - mental state examination ( mmse ) score < 10 points out of 30 . 
 we excluded patients with a mmse < 10 because it can be quite difficult to distinguish between features of severe dementia and delirium at admission as well as to measure improvement of cognitive function in this group . 
 written informed consent was obtained from all participants . in case of delirium or cognitive impairment at the time of admission , informed consent was obtained from a representative of the patient . 
 all participants were observed daily by the nursing and medical staff and by members of the research team until discharge . to screen for a change in behavior , the 13-item delirium observation screening scale was used during the first 5 days of admission . 
 the diagnosis of delirium was made by a geriatrician , according to the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv ) , and was based on the psychiatric examination of the patient , the medical and nursing records , including the delirium observation screening scale scores , and information given by the patient 's closest relative . 
 when the diagnosis of delirium was doubtful , the case was discussed with the geriatric consultation team to gain consensus . 
 cognitive functioning was assessed in absence of delirium using the mmse . when it was impossible to score the mmse during admission because the patient was too ill , cognitive function was discussed with a clinician or assessed with information from the available medical records . 
 when the clinical opinion was that the patient would have a mmse score 10 , the patient was not excluded from the study . 
 this index encompasses 19 medical conditions , including dementia , and each condition is weighted with a score of 1 - 6 by severity . 
 physical functionality was assessed using the 6-item katz activities of daily living scale and the barthel index . 
 instrumental functionality was assessed using the 7-item older americans resource scale for instrumental activities of daily living . 
 blood samples of all patients were collected within 48 h after admission . when a patient developed delirium during the hospital stay , new blood samples were collected within 24 h after the onset of the delirium and 
 non - fasting blood was collected preferably between 8 and 10 a.m. in an 8-ml tube containing ethylene diamine tetraacetic acid . 
 after blood sampling , the tubes were stored at room temperature to prevent changes in the transfer of amino acids between plasma and blood cells . within 3 h , 
 plasma amino acid levels were determined by high - performance liquid chromatography with automated pre - column derivatization with ortho - phthalaldehyde as previously described . 
 plasma hva levels were determined by reversed - phase high - performance liquid chromatography and electrochemical detection , as previously described for the determination of serotonin . 
 medians and interquartile ranges were determined for continuous participant characteristics and proportions for categorical characteristics . 
 biochemical parameters with a skewed distribution were logarithmically transformed ( all amino acids , amino acid ratios and hva ) . 
 univariate one - way analysis of variance was used to investigate the association between mean levels of amino acids , amino acid ratios and hva ( dependent variables ) and the presence of delirium 
 additional analyses were performed for all amino acids , amino acid ratios and hva after also adding mmse score to the models . 
 a two - tailed p < 0.05 was defined as statistically significant . statistical package for the social sciences , version 21.0 ( spss inc . , 
 the present study was performed within the delirium in the old ( dito ) study in which mean levels of neopterin , interleukin-6 and insulin - like growth factor-1 were compared between patients with and without delirium . in the dito study , a cross - sectional study , we included patients who were admitted to the wards of internal medicine and geriatrics of the erasmus university medical center and the ward of geriatrics of the havenziekenhuis , rotterdam , the netherlands . 
 exclusion criteria were a diagnosis of lewy body dementia , parkinson 's disease , neuroleptic malignant syndrome , tardive dyskinesia , ongoing treatment with antipsychotics or other psychiatric medications except haloperidol and benzodiazepines , aphasia , insufficient understanding of the dutch language and a mini - mental state examination ( mmse ) score < 10 points out of 30 . 
 we excluded patients with a mmse < 10 because it can be quite difficult to distinguish between features of severe dementia and delirium at admission as well as to measure improvement of cognitive function in this group . 
 written informed consent was obtained from all participants . in case of delirium or cognitive impairment at the time of admission , informed consent was obtained from a representative of the patient . 
 all participants were observed daily by the nursing and medical staff and by members of the research team until discharge . to screen for a change in behavior , the 13-item delirium observation screening scale was used during the first 5 days of admission . 
 the diagnosis of delirium was made by a geriatrician , according to the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv ) , and was based on the psychiatric examination of the patient , the medical and nursing records , including the delirium observation screening scale scores , and information given by the patient 's closest relative . 
 when the diagnosis of delirium was doubtful , the case was discussed with the geriatric consultation team to gain consensus . 
 when it was impossible to score the mmse during admission because the patient was too ill , cognitive function was discussed with a clinician or assessed with information from the available medical records . 
 when the clinical opinion was that the patient would have a mmse score 10 , the patient was not excluded from the study . 
 this index encompasses 19 medical conditions , including dementia , and each condition is weighted with a score of 1 - 6 by severity . 
 physical functionality was assessed using the 6-item katz activities of daily living scale and the barthel index . 
 instrumental functionality was assessed using the 7-item older americans resource scale for instrumental activities of daily living . 
 blood samples of all patients were collected within 48 h after admission . when a patient developed delirium during the hospital stay , new blood samples were collected within 24 h after the onset of the delirium and 
 non - fasting blood was collected preferably between 8 and 10 a.m. in an 8-ml tube containing ethylene diamine tetraacetic acid . 
 after blood sampling , the tubes were stored at room temperature to prevent changes in the transfer of amino acids between plasma and blood cells . within 3 h , 
 plasma amino acid levels were determined by high - performance liquid chromatography with automated pre - column derivatization with ortho - phthalaldehyde as previously described . 
 plasma hva levels were determined by reversed - phase high - performance liquid chromatography and electrochemical detection , as previously described for the determination of serotonin . 
 medians and interquartile ranges were determined for continuous participant characteristics and proportions for categorical characteristics . 
 biochemical parameters with a skewed distribution were logarithmically transformed ( all amino acids , amino acid ratios and hva ) . 
 univariate one - way analysis of variance was used to investigate the association between mean levels of amino acids , amino acid ratios and hva ( dependent variables ) and the presence of delirium 
 additional analyses were performed for all amino acids , amino acid ratios and hva after also adding mmse score to the models . 
 table 1 presents the baseline characteristics of the 86 participants who were included in the study . of the 23 patients diagnosed with delirium , 21 
 the mean levels and corresponding 95% confidence intervals ( cis ) of the investigated biochemical parameters in patients with and without delirium are presented in tables 2 and 3 . in adjusted models , 
 mean levels of arginine were significantly lower in patients with delirium ( 34.8 mol / l , 95% ci 28.8 - 42.0 ) than in those without ( 45.2 mol / l , 95% ci 40.6 - 50.5 ) ( p = 0.022 ) ( fig . 
 mean phe / tyr ratios were significantly higher in patients with delirium ( 1.34 , 95% ci 1.19 - 1.51 ) than in patients without delirium ( 1.14 , 95% ci 1.06 - 1.22 ) ( p = 0.028 ) ( fig . 
 1 ) . in addition , mean trp / lnaas ratios were significantly lower in patients with delirium ( 4.90 , 95% ci 4.19 - 5.74 ) than in those without ( 6.12 , 95% ci 5.58 - 6.71 ) ( p = 0.021 ) ( fig . 
 no associations between the other amino acids and ratios and delirium were found , although citrulline ( fig . 1 ) and trp levels were at the border of significance lower in patients with delirium than in those without ( p = 0.052 and p = 0.067 , respectively ) . 
 n = 1 [ not enough plasma ] ; no delirium , n = 1 [ measurement failed ] ) . 
 mean hva levels were not statistically significantly different between patients with delirium ( 123.0 nmol / l , 95% ci 93.3 - 162.6 ) and patients without delirium ( 93.3 nmol / l , 95% ci 79.4 - 109.4 ) ( p = 0.098 ) . 
 in the models additionally adjusted for mmse score , the association between arginine and delirium did not reach statistical significance ( delirium : mean 36.0 mol / l , 95% ci 28.7 - 45.1 vs. no delirium : mean 44.8 mol / l , 95% ci 39.8 - 50.5 , p = 0.107 ) . 
 mean phe / tyr ratios remained borderline significantly higher in patients with delirium ( 1.34 , 95% ci 1.16 - 1.55 ) than in those without ( 1.15 , 95% ci 1.07 - 1.24 ) ( p = 0.089 ) and mean trp / lnaas ratios remained borderline significantly lower in patients with delirium ( 5.00 , 95% ci 4.15 - 6.01 ) compared to those without ( 6.15 , 95% ci 5.58 - 6.78 ) ( p = 0.062 ) . 
 table 1 presents the baseline characteristics of the 86 participants who were included in the study . of the 23 patients diagnosed with delirium , 21 
 the mean levels and corresponding 95% confidence intervals ( cis ) of the investigated biochemical parameters in patients with and without delirium are presented in tables 2 and 3 . in adjusted models , 
 mean levels of arginine were significantly lower in patients with delirium ( 34.8 mol / l , 95% ci 28.8 - 42.0 ) than in those without ( 45.2 mol / l , 95% ci 40.6 - 50.5 ) ( p = 0.022 ) ( fig . 
 mean phe / tyr ratios were significantly higher in patients with delirium ( 1.34 , 95% ci 1.19 - 1.51 ) than in patients without delirium ( 1.14 , 95% ci 1.06 - 1.22 ) ( p = 0.028 ) ( fig . 
 1 ) . in addition , mean trp / lnaas ratios were significantly lower in patients with delirium ( 4.90 , 95% ci 4.19 - 5.74 ) than in those without ( 6.12 , 95% ci 5.58 - 6.71 ) ( p = 0.021 ) ( fig . 
 no associations between the other amino acids and ratios and delirium were found , although citrulline ( fig . 1 ) and trp levels were at the border of significance lower in patients with delirium than in those without ( p = 0.052 and p = 0.067 , respectively ) . 
 hva data were missing for 2 patients ( delirium , n = 1 [ not enough plasma ] ; no delirium , 
 mean hva levels were not statistically significantly different between patients with delirium ( 123.0 nmol / l , 95% ci 93.3 - 162.6 ) and patients without delirium ( 93.3 nmol / l , 95% ci 79.4 - 109.4 ) ( p = 0.098 ) . in the models additionally adjusted for mmse score , the association between arginine and delirium did not reach statistical significance ( delirium : mean 36.0 mol / l , 95% ci 28.7 - 45.1 vs. no delirium : mean 44.8 mol / l , 95% ci 39.8 - 50.5 , p = 0.107 ) . 
 mean phe / tyr ratios remained borderline significantly higher in patients with delirium ( 1.34 , 95% ci 1.16 - 1.55 ) than in those without ( 1.15 , 95% ci 1.07 - 1.24 ) ( p = 0.089 ) and mean trp / lnaas ratios remained borderline significantly lower in patients with delirium ( 5.00 , 95% ci 4.15 - 6.01 ) compared to those without ( 6.15 , 95% ci 5.58 - 6.78 ) ( p = 0.062 ) . 
 in the present study we found disturbed serotonergic neurotransmission and an increased status of oxidative stress in patients with delirium when compared to patients without delirium . as far as we are aware , this is the first delirium study investigating bh4 status and levels of arginine and citrulline in acutely ill elderly hospitalized patients . in order to assess the bh4 status , we measured the phe / tyr ratio . in patients with delirium we found an increased ratio , suggesting a deficiency in the essential cofactor bh4 for the production of serotonin , dopamine and no 
 . decreased bh4 availability has already been found in other neuropsychiatric disorders such as alzheimer 's disease , parkinson 's disease   and schizophrenia . 
 our finding is not in agreement with the results of a previous delirium study which showed that levels of bh4 and phe / tyr ratios did not differ between patients with and without delirium . 
 however , that study included a relatively younger group of patients undergoing elective cardiac surgery . in the present study , 
 serotonergic neurotransmission was investigated with the trp / lnaas ratio and the phe / tyr ratio . 
 we found that patients with delirium had a decreased trp / lnaas ratio , which might suggest reduced serotonin production in the central nervous system . 
 this hypothesis is strengthened by the finding that patients with delirium had an elevated phe / tyr ratio , which might suggest deficiency in the essential cofactor bh4 in the production of serotonin . in previous studies , controversial results have been reported . 
 several studies found a reduced trp / lnaas ratio during delirium , whereas two studies reported no difference in this ratio between patients with and without delirium . 
 the study performed by flacker and lipsitz   included only patients with mild illnesses not requiring hospitalization . 
 therefore , the findings may not be generalizable to acutely ill patients who needed medical care in hospital . the study performed by van der cammen et al .   included delirium patients with alzheimer 's disease . 
 it might be possible that in those patients a disturbance in cholinergic neurotransmission played a more important role in the development of delirium than disturbances in other pathophysiological pathways . 
 furthermore , we found no differences in phe / lnaas ratios , tyr / lnaas ratios and hva levels between patients with and without delirium , suggesting that dopaminergic neurotransmission is not impaired during delirium . 
 the finding that plasma hva levels are not significantly increased in patients with delirium compared to patients without delirium is not in agreement with earlier results . 
 however , those studies were performed in patients with alzheimer 's disease   and patients undergoing cardiac surgery , and therefore the results may not be generalizable . 
 ramirez - bermudez et al .   found that cerebrospinal fluid hva levels correlated with psychotic symptoms of delirium ( hallucinations and delusions ) in neurological patients . 
 it might also be possible that we did not find an association between the dopaminergic markers and the presence of delirium because we included patients both with and without psychotic features . in our study , we also found reduced plasma arginine levels and borderline statistically significantly reduced citrulline levels in patients with delirium . considering the cross - sectional study design 
 first , it is possible that patients with delirium had a pre - existing arginine deficiency which might have resulted in a reduced production of citrulline by nos ( fig . 
 second , when bh4 is partially deficient , as our results do suggest , some cellular sources of nos may generate o2 instead of citrulline and no from arginine ( fig . 
 if this latter scenario is true for delirium , this would also suggest an increased status of oxidative stress , since it favors peroxynitrite formation ( fig . 
 if peroxynitrite is not scavenged by antioxidants , it may cause oxidative damage to cellular macromolecules , which has already been hypothesized to occur in alzheimer 's disease . 
 however , both amino acids have been investigated previously by osse et al .   in patients with delirium 
 after cardiac surgery , but they found no differences in arginine and citrulline levels between patients with and without delirium . since they also reported no difference in bh4 status between patients with and without delirium , 
 first , our findings were obtained in a relatively small group of patients ; therefore , confirmation in a larger population is recommended . 
 second , it might be speculated that the degree of the patients ' cognitive functioning influenced the mean levels of the biochemical parameters . in this study , we adjusted for the charlson comorbidity index , which includes dementia , and our estimates remained statistically significant . however , for our additional analysis mmse scores were not available for all patients ; therefore , we can neither confirm nor deny that the presence of a comorbid cognitive disturbance , not diagnosed as dementia ( yet ) , was a confounding factor . 
 it is possible that the levels of biochemical markers are dependent on delirium duration and severity or even fluctuate during the day in patients with delirium , just like delirium symptoms . in the present study , 
 blood sampling and delirium occurred on the same day , but there is a possibility that the patients had no delirium symptoms at the moment of blood sampling . 
 finally , some potential participants were not included in the study and this may have resulted in some selection bias ; however , since this was random and occurred in both patients with and without delirium , we think that our results are only minimally influenced by this . 
 first , the intensive monitoring of clinical symptoms of patients with delirium until discharge and the dsm - iv diagnosis by a geriatrician makes it less likely that we missed delirium or misdiagnosed symptoms . 
 second , we did not focus on one but on several possible pathways that might lead to delirium as it has been suggested that the pathophysiology is multifactorial . 
 first , our findings were obtained in a relatively small group of patients ; therefore , confirmation in a larger population is recommended . 
 second , it might be speculated that the degree of the patients ' cognitive functioning influenced the mean levels of the biochemical parameters . in this study , we adjusted for the charlson comorbidity index , which includes dementia , and our estimates remained statistically significant . however , for our additional analysis mmse scores were not available for all patients ; therefore , we can neither confirm nor deny that the presence of a comorbid cognitive disturbance , not diagnosed as dementia ( yet ) , was a confounding factor . 
 it is possible that the levels of biochemical markers are dependent on delirium duration and severity or even fluctuate during the day in patients with delirium , just like delirium symptoms . in the present study , 
 blood sampling and delirium occurred on the same day , but there is a possibility that the patients had no delirium symptoms at the moment of blood sampling . 
 finally , some potential participants were not included in the study and this may have resulted in some selection bias ; however , since this was random and occurred in both patients with and without delirium , we think that our results are only minimally influenced by this . 
 first , the intensive monitoring of clinical symptoms of patients with delirium until discharge and the dsm - iv diagnosis by a geriatrician makes it less likely that we missed delirium or misdiagnosed symptoms . 
 second , we did not focus on one but on several possible pathways that might lead to delirium as it has been suggested that the pathophysiology is multifactorial . 
 in this study in older , acutely ill hospitalized patients , we found that patients with delirium had higher phe / tyr ratios , lower trp / lnaas ratios and lower levels of arginine and citrulline than patients without delirium . 
 these findings might suggest that decreased bh4 availability , disturbed serotonergic neurotransmission and an increased status of oxidative stress may have played a role in the pathogenesis of delirium in our patient group . since as far as we know this is the first delirium study investigating bh4 status and levels of arginine and citrulline in acutely ill elderly hospitalized patients , confirmation of our results in a larger , comparable population is recommended . 
 moreover , more research is needed to explore the potential differences in the pathophysiology of delirium in patients with and without cognitive disorders . 
 ","backgroundoxidative stress and disturbances in serotonergic and dopaminergic neurotransmission may play a role in the pathophysiology of delirium.aimsin this study , we investigated levels of amino acids , amino acid ratios and levels of homovanillic acid ( hva ) as indicators for oxidative stress and disturbances in neurotransmission.methodsplasma levels of amino acids , amino acid ratios and hva were determined in acutely ill patients aged 65 years admitted to the wards of internal medicine and geriatrics of the erasmus university medical center and the ward of geriatrics of the havenziekenhuis , rotterdam , the netherlands . 
 differences in the biochemical parameters between patients with and without delirium were investigated by analysis of variance in models adjusted for age , gender and comorbidities.resultsof the 86 patients included , 23 had delirium . 
 in adjusted models , higher mean phenylalanine / tyrosine ratios ( 1.34 vs. 1.14 , p = 0.028 ) , lower mean tryptophan / large neutral amino acids ratios ( 4.90 vs. 6.12 , p = 0.021 ) and lower mean arginine levels ( 34.8 vs. 45.2 mol / l , p = 0.022 ) were found in patients with delirium when compared to those without . 
 no differences were found in hva levels between patients with and without delirium.conclusionthe findings of this study suggest disturbed serotonergic neurotransmission and an increased status of oxidative stress in patients with delirium .",pubmed
"overweight and obesity prevalence rates are increasing worldwide . about 1.4 billon adults were overweight in 2008 , of whom 200 million men and 300 million women were considered to be obese . in the netherlands , prevalence rates of overweight ( and obesity ) are 60% ( 14% ) in men and 44% ( 13% ) in women . 
 high prevalence rates have been found specifically among ethnic minorities and low socioeconomic status ( ses ) groups [ 1 , 3 ] . 
 these health risks include cancer , cardiovascular diseases like hypertension and diabetes , and problems with mobility [ 57 ] . 
 the who   recommends adults aged 1864 to engage in moderate - intensity aerobic physical activities such as walking , cycling , or doing household chores for at least 150 minutes a week . active transport ( i.e. , walking or cycling from a to b ) can thus help achieve sufficient levels of physical activity [ 9 , 10 ] . 
 this review of reviews showed that age and overweight were inversely correlated with physical activity , whereas the male gender and ethnic origin ( white ) were positively correlated with being active . 
 generally , these correlates were also found for active transport among low - income populations . in the past decade 
 particularly , studies from the usa and australia have found positive relations between physical activity and neighbourhood walkability [ 13 , 14 ] . 
 most of these studies , however , have been performed among high - ses populations who tend to live in neighbourhoods with relatively high walkability . moreover , high - ses populations have shown higher levels of physical activity and lower prevalence rates of overweight compared to low - ses populations . 
 it is not yet clear whether the findings from these studies are also valid for low - ses populations living in deprived neighbourhoods . 
 detailed information on the relation between neighbourhood characteristics and physical activity among subgroups within this population is currently lacking . 
 it is hypothesized that more favourable neighbourhood characteristics are associated with higher levels of activity . 
 past studies that focused on the effect of neighbourhood walkability on active transport have shown that associations are reduced after controlling individual demographic characteristics . however , in addition to the confounding role of demographic characteristics , knowledge about interactions between demographic variables and neighbourhood characteristics is required in order to distinguish subgroups that are more vulnerable or responsive to environmental features than others . 
 inhabitants of dutch deprived neighbourhoods vary in ethnic background and are often characterized by a low socioeconomic status . as a result , 
 the prevalence rate of overweight is higher , and level of physical activity is lower compared to those of the average dutch population [ 17 , 18 ] . 
 this makes inhabitants of dutch deprived districts an important target group for physical activity interventions . 
 furthermore , previous research has shown that it is difficult to address ethnic minorities , and guidelines for this specific group are lacking . 
 more insight into the personal and neighbourhood factors that are associated with active transport could contribute to the development of effective interventions targeting ethnic minorities . 
 therefore , this study investigates which personal and neighbourhood environmental characteristics are associated with active transport among inhabitants of dutch deprived neighbourhoods . in addition , interactions between personal and neighbourhood environmental characteristics will be tested to explore differential associations of neighbourhood walkability with active transport within subgroups . 
 briefly , urban40 is a longitudinal study that evaluates the health impact of area - based interventions to improve housing , employment , education , social integration , and safety in the 40 most severely deprived neighbourhoods in the netherlands , also known as the dutch district approach . in 2007 , 
 the dutch government made a list of the 140 most deprived neighbourhoods in the netherlands , based on scores on eighteen different registry - based indicators of physical and socioeconomic deprivation as well as physical and social problems reported by residents . from this list 
 , twenty neighbourhoods were selected that belonged to the 140 most deprived dutch neighbourhoods . in each district , 250 randomly selected adults aged 18 and older received a letter in which they were invited to participate , including a reply card that they could return free of charge . trained interviewers contacted the residents who sent a reply card and arranged home visits for the interview . up to two reminder letters were sent when people did not respond via the reply card . in addition , trained interviewers from an interview agency specialized in research among multicultural populations recruited participants by unplanned home visits ( ringing doorbell at the addresses on the original list ) . 
 the trained interviewers were tailored to gender and ethnicity ( spoken language , e.g. , dutch , english , turkish , classical arabic , or arab - berber ) of the residents . 
 financial constraints limited recruitment of respondents by unplanned home visits in all districts , favouring larger districts . 
 respondents filled out the questionnaire by themselves , but they could also ask the assistant that brought the questionnaire to help them , if needed in their own language ( dutch , english , turkish , classical arabic , or arab - berber ) . 
 in particular the participants that were of non - dutch origin made use of this option ; they were orally interviewed and the assistant filled in the answers . 
 a total of 5000 respondents were invited to participate . of the 374 participants that returned the reply card , 299 filled out the questionnaire . 
 this resulted in a total study population of 740 adults that were recruited between may 2010 and november 2011 . 
 after deletion of cases without data on key variables ( n = 118 ) , a final sample of 622 participants remained . 
 active transport was defined as the minutes per week spent on walking or cycling from a to b. the validated squash questionnaire for physical activity measured active transport by asking respondents to think about a regular week in the past months . 
 respondents then indicated how many days per week they engaged in several forms of physical activity and how many minutes they engaged in them . 
 subsequently , they could fill in the days , hours , and minutes spent on active transport . 
 total minutes of walking for active transport per day were calculated by multiplying the number of hours reported with sixty and adding them to the minutes reported . 
 subsequently , minutes of active walking per week were calculated by multiplying the minutes per day with the reported number of days per week . 
 this number of minutes of walking for active transport per week was used in the analyses . 
 minutes of cycling for active transport per week were calculated according to the same steps . 
 neighbourhood environmental characteristics were assessed by use of the neighbourhood walkability scale ( news ) . 
 three scales with twelve questions in total investigated the access to services ( three items ) (  = 0.71 ) ( e.g. , shops are within easy walking distance from my home ) , neighbourhood surroundings ( four items ) (  = 0.66 ) ( e.g. , the natural surroundings in my neighbourhood are beautiful ) , and safety from crime ( five items ) (  = 0.66 ) ( e.g. , because of criminality it is unsafe to walk in my neighbourhood during the day ) . three items of the  safety from traffic  scale were included separately in the analyses because of the low reliability of the scale (  < 0.6 ) . 
 the items were  there is much traffic on nearby streets which make walking in the neighbourhood difficult or unpleasant ;   the speed of traffic in nearby streets is normally low ( 30  km / h or less ) ;  and  most car drivers exceed maximum speed when driving through the neighbourhood . 
 the answers were given on a four - point likert scale (  completely disagree 
 a higher score corresponded with better access to services , better neighbourhood surroundings , and more safety from crime and traffic . 
 personal characteristics that were taken into account were age , sex , ethnicity , and body mass index ( bmi ) . 
 ethnicity of the respondent was operationalized by assessing the country of birth of both parents . 
 if a respondent had at least one parent who was born outside the netherlands , he or she was considered to be a migrant . 
 neighbourhood status was included as a confounder , to correct variations in degree of deprivation among neighbourhoods . 
 neighbourhood status was determined using the neighbourhood status score ( nss ) of the dutch social cultural planning office ( scp ) . 
 this score is based on the income , education level , and employment status of the inhabitants and indicates the degree of social deprivation per neighbourhood . 
 lower scores correspond with a larger degree of social deprivation , whereas high scores indicate a more favourable socioeconomic status .   characteristics of the target population were investigated by means of descriptive analyses . 
 independent samples t - tests were performed to investigate the difference in scores on the news and in minutes of active transport per week between the following subgroups : respondents equal to or younger than the median age of 43 years and respondents older than 43 years : men and women ; native dutch respondents and migrants ; and respondents with a bmi lower than 25 and respondents with a bmi higher than 25 . 
 bivariate correlations were calculated to explore the associations between personal characteristics , and minutes per week spent in active transport and the associations between news scales and minutes spent engaging in active transport . in order to gain more insight into these correlations and to adjust for the difference in effect between baseline characteristics multivariate 
 linear regression analyses were performed . the full main effects model consisted of nss , the personal characteristics ( age , sex , ethnicity , and bmi ) , and the neighbourhood characteristics . 
 interactions between personal characteristics and neighbourhood variables were investigated by adding each interaction term separately to the full main effects model . 
 most of the participants were dutch , the migrants were mainly non - western ( 87.45% ; not tabulated ) . 
 the nss of the twenty participating districts varied between 3.64 ( highly deprived ) and 0.15 ( moderately deprived ) with a mean of 1.82 and a sd of 1.03 . 
 table 2 shows the scores for the neighbourhood characteristics and the minutes of active transport per week for subgroups of the population . on average , 
 residents of deprived neighbourhoods walked 33 minutes and cycled for 32 minutes per week for active transport . 
 the younger participants were found to be more active in terms of walking as well as cycling than the older participants . 
 migrants were more active in terms of walking , whereas the dutch biked more often . 
 participants with a bmi below 25 were more active than those overweight , with a significant difference in activity levels for cycling . 
 respondents older than 43 years perceived the neighbourhood surroundings as more positive than the younger respondents . women experienced better access to services than men . 
 native dutch participants perceived better access to services , better neighbourhood surroundings , and felt safer from crime than migrants . 
 furthermore , they experienced less traffic on nearby streets that made walking difficult than migrants . 
 participants who lived in a neighbourhood with a relatively higher status within this population perceived all neighbourhood characteristics as more favourable than the people from districts with a lower nss . 
 multivariate regression analyses showed that minutes of walking decreased significantly by one minute for each year increase in age . 
 nss was also found to be a significant associate of walking ; respondents from higher status neighbourhoods walked less than respondents from lower status neighbourhoods . 
 perceived speed of traffic was the only neighbourhood variable found to be a statistically significant associate of walking . 
 interactions between ethnicity and the perception of traffic exceeding maximum speed (  : 0.40 ; p < 0.001 ) ; sex and the perception of traffic exceeding maximum speed (  : 0.26 ; p < 0.05 ) ; and between age and speed of traffic (  : 0.53 ; p < 0.01 ) were statistically significant . 
 subgroup analyses indicated that migrants and women reported more walking for transport when cars were perceived to not exceed maximum speed (  : 0.18 ; p < 0.01 and  : 0.13 ; p < 0.05 , resp . ) , whereas this perceived environmental factor was unrelated to walking of native dutch and male respondents (  : 0.06 ; ns and  : 0.04 ; ns , resp . ) . 
 stratification for age showed that low perceived speed of traffic was significantly associated with more walking in respondents of 43 years or younger (  : 0.18 ; p < 0.01 ) but not in respondents older than 43 (  : 0.06 ; ns ) . 
 every year of increase in age was associated with a decrease of one minute of cycling per week ( table 3 ) . 
 the native dutch cycled more compared with the migrants (  : 0.11 ; p  0.01 ) . 
 the interaction between ethnicity and neighbourhood surroundings was found to be significant (  : 0.50 ; p  0.01 ) . 
 more attractive neighbourhood surroundings were significantly associated with more cycling per week among the migrants (  : 0.23 ; p  0.001 ) , but not in the dutch respondents (  : 0.02 ; ns ) . 
 this study investigated which personal and neighbourhood environmental characteristics were associated with active transport among inhabitants of dutch deprived neighbourhoods . 
 this finding is supported by a study of ogilvie et al .   and could be a result of differences in fitness between the younger and the older respondents . 
 people tend to perceive more barriers for physical activity as they age , such as physical disabilities and poorer perceived health . 
 migrants walked more minutes per week compared with the dutch , probably because walking is considered to be an easy and cheap mode of transportation , which is less sensitive to cultural habits compared to cycling . 
 the dutch cycled more than the migrants , probably because they are more used to cycling and have more access to this mode of transportation . 
 higher scores on bmi correlated with lower levels of active transport , which confirms findings of pucher et al . . 
 perceived speed of traffic was the only neighbourhood environmental characteristic that was associated with walking for transport . 
 when the average speed of traffic was perceived as low , people reported more walking for active transport . 
 a review of previous studies reported mixed evidence with regard to safety and active transport , which illustrates a need for clear definitions of safety in this field of research . 
 our measurements confirm this need for proper measurements of safety from traffic , since we were forced to include the relevant questions separately in the analyses as a result of low reliability of the scale . 
 interactions showed that with regard to ethnicity , migrants appeared to be more responsive to neighbourhood walkability than the dutch respondents . 
 speed of traffic ( walking ) and aesthetics ( cycling ) was found to be more strongly associated with active transport among migrants than among native dutch . 
 this could be a result of cultural differences ; migrants seem to be more in need of a safe , attractive , and stimulating environment to induce active transport . 
 an investigation of transportation among dutch migrants showed that migrants cycled more when high quality cycle infrastructure was available and if a strong cycle culture was present in the neighbourhood . 
 in addition , female and younger respondents were subgroups that appeared to be somewhat more responsive to traffic safety than male and older respondents . a dutch study on traffic safety found that higher levels of neighbourhood traffic safety correlated with increased odds of being active , especially in women and people aged 35 to 59 , which partly confirms our findings . 
 personal characteristics have been shown to be stronger associates of active transport than neighbourhood characteristics , a finding which is supported by multiple other studies [ 33 , 34 ] . 
 nevertheless , there were also studies that did find associations between neighbourhood walkability and active transport [ 13 , 14 ] . 
 this could be explained by differences in levels of transportation facilities in the countries under study . 
 most of these studies were conducted in the us or australia , countries that sometimes lack proper walking or cycling trails . 
 however , the netherlands especially is already well - equipped for facilitation of walking and cycling , so relative differences in neighbourhood characteristics may only have a minimal impact on active transport . 
 this implies that results of these studies are likely to be only valid for the population and country in which it was conducted , as was suggested in previous studies [ 12 , 34 ] . 
 the low variability of neighbourhood characteristics within the deprived districts in our study may have decreased the possibility to find strong environmental relationships even more . 
 it is also possible that the characteristics investigated in this study did not cover all environmental aspects that might be relevant in active transport . 
 nevertheless , interaction effects indicated that within some groups of the population under study , neighbourhood characteristics are important and need to be considered in efforts to promote physical activity . 
 since the data collected for this study was cross - sectional , statements about causality can not be made . 
 second , the overall response rate was low , although it was equivalent to those of comparable studies of the past years [ 10 , 12 , 35 ] . 
 third , self - reported measures of active transport were used , which could have been prone to recall bias and socially desirable answers . 
 fourth , an abbreviated version of the news questionnaire was used , which may be the reason for the one scale on safety from traffic being considered unreliable .   despite some limitations , this study adds valuable information to current evidence in particular because of the focus on a low - ses population , on the understudied cycling behaviour , and the ethnic composition of the study population . 
 participants were hard to reach and are therefore often neglected in research , among others because of possible cultural and language differences between the interviewer and respondents . 
 intensive recruitment strategies such as matching of interviewer and participant and executing the study in the respondents ' mother tongue were used to overcome this difficulty . 
 the setting of deprived neighbourhoods was also a valuable characteristic , since they are the focus of governmental policies and interventions . 
 insight into the associations between neighbourhood characteristics and patterns of physical activity and their determinants in these districts could provide more information about the contents and implementation of such policies and interventions . 
 results of the current study may be used to design experimental research in order to test causality of the findings . 
 eventually , this may lead to evidence for effective intervention development for the subgroups most in need of interventions to increase active transport and subsequently levels of physical activity .","background . 
 active modes of transport like walking and cycling have been shown to be valuable contributions to daily physical activity . the current study investigates associations between personal and neighbourhood environmental characteristics and active transport among inhabitants of dutch deprived districts 
 . method . 
 questionnaires about health , neighbourhoods , and physical activity behaviour were completed by 742 adults . 
 data was analysed by means of multivariate linear regression analyses . results . 
 being younger , female , and migrant and having a normal weight were associated with more walking for active transport . 
 being younger , male , and native dutch and having a normal weight were associated with more cycling for active transport . 
 neighbourhood characteristics were generally not correlated with active transport . 
 stratified analyses , based on significant person - environment interactions , showed that migrants and women walked more when cars did not exceed maximum speed in nearby streets and that younger people walked more when speed of traffic in nearby streets was perceived as low . among migrants , more cycling was associated with the perceived attractiveness of the neighbourhood surroundings . 
 discussion and conclusion . 
 results indicated that among inhabitants of dutch deprived districts , personal characteristics were associated with active transport , whereas neighbourhood environmental characteristics were generally not associated with active transport . 
 nevertheless , interaction effects showed differences among subgroups that should be considered in intervention development .",pubmed
"optic neuritis related to ms is an acute optic neuropathy occurring in young people , especially in women . 
 the clinical picture shows a decline in visual acuity , a pain which increases with movements of the eye , a decline in contrast sensitivity , dyschromatopsia and changes in the visual field ( 1 ) . 
 it occurs more often unilaterally in young people , predominantly in women with an incidence of 1 - 5 per 100,000 annually ( 2 ) . 
 the main aim of this study was to determine the onset of optic neuritis as the first ms clinical presentation . 
 we also wanted to analyze the role and significance of morphometric changes in the optic nerve and visual field by analyzing parameters of optic coherent tomography ( oct ) and computer perimetry in these patients . 
 one of the aims was to determine the efficiency of pulse corticosteroid therapy in those patients . 
 a clinical , retrospective study was conducted at the eye clinic and the neurological clinic , sarajevo university clinical centre ( succ ) at the time of the march 20092011 . after the first clinical signs follower is a detailed examination of patients by neurologists and ophthalmologists . 
 here we did not analyzed cases of ms relapses . in all analyzed patients we found a unilateral optic neuritis . 
 we approached ms verification according to paty or brakhof modern clinically defined multiple sclerosis ( cdms ) criteria ( 3 , 4 ) . 
 first criterion : at least two separate clinical episodes of the disease that occurred at different times and at least two fields of demyelination . of radiological tests , 
 mri of the brain and spinal cord were analyzed. second criterion : two ms clinical episodes , one mri manifest lesion and paraclinical symptoms. laboratory - supported ms diagnosis: two episodes , one clinically and paraclinically confirmed lesion and immunoglobulin abnormalities of cerebrospinal fluid. one episode , two clinically separate lesions and cerebrospinal abnormalities . 
 first criterion : at least two separate clinical episodes of the disease that occurred at different times and at least two fields of demyelination . of radiological tests , 
 second criterion : two ms clinical episodes , one mri manifest lesion and paraclinical symptoms . 
 laboratory - supported ms diagnosis :  two episodes , one clinically and paraclinically confirmed lesion and immunoglobulin abnormalities of cerebrospinal fluid . 
 one episode , two clinically separate lesions and cerebrospinal abnormalities . of neurological parameters we analyzed clinical manifestations of motor , sensory and sensitive disturbances , relevant to the confirmation of ms diagnosis . of ophthalmological parameters 
 , we analyzed : visual acuity ( va ) by snellen charts , visual field ( vf ) by octopus 100 , and thickness of the peripapillary rim nerve fibres by stratus zeiss optic coherent tomography ( oct ) . 
 . age structure of ms patients with verified optic neuritis gender structure of ms patients with verified optic neuritis the age structure analysis of the sample shows that optic neuritis was most often present in the patients aged 18 to 30 years . in the entire sample , onset of optic neuritis 
 was more frequent in the female patients ( 70% ) compared to the male patients . 
 the first clinical manifestation of ms in our sample statistically significant tests ( x=9,7 p=0,01 ) we have confirmed that the optic neuritis is one of the first clinical manifestation of ms , compared to the other motor , sensory and sensory events in our sample . 
 ophthalmological parameters we followed after 7 and 15 days , after 1 , 3 , 6 and 12 months . analyzing disturbances in the computerised visual field : 
 centrocecal and paracentral scotoma and diffuse sensitivity , we find most frequent disturbances in the centrocecal region in 50% of the cases . 
 different kinds of scotoma in visual field in ms patients with optic neuritis by analysis of oct results in part of the nerve fibre thickness of papillary and parapapillary layers ( of the neuroretinal rim ) , we had , after three months , results showing the thinning of the nerve fibres in the whole circumference , most often in the upper quadrant . 
 retinal nerve fibre layer ( rnfl ) by optic disc visual acuity on first examination and one month after corticosteroid therapy visual acuity in all patients with optic neuritis at the first examination ranged from 0.1 to 0.3 with correction . 
 after the administration of pulse corticosteroid therapy , all patients with optic neuritis had a significant improvement of va , and a month upon administration it varied from 0.6 to 1.0 . 
 in our sample of 89 patients with the verified ms diagnosis , 10 patients had a clinical picture of optic neuritis , as a first clinical sing of disease , it was statistically significant ( x= 9,7 p=0,01 ) . 
 the optic neuritis study group describes the changes of visual functions in ms patients , which usually present as a decline in vision , blurring , decline in contrast sensitivity , color vision disturbances , as well as disturbances in the visual field ( 1 - 5 ) . in their studies , allanore y. and deretzi g.confirm the genetic impact on ms development as an autoimmune disease in some families ( 6 ) . 
 studies were performed to analyze the influence of stress , viral and bacterial infections of respiratory tract , urinary and gastrointestinal systems on the appearance and occurrence of ms exacerbations ( 7 , 8) . in our study , the disease appeared most often in the patients aged 18 - 30 years . 
 population get results which show that the ratio of the incidence of occurrence of ms ranged from 1.9 : 1 to 3.6:1 in favor of women ( 9 ) 
 . a large number of studies confirm the ms and on occurrence at a younger age and more often in women ( 1 , 2 , 5 , 10 ) . 
 balashov ke . finds a higher on incidence in the spring months in the patients without verified demyelisating lesions , which has been observed in our past practice too , and which could be an aim of our future studies ( 11 ) . 
 cigarette smoking is also a risk factor in the development of ms and on ( 12 ) . in 50% of our subjects we had centrocecal scotoma in the visual field , and sensitivity depression in 30% . 
 disturbances in the central areas of the visual field were verified in other studies as well ( 1 , 13 ) . 
 visual acuity in all patients with optic neuritis at the first examination ranged from 0.1 to 0.3 with correction . 
 all patients with an on and verified ms diagnosis monitored in this study were treated with pulse corticosteroid therapy in a dosage of 1,000 mg for three days , followed by 8 days of 1mg / kg b.w . 
 a month after pulse corticosteroid therapy , visual acuity in all patients with on ranged from 0.6 to 1.0 . 
 corticosteroids administered intravenously by the pulse therapy scheme prevent on relapse , but they have the same effect on definite va as corticosteroids orally administered . 
 ontt finds that the on patients treated with prednisolone orally administered in a dosage of 1mg / kg b.w . for 14 days 
 ontt shows that intravenously administered corticosteroids inhibit ms development for a 2-year period , bur after three years that effect disappears ( 14 , 15 ) . 
 three months after the first on presentations , the oct analysis of retinal nerve fibre thickness and neuroretinal rim confirmed the nerve fibre thinning in all subjects . 
 ontt shows that 50% of the patients with on develop ms over a 15-year period . 
 ontt also finds that in on patients without mri changes , ms develops in 25% of the cases , and in the cases with one or more cns lesions that is 75% . 
 all patients had oct - verified nerve fibre atrophy after on ( 1 , 14 ) . according to other researchers , by oct analysis 
 it can discover in vivo atrophy of nerve fibres in patients with ms as structural damages of axons of afferent fibres of the visual pathway ( 15 , 16 ) . 
 oct rnfl thickness average analysis studies show , by morphometric analysis of the structure of nerve fibres by oct , that there is fibre atrophy in the ms patients with and without on 
 . however , retinal nerve fibre atrophy is more significant in the patients with on ( 16 ) . 
 in our sample with the predominantly female gender ranging from 18 to 30 years of age , on occurred as the first clinical presentation of ms . 
 the analysis of the computerised visual field confirmed centrocecal disturbances as the most common , and optic coherent tomography registered the thinning of the nerve fibres of the neuroretinal rim in all subjects . a month after pulse corticosteroid therapy ,","aim : to analyze the clinical signs of multiple sclerosis ( ms ) and show that optic neuritis is one of the first event , which indicates the development of disease.patients and methods : the study involved 89 cases in which it confirmed ms at the time of the march 20092011 . since ophthalmological parameters were analyzed visual acuity ( va ) , visual field ( vf ) , and retinal nerve fibre layer ( rnfl ) thickness of peripapillary rim by optic coherent tomography ( oct).results : ten(10 ) patients had on as the first clinical manifestation of the disease which was statistically significant ( x2 = 9,7 p=0,01 ) compared to the manifestation of other clinical signs of disease . in vf , centrocecal scotomas were predominant in 50% of the subjects ; the rnfl thinning of the neuroretinal rim was verified in all patients , most often in the upper quadrant . a month after pulse corticosteroid therapy , visual acuity in all patients with on ranged from 0.6 to 1.0.conclusion:on is one of the first ms clinical manifestation . in vf , 
 the most common disturbances are in the centrocecal area . 
 the rnfl thinning was verified in all patients with oct .",pubmed
"diabetes can cause acute and long - term complications , and is as well , a global risk factor for cardiovascular disease.1 lifestyle and behavior , as well as individual genetic predisposition , influence the risk of diabetes and prevalence in a community.2 public knowledge is the basis for lifestyle and behavior in a given community.3 therefore , the knowledge particular to diabetes is a key determinant of the lifestyle of a community , and can discriminate those who are at risk from those who are free of risk . 
 diabetes is increasingly prevalent in ethnic minority groups globally.2,4,5 recently , lorga et al6 reported that 16.7% of the karen rural community had an abnormally high blood glucose level ( > 100 mg / dl ) , and that there was a considerable proportion of prediabetic persons ( 13.04% ) in a cross - sectional survey among the karen minority . 
 the karen ethnic rural community along the thai  myanmar border is a hard - to - reach population for health promotion services . 
 karen people use their native language and most of the senior adult population in the community can not read thai script . 
 moreover , in a district like thasongyang , the population pattern is very dynamic and unstable because of a large migration across the border.7 karen people are indigenous to , and tend to stay along the thai - myanmar border such as area around tak province . 
 there is limited literature to guide the assessment of health literacy and knowledge of diabetes in such a community.8 the objective of the study was to assess the knowledge of diabetes of the rural karen residents , and compare the diabetes knowledge between male and female respondents . 
 this study was approved by the ethical board of the boromrajonani college of nursing nakhon lampang , lampang , thailand , and conducted with the verbal informed consent of participants . 
 it was conducted in thasongyang , which is a district in the far northwest of thailand , by the narrow moei river on the border of myanmar . 
 all participants were thai citizens but most of the participants could not read thai script . none had any prior known diagnosis of diabetes . 
 diabetes knowledge was assessed by a questionnaire that was developed by consensus between native researchers and researchers from the boromrajonani college of nursing nakhon lampang . 
 the questionnaires were delivered in the karen language by researchers speaking both thai and the karen language , and were completed by the interviewers . 
 fasting plasma glucose ( fpg ) measurement was preceded by overnight fasting for 8 hours before the test . 
 body weight and height were measured by the public health officers using a standard measuring scale . 
 body mass index was calculated by the ratio of body weight in kilograms divided by the square of the height in meters . 
 continuous variables were summarized as median , maximum , and minimum values , as the distribution was not normal . the nonparametric test for trend analysis 
 was run by  nptrend  in stata version 11 ( stata corp , college station , tx ) .  yes ,   no ,  and  not sure  answers in the questionnaires were recoded as categories in order to compare by rank sum test . 
 a correct answer was coded to be 1 , and an incorrect answer , or the answer of  not sure ,  was coded as zero . 
 statistical significance was defined as p value less than 0.05 , with a 95% confidence interval . 
 diabetes knowledge was assessed by a questionnaire that was developed by consensus between native researchers and researchers from the boromrajonani college of nursing nakhon lampang . 
 the questionnaires were delivered in the karen language by researchers speaking both thai and the karen language , and were completed by the interviewers . 
 fasting plasma glucose ( fpg ) measurement was preceded by overnight fasting for 8 hours before the test . 
 body weight and height were measured by the public health officers using a standard measuring scale . 
 body mass index was calculated by the ratio of body weight in kilograms divided by the square of the height in meters . 
  
 continuous variables were summarized as median , maximum , and minimum values , as the distribution was not normal . the nonparametric test for trend analysis was run by  nptrend  in stata version 11 ( stata corp , college station , tx ) .  yes ,   no ,  and  not sure  answers in the questionnaires were recoded as categories in order to compare by rank sum test . 
 a correct answer was coded to be 1 , and an incorrect answer , or the answer of  not sure ,  was coded as zero . 
 statistical significance was defined as p value less than 0.05 , with a 95% confidence interval . 
 we assessed the diabetes knowledge briefly in three parts ( 1 ) general knowledge , ( 2 ) knowledge about the risk factors , and ( 3 ) knowledge about the symptoms , diagnostic signs , and complications of diabetes mellitus . 
 one - third of the population answered correctly ( range 27.09%41.81% ) , and another one - third gave no answer to these questions ( range 33.44%39.80% ) , whereas another one 
 third gave a wrong answer . among the karen community , 36.45% knew that old age is a risk factor for diabetes , 37.12% knew that they had diabetic relatives , 39.80% knew that obesity can lead to diabetes , 37.12% answered that exercise can prevent diabetes , 25.09% reported that pregnant women can have diabetes , and 41.81% said they should not eat excess fatty food for fear of diabetes . regarding the symptoms , diagnosis and complications of diabetes , less than 30% knew that fasting blood glucose level should be less than 100 mg% . 
 a few of the rural karen ethnic residents could correctly answer questions in the third part of the questionnaire . among the respondents , 33.78% answered that diabetic patients could have thirst and polyuria , 34.78% knew that numbness of the limbs might link to diabetes , 35.12% recognized the importance of foot care in people living with diabetes , 32.78% answered that untreated diabetes could lead to blindness , and 34.78% knew that slow wound healing might be due to diabetes . 
 overall , one - third of the sample population gave the correct answers ( range 32.78%34.78% ) while the other two - thirds were either unsure or gave wrong answers . 
 overall , public knowledge of diabetes among karen ethnic rural community is at the lower extreme ( figure 1 ) . 
 diabetes knowledge was compared between male and female by median sum of the knowledge scores . 
 overall knowledge level in female participants was also lower than in male participants ( figure 2 ) . 
 the median sum of the knowledge scores was 6 ( interquartile range : 211 ) among males , and 3 ( interquartile range : 18 ) among the females ( p for trend = 0.009 ) . there was a significant difference in level of knowledge among females and males . 
 we assessed the diabetes knowledge briefly in three parts ( 1 ) general knowledge , ( 2 ) knowledge about the risk factors , and ( 3 ) knowledge about the symptoms , diagnostic signs , and complications of diabetes mellitus . 
 one - third of the population answered correctly ( range 27.09%41.81% ) , and another one - third gave no answer to these questions ( range 33.44%39.80% ) , whereas another one 
 third gave a wrong answer . among the karen community , 36.45% knew that old age is a risk factor for diabetes , 37.12% knew that they had diabetic relatives , 39.80% knew that obesity can lead to diabetes , 37.12% answered that exercise can prevent diabetes , 25.09% reported that pregnant women can have diabetes , and 41.81% said they should not eat excess fatty food for fear of diabetes . regarding the symptoms , diagnosis and complications of diabetes , less than 30% knew that fasting blood glucose level should be less than 100 mg% . 
 a few of the rural karen ethnic residents could correctly answer questions in the third part of the questionnaire . among the respondents , 33.78% answered that diabetic patients could have thirst and polyuria , 34.78% knew that numbness of the limbs might link to diabetes , 35.12% recognized the importance of foot care in people living with diabetes , 32.78% answered that untreated diabetes could lead to blindness , and 34.78% knew that slow wound healing might be due to diabetes . 
 overall , one - third of the sample population gave the correct answers ( range 32.78%34.78% ) while the other two - thirds were either unsure or gave wrong answers . 
 overall , public knowledge of diabetes among karen ethnic rural community is at the lower extreme ( figure 1 ) . 
 diabetes knowledge was compared between male and female by median sum of the knowledge scores . 
 overall knowledge level in female participants was also lower than in male participants ( figure 2 ) . 
 the median sum of the knowledge scores was 6 ( interquartile range : 211 ) among males , and 3 ( interquartile range : 18 ) among the females ( p for trend = 0.009 ) . there was a significant difference in level of knowledge among females and males . 
 the number of diabetes cases is rising worldwide , and the burden of this is more severe in low- and middle - income countries.9,10  belonging to an ethnic minority  has been reported as a risk factor for the development of type 2 diabetes.2 disadvantaged people in these groups are more prone to the consequences of a silent epidemic.11 it is important to assess the public knowledge of diabetes , especially in minority ethnic populations . today , rising public health education and reduced disparity between groups are compulsory for the prevention and care of diabetes in all settings.12 risk perception of a community is the basis for healthy behavioral changes within a community.13 perception of risk among people at risk can prevent the disease 
 thus , we assessed the perception of diabetes risk factors among the karen ethnic rural community . 
 we found only one - third of the study sample had knowledge about diabetes risk factors ( table 2 ) . 
 most in the karen community did not know that elderly age , being overweight , and physical inactivity are risk factors for diabetes . 
 initiation of measures to educate this minority population , including use of mass media , and printed materials in the local language , might be a useful strategy for risk communication . 
 knowledge of diabetes symptoms is important for early diagnosis and accessing care . recognizing the symptoms of diabetes and its complications may bring the diabetic individuals to seek health care service . 
 symptoms of diabetes complications are common presentations , even though these might be indicators of late diagnosis , or poor glycemic control . 
 individuals lacking knowledge of these may not seek medical care in time , and consequently will have complications of diabetes.14 we assessed such knowledge in karen ethnic community ( table 2 ) . 
 the histogram of knowledge scoring was skewed to the left and peaked at lower end of the scoring ( figure 1 ) . 
 in contrast , the similar histogram for public knowledge of diabetes in singapore , was reported as totally skewed to the right side.15 the knowledge distribution among the karen ethnic sample was at the lower end , and pointed out a need for intervention . 
 in fact , diabetes and its consequent cardiovascular disease risk are higher in women.7 there is also increasing concern about gestational diabetes;16 however , our observations pointed out that diabetes knowledge among female karen participants was lower than among males . 
 this type of health inequality has also been reported in many other diseases.17 type 2 diabetes is more common in disadvantaged groups of women , such as those with least income and who are least educated.18 risk behaviors for type 2 diabetes have been highly prevalent even in the developed setting;19 moreover , poor women in the developing world tend to delay seeking care until symptoms are too severe.17 in the particular setting of this study , it is likely that women usually work in the home at housekeeping , and become housebound and sedentary . 
 a television education program in the local language might reach these women.19 lack of diabetes knowledge can cause missed diagnosis , late diagnosis , and higher incidence of diabetes complications . 
 creation of a peer - group gathering place for women , such as a health education club for mothers , and exercise club for housewives , may promote women s knowledge of diabetes and sharing of information . 
 in contrast to the current study findings , in a previous study , the public knowledge among thai people was reported to be fairly high and there was no gender gap between thai men and women.20 it is obvious that diabetes health education is poor among the karen ethnic community . 
 this might be due to a language barrier , which is a major obstacle to educating minority groups , as in other parts of asia.21 existing evidence suggests that such a situation calls for a culturally tailored and well - designed health education strategy.22 
 as this was a pilot study swiftly conducted to provide important information , there were weaknesses in the current study . 
 the questionnaire used in this study was made easily comprehensible for rural karen residents , and study findings may not be generalizable to other settings . albeit , the findings are the first of their kind to our knowledge , and are meant to assist in guiding further studies . 
 despite these limitations , this investigation contributed to our understanding of the health disparity in an ethnic minority population . 
 it provided insights into the need for health education interventions for the prevention of diabetes in a rural ethnic community . 
 the result of this study showed that karen ethnic men and women were less knowledgeable about all aspects of diabetes . 
 public education about diabetes in this setting should be started urgently and carefully , to overcome language and cultural barriers and prevent diabetes epidemic among these rural people . 
 our study finding is expected to be a step forward , in minimizing the disparity in diabetes health education .","background and purposethe public knowledge of diabetes is important for prevention of disease . this study aimed to evaluate knowledge of diabetes , risk factors , and the common warning signs of diabetes and complications among community participants in a rural karen ethnic community.methodsparticipants 
 were asked to answer a questionnaire regarding their knowledge of diabetes . 
 fasting blood glucose testing , blood pressure measurement , and body mass index ( bmi ) assessment were provided to the participants . 
 the study was conducted at thasongyang district , tak province , thailand.resultsa total of 299 karen rural residents were included in the study . 
 the median age was 45 years and median fasting blood glucose was 88 mg / dl . 
 the response rate to the questionnaires was 91.97% . 
 half of the participants knew diabetes is a noncommunicable disease needing lifelong treatment . 
 overall , one - third of the community participants could correctly answer the knowledge assessment questions regarding risk factors and common features of diabetes . whereas the other two - thirds either gave a wrong answer or were  not sure  . 
 female participants had poorer diabetes knowledge than the males.conclusionthe public knowledge of diabetes , as represented by this sample of the karen ethic community , is alarmingly low . 
 there is significant gender difference in knowledge level . culturally tailored and gender - sensitive diabetes health education interventions are urgently needed in this minority ethnic community .",pubmed
"it is well established that individuals with serious mental illness ( smi ) , which include diagnoses like schizophrenia and bipolar disorder , have a much higher rate of morbidity and mortality in comparison to the general population . 
 specifically , diabetes impacts approximately 25% of individuals with smi , 2 to 3 times the rate than the general population . while this increased rate in diabetes is a multifaceted issue , factors such as lower utilization of preventive care and use of newer antipsychotic medications are likely to be associated with higher prevalence of diabetes in this population . with a growing emphasis on preventive and population - based care set forth by the affordable care act ( aca ) , new avenues of health care delivery are being promoted . through the aca , under section 2703 , 
 these health home programs were designed to provide services to reduce barriers to care for those with chronic conditions . given the chronic impairments and difficulties faced by those with smi , it has been hypothesized that the use of the health home will improve early recognition of developing problems and provide access to much needed preventative somatic care . 
 furthermore , given the psychiatric nature of the impairments faced by those with smi , it was hypothesized that placing the health home within the mental health clinic these individuals receive the majority of their care would be the ideal setting to maximize exposure to health home services . 
 this case study presents some preliminary results on a new  behavioral health home  ( bhh ) , which focused on the use of population - based care to identify abnormal hemoglobin a1c ( hba1c ) levels and clarify the severity of such findings thus permitting enhanced , individualized interventions for individuals with smi . 
 it was hypothesized that through the use of bhh services , patients would receive better quality of somatic care and that gaps in care would be readily identified . 
 this bhh was embedded in the division of community psychiatry at the university of maryland school of medicine . 
 the division consists of 2 large outpatient mental health clinics , adult and child assertive community treatment ( act ) team , and an adult psychiatric rehabilitation program ( prp ) . in total , these settings serve 2000 individuals annually . although efforts to enhance somatic care for patients with smi had been a top priority of the division since 2005 , it was in 2013 that the state of maryland approved funding that allowed for the creation of the bhh . 
 behavioral health home referrals were made to patients who were considered to require the most assistance . 
 this was determined by selecting persons with smi who were enrolled in the more intensive act or prp services of the division . 
 individuals in these intensive services were referred to the bhh staff by their mental health provider , and for those who consented , an intake was conducted by bhh initiate services . once enrolled , the bhh provided a minimum of 2 bhh services per month , coordinated by a nurse case manager . a wide variety of services qualified for bhh services , including care coordination , comprehensive transitional care , and health promotion . 
 one service per month was also allowed to be delivered in a group format . given this allowance , a population - based health management program was created to target highly prevalent somatic conditions for individuals with smi ( eg , diabetes and hypertension ) . while initially designed to alternate the somatic condition being tracked every month ( eg , may  diabetes , june  hypertension ) , it was quickly realized that the high prevalence of these conditions required more monitoring time for a more comprehensive assessment . 
 it was determined that monitoring a specific health condition for several consecutive months provided a better understanding of the population s care needs and impact of interventions that included additional education about the illness , nutritional guidelines , and value of regular exercise . 
 patients who did not have their hba1c level drawn within 6 months of the monthly chart review were identified to be not in compliance with clinic recommendations for individuals prescribed atypical antipsychotics . 
 changes in , as well as severity of , hba1c levels ( eg , normal , prediabetes , or type 2 diabetes ) were assessed during monthly chart reviews . 
 this bhh was embedded in the division of community psychiatry at the university of maryland school of medicine . 
 the division consists of 2 large outpatient mental health clinics , adult and child assertive community treatment ( act ) team , and an adult psychiatric rehabilitation program ( prp ) . in total , these settings serve 2000 individuals annually . although efforts to enhance somatic care for patients with smi had been a top priority of the division since 2005 , it was in 2013 that the state of maryland approved funding that allowed for the creation of the bhh . 
 behavioral health home referrals were made to patients who were considered to require the most assistance . 
 this was determined by selecting persons with smi who were enrolled in the more intensive act or prp services of the division . 
 individuals in these intensive services were referred to the bhh staff by their mental health provider , and for those who consented , an intake was conducted by bhh initiate services . once enrolled , the bhh provided a minimum of 2 bhh services per month , coordinated by a nurse case manager . a wide variety of services qualified for bhh services , including care coordination , comprehensive transitional care , and health promotion . 
 one service per month was also allowed to be delivered in a group format . given this allowance , a population - based health management program was created to target highly prevalent somatic conditions for individuals with smi ( eg , diabetes and hypertension ) . while initially designed to alternate the somatic condition being tracked every month ( eg , may  diabetes , june  hypertension ) , it was quickly realized that the high prevalence of these conditions required more monitoring time for a more comprehensive assessment . 
 it was determined that monitoring a specific health condition for several consecutive months provided a better understanding of the population s care needs and impact of interventions that included additional education about the illness , nutritional guidelines , and value of regular exercise . 
 patients who did not have their hba1c level drawn within 6 months of the monthly chart review were identified to be not in compliance with clinic recommendations for individuals prescribed atypical antipsychotics . 
 changes in , as well as severity of , hba1c levels ( eg , normal , prediabetes , or type 2 diabetes ) were assessed during monthly chart reviews . 
 a total of 120 ( 73 males and 47 females ) patients with smi were enrolled and receiving population - based health management services within 10 months of starting the bhh . 
 the average age of the population was 50 years ( standard deviation = 12.75 years ) . of the 120 patients , 54 ( 45% ) 
 were identified who did not have hba1c levels drawn per the clinic s recommended guideline of having hba1c drawn every 6 months for those prescribed antipsychotic medications . 
 thirty - three patients had a diagnosis of type 2 diabetes , 4 of which were newly diagnosed as a result of the population - based health management ( phm ) initiative . 
 another 39 patients were identified as having prediabetes ( hba1c levels between 5.7 and 6.4 ; table 1 ) . 
 abbreviations : bhh , behavioral health home ; hba1c , hemoglobin a1c . of the 33 patients identified as having type 2 diabetes , 
 of the 16 patients , 4 within their target goal achieved this range after initiating in bhh services . 
 two patients who were originally in the prediabetes range when starting bhh services had higher hba1c levels and were in the type 2 diabetes after 10 months . 
 another 7 patients had improvements in hba1c but remained above their target goal , and 4 patients had elevated hba1c since initiating bhh services . 
 behavioral health home services were able to identify 54 individuals who did not have their hba1c levels checked according to the program s recommended guidelines for individuals being prescribed antipsychotic medications . 
 this screening is particularly important for individuals with smi because , consistent with the literature , approximately 27% of this sample had a diagnosis of diabetes . 
 also , another 4 patients were identified to be within diabetic range but did not have any diagnosis of diabetes . 
 preliminary data indicate that the use of bhh services may be particularly useful in identifying gaps in care such as laboratory monitoring or missed diagnoses and allow for the delivery of preventative and individualized proactive care . in regard to health improvements 
 , the data for the 33 patients who were within the type 2 diabetes range are mixed . 
 slightly over 12% ( n = 4 ) of patients improved to be within the recommended guidelines for hba1c levels . 
 another 21% ( n = 7 ) showed improvements in their hba1c levels , albeit still above the target goal . 
 however , 6% ( n = 2 ) of individuals who were originally considered to be in the prediabetes range moved within the diabetic range , and another 12% ( n = 7 ) had higher hba1c levels in comparison to when they started receiving services . 
 given identified problems with traditional primary care settings for this population , new models of care that can deliver  patient - centered  primary care within the mental health setting are needed to decrease the high rates of mortality and morbidity . 
 while more empirical data are needed , preliminary results indicate that the use of a bhh may be particularly useful in understanding the unique characteristics of a specific population . 
 this allows for earlier recognition of common medical disorders through more careful monitoring according to program guidelines and promotes proactive preventative care to prevent advancement of disease . as a result of these findings , 
 a center for disease control and prevention diabetes prevention program group has been started within the mental health clinic and closer monitoring of clinician practice with regard to monitoring and referral to specific services is being done . 
 it is important to note that the current report is limited by the fact that not all patients received equal amounts of phm services , given that enrollment occurred throughout the entirety of the 10 months . 
 also , given the lack of a comparison group , meaningful statistical tests could not be conducted . 
 future research examining the utility of bhh services would benefit from investigating both a clinical and a control sample . furthermore , given that these services are designed to provide more patient - centered care for individuals with smi , an examination of patient satisfaction with bhh and phm services is warranted . 
 while the current data are mixed , the bhh may provide a cost - effective way to provide patient - centered care that helps identify gaps in somatic care for individuals with smi and provide this population with much need interventions .","objective : individuals with serious mental illness ( smi ) have higher rates of preventable diseases such as diabetes in comparison to the general population . 
 while multifaceted , these high rates of preventable diseases in the population with smi may be partially attributed to limited access to primary care . a new program , the behavioral health home ( bhh ) , which allows for the delivery of somatic care coordination and population - based care , may provide this population with the much needed somatic coordination and education it requires.methods:the impact of the population - based health management program of the bhh identification and severity rating of glucose metabolism disorders was assessed during the initial 10 months of the bhh.results:multiple patients were identified who either were not having hemoglobin a1c ( hba1c ) levels drawn per recommended guidelines for individuals prescribed antipsychotic medications or were within diabetic range but did not have a diagnosis of diabetes . 
 mixed results occurred in regard to patients hba1c levels while engaging in the bhh.conclusion:this case study provides some initial evidence for the utility of the bhh in regard to identifying patients who need preventive care .",pubmed
"the american demographer and sociologist valerie oppenheimer wrote a series of influential articles in which she emphasized the role of men s socioeconomic position in demographic change , in particular in the declining rates of marriage and the underlying tendency to increasingly postpone and perhaps even forego marriage ( oppenheimer 1988 , 2000 , 2003 ; oppenheimer et al . 
 i review oppenheimer s original theoretical study , i discuss how her study was held up in empirical research in europe , and i provide a new test of the theory for the european setting . in doing so , i try to resolve some remaining gaps in the empirical literature , and i evaluate whether the theory is equally valid in different countries that make up the european context . given the recent economic crisis in the united states and in europe , and the growing concerns about economic inequality , the influence of men s economic position on marriage and family formation remains a vital concern . at the time oppenheimer began writing her articles on 
 how men s economic position influenced marriage formation  in the late 1980s and early 1990s  this was generally not a popular idea . 
 the declining rates of marriage and increasing rates of divorce were typically conceptualized in terms of an "" erosion of marriage . "" 
 one theory looked for the culprit in the growing economic role of women in society . 
 this theory was voiced by demographers and economists working from a micro - economic perspective ( becker 1981 ; espenshade 1985 ; farley 1988 ) , although , as oppenheimer noted ( 1988 , p. 575 ) , it bore a strong resemblance to classic sociological theories formulated by functionalists like talcot parsons ( parsons 1949 ) . 
 the explanation basically argued that more symmetrical economic roles of men and women would lead to a decline in the gains to marriage , or to put it in parsonian terms , would undermine marital solidarity . 
 the second explanation argued that the decline of marriage was related to value change , and in particular to the increasing need for individual autonomy on the one hand , and the ideological condemnation of traditional institutions like marriage on the other . 
 this second perspective was expressed more strongly by european demographers like lesthaeghe and van de kaa although it was also used by the influential american demographers at the time ( bumpass 1990 ; rindfuss and van den heuvel 1990 ) . in their second demographic transition theory , lesthaeghe and van de kaa argued that ideological change in combination with secularization was driving not only the postponement of marriage , but also the increase in cohabitation , the rise in divorce , and the decline of fertility ( lesthaeghe 1983 ; lesthaeghe and meekers 1986 ; lesthaeghe and surkuyn 1988 ; van de kaa 1987 ) . 
 while the first explanation saw the engine of the demographic transition in economic change , the second emphasized the primacy of cultural change . 
 both theories , however , were pessimistic about the future of marriage : the economic perspective saw marriage as incompatible with symmetrical gender roles , the second saw it as incompatible with individualistic values . while there was a considerable debate between the proponents of economic and cultural explanations , oppenheimer criticized both perspectives . 
 for example , she noted that there were no signs of a so - called independence effect . 
 women with attractive economic resources were not less likely to enter marriage , as would be predicted from the micro - economic perspective ( oppenheimer and lew 1995 ) . 
 although women s employment and education had an effect on fertility and divorce , this did not appear to be the case for marriage timing ( oppenheimer 1997 ) . 
 oppenheimer also had empirical critique on the cultural perspective . when looking at simple descriptive statistics on what people want for themselves  on people s hopes and desires  she noted that the majority of both single men and women still wanted to be married ( oppenheimer 1994 ) . 
 the anti - marriage ideology may have existed in feminist circles or in the pop culture of the sixties , but it had not spread to a larger audience in the way that , for example , egalitarian gender norms had done . 
 oppenheimer also had theoretical criticisms of the two explanations ( oppenheimer 1994 , 1997 ) . 
 first , she believed that the theories were basically about nonmarriage and not about delays in marriage . 
 as other demographers also had observed , the declining marriage rate was primarily driven by increases in the age at marriage , and not so much by a decline in the proportion of persons who marry eventually , although the latter could of course not yet be observed in the late 1980s . 
 this seems by and large correct now , although the proportion of the marrying persons among the lower educated in the united states did appear to decline ( goldstein and kenney 2001 ) . 
 a second part of her theoretical critique was against the micro - economic model of specialization . quoting historical demographic work , oppenheimer noted that wives in the past had always worked for pay when circumstances required this . 
 wives worked to make ends meet when the husband was not making enough money , when he was unemployed , or when household costs were temporarily pressing ( oppenheimer 1982 ) . 
 oppenheimer argued that specialization in marriage is an inflexible and risky strategy in many different societal contexts . 
 if marriage was not based on a model of full specialization in the more distant past , oppenheimer argued , why would it then cease to exist in the modern era in which wives began to work ? 
 oppenheimer not only criticized the then dominant perspectives on demographic change , she also presented an alternative . 
 her explanation can be placed in the economic rather than the cultural camp , but it was different in that it focused on men rather than women . during the 1980s and 1990s , young men 
 s economic position in the united states had deteriorated quickly , especially for those with little schooling . in the poor and uncertain economic prospects of young men 
 , oppenheimer saw an important potential for understanding the decline of marriage . because the earlier explanation had focused more on women  especially through arguments about women s economic independence 
 one could say that oppenheimer was in fact "" bringing men back into the debate . "" 
 first , she reinstated older malthusian ideas about the economic costs of marriage ( hajnal 1965 ; easterlin 1980 ) . as setting up and running a household costs money , men who are unable to fulfill 
 oppenheimer recognized that this traditional male - breadwinner hypothesis may have lost some of its force when gender roles become more symmetrical . 
 nonetheless , she argued that it would also be naive to expect men s economic resources to become unimportant in influencing marriage prospects : this would be "" throwing out the baby with the bathwater . 
 "" the second way in which she brought men back in the debate was through her uncertainty hypothesis ( oppenheimer 1988 ) . 
 the argument is that unstable careers , as indicated by low - status jobs , nonemployment , and irregular and temporary employment , signal uncertainty . 
 this uncertainty applies not only to whether the husband will be able to provide in the future , but also to the type of life he will lead . work structures the lifestyle a person will develop , and when men have not yet settled in their career it is difficult to predict what married life will be like . in this way 
 . an important difference between the breadwinner and the uncertainty hypotheses is that the former focuses primarily on the financial aspects of employment whereas the latter is also concerned with its social consequences . 
 an implication is that the neo - malthusian argument would be fully covered by effects of income , whereas the uncertainty argument would also be reflected in indicators like irregular attachment to the labor market , the amount of work experience , career trajectories , and temporary employment . in a separate article 
 , oppenheimer also developed and operationalized the concept of a stopgap job , i.e. , a job that is not a reflection of an employee s educational credentials and that is meant as temporary by both employer and employee ( oppenheimer and kalmijn 1995 ) . 
 men in such stopgap jobs would postpone marriage because they are not settled in their career and therefore can not yet make a suitable match in the marriage market . 
 compared to the other two perspectives , oppenheimer s theory has a more optimistic implication for the future of marriage . 
 after all , female labor force participation was unlikely to decline in the future and individualism did not appear to be receding . in oppenheimer 
 s theory , the economic position of young men largely depends on macro - economic conditions . because unemployment rates tend to have cyclical rather than linear trend patterns , the economic position of young men could improve and this would then have positive repercussions for marriage . 
 moreover , the theory only implies the postponement of marriage until men accumulate more work experience and become settled in their career , and not an erosion of the institution of marriage , as the other theories seem to imply . 
 oppenheimer 's explanation had a second attractive feature : it could also explain another important demographic trend , namely the rise in cohabitation ( oppenheimer 2003 ) . 
 oppenheimer argued that a man s failure to provide economically would be less of a problem for cohabitation than for marriage . 
 for many couples , cohabitation is a trial stage before marriage , and it may be that uncertainty about a young man s position is more tolerable during the cohabiting stage than it would be for a long - term commitment to marriage . 
 assuming that the costs of breaking up a cohabiting union are lower , cohabitation can therefore provide a way for couples to reduce uncertainty about future career prospects . in a sense , oppenheimer argued that a cultural innovation like cohabitation before marriage ( on a massive scale ) was the outcome of economic needs rather than the result of ideological change . in line with this , other authors even argued that cohabitation is a rational response to uncertainty : a flexible partnership well - suited for a flexible labor market ( mills et al . 
 american research generally supports the view that poor economic prospects for men are associated with a delay in marriage . 
 this has been demonstrated for a range of indicators , including employment per se , unstable employment , low earnings , and other indicators of career "" immaturity "" ( clarkberg 1999 ; lichter et al . 
 1992 ; lloyd and south 1996 ; mare and winship 1991 ; oppenheimer 2003 ; oppenheimer et al . 
 1997 ; sassler and schoen 1999 ; smock and manning 1997 ; sweeney 2002 ; xie et al . 
 there is also evidence in the united states that cohabitation is less strongly influenced by men s economic position than marriage , although there is no clear reverse income effect , i.e. , that the poor are being selected into cohabiting unions . 
 furthermore , in the united states , the income effect on marriage timing appears to be stable over time . sweeney ( 2002 ) 
 compared two cohorts in the united states and found that in the cohort marrying during the 1980s and 1990s , men s income had an equally strong positive effect on the entry into marriage as in the cohort marrying during the 1960s and 1970s ( sweeney 2002 ) . 
 in this article , i develop a new test of oppenheimer s theory for the european context . 
 first , the demographic trends that occurred in europe were similar , although sometimes less dramatic and sometimes occurring later . 
 the age at marriage has risen , the rate of marriage has declined , and cohabitation has increased as well ( kiernan 2002 ; lesthaeghe 1983 ; van de kaa 1987 ) . 
 second , many european countries experienced economic problems that were similar to those in the united states . 
 they were especially salient for outsiders on the labor market , such as young adults , ethnic minorities , and women . 
 several authors argued that partly in response to economic globalization , young european men ( and women ) faced increasing levels of economic uncertainty in their transition from school to work ( blossfeld et al . 2005 ) . in many european countries , especially in southern europe , 
 levels of youth unemployment were even higher than in the united states , a phenomenon which has often been linked to the higher degrees of employment protection in europe ( mller and gangl 2003 ; nickell 1997 ) . 
 there are also reasons to believe that the theory would be less applicable to europe . 
 one counter argument lies in the role of the welfare state . in several european societies , and particularly in social - democratic welfare states like sweden , denmark , and the netherlands , social security is more generous and more universally provided than it is in the united states ( arts and gelissen 2002 ; esping - andersen 1993 ) . 
 this means that in many european countries , young men receive unemployment benefits when they are out of work . 
 moreover , for those who have never worked , basic welfare is provided , albeit at a minimum level . as a result 
 following the neo - malthusian argument , it could thus be argued that employment problems do not per se lead to marriage postponement in europe . 
 a rebuttal of this point is that oppenheimer s argument about uncertainty and assortative mating , which is not only about money , but also about stability and predictability , could still apply to europe . 
 a young man who is on unemployment benefits remains an uncertain candidate on the marriage market even if he has the financial means to support a household at that point in time . 
 another important difference between the american and the european case lies in the degree of heterogeneity . 
 although the united states is certainly not a homogeneous country  there are important ethnic , racial , and regional differences  it is fair to say that europe is more heterogeneous ( at least regionally ) than the united states . in comparative studies , it has often been argued that european countries can be rated on a continuum from more traditional societies such as spain , greece , and italy on the one hand , to more modern and more economically developed societies such as sweden and the netherlands on the other ( hagenaars et al . 
 these differences are expressed in a number of social and cultural domains , including differences in marriage and family living . 
 for example , in more traditional european societies , cohabitation and divorce are less common and less accepted , marriage has a higher social status , gender roles in marriage are more unequal , female labor force participation is lower , and extended family ties are stronger ( hans - peter blossfeld and hakim 1997 ; gelissen 2003 ; kalmijn 2003 ; knudsen and waerness 2008 ; reher 1998 ) . 
 these indicators are strongly correlated , both with each other and with the level of economic development in a country ( gdp ) . 
 this degree of heterogeneity suggests that oppenheimer s theory may not apply equally to all european countries . 
 for example , in contexts where gender roles are more egalitarian , men s economic situation could be less important for the entry into marriage and cohabitation . in these settings , men are not the only breadwinners and women s economic resources should be of growing importance , making men s economic resources less important . what has the evidence in europe shown so far ? 
 perhaps the most important source of evidence comes from a large multi - nation project initiated by the german sociologist blossfeld and his colleagues ( blossfeld et al . 
 in this project , blossfeld brought together a number of demographers and sociologists from different parts of the world ( with an overrepresentation of european countries ) , with the aim of examining the effect of men and women s individual economic resources on the timing of marriage and parenthood . 
 while the authors used their own country - specific longitudinal data , they used similar methods and variables , leading to a reasonably uniform and comparable set of outcomes . 
 the project s goal was to test the exact same set of hypotheses in each country . 
 the hypotheses were borrowed in part from oppenheimer s work but they were translated by mills and blossfeld to make them fit for a broader societal setting ( mills and blossfeld 2005 ) . 
 the articles were combined in a volume for which oppenheimer wrote the foreword ( blossfeld et al . 
 the articles in blossfeld s volume provide generally positive evidence for the theory in the european countries studied ( germany , the netherlands , france , sweden , hungary , great britain , italy , and spain ) . in 
 virtually all countries , school enrollment  one of the indicators of uncertainty  negatively affected the entry into marriage . 
 more importantly , men s unemployment appeared to lower the chances of entering marriage in most countries ( bernardi and nazio 2005 ; kieffer et al . 
 2005 ; liefbroer 2005 ; noguera et al . 2005 ; robert and bukodi 2005 ) . in britain , 
 an effect of unemployment was observed only on the transition from cohabitation to marriage , and not on the transition from being single to living together ( francesconi and golsch 2005 ) . in sweden , only unemployment after leaving school appeared to delay marriage formation , not unemployment after a period of employment ( bygren et al . 2005 ) . 
 some evidence was also found for the effect of temporary contracts . in italy , spain , 
 france , and the netherlands , it was shown that men who were employed temporarily were less likely to enter marriage than men who had permanent employment . in germany and hungary 
 , there was no effect of temporary work , however , and in several other countries , the effect could not be studied . 
 a recent analyses of fertility in europe has also pointed to the delaying effect of temporary contracts ( adsera 2011 ) . outside the blossfeld project 
 , there were a number of important individual articles in which aspects of oppenheimer s theory were tested . 
 for example , in sweden , it was found that men s employment increased the chances of union formation while it did not affect the chances of marriage after cohabitation ( bracher and santow 1998 ) . in norway , men s employment increased the chances of marrying after being single and the chance of marrying after living together ( kravdal 1999 ) . in the netherlands , 
 men s employment had a stronger effect on direct marriage than on cohabitation but there was no effect of employment on marriage after cohabitation ( kalmijn and luijkx 2005 ; cf . 
 the evidence outside europe ( i.e. , israel ) has been supportive as well ( raz - yurovich 2010 ) , as has been the evidence in central and eastern europe , a region not included in the present article ( gerber and berman 2010 ) . while the role of employment has often been studied in europe , less 
 is known about how men s income and earnings affect union formation . many of the studies discussed above were based on retrospective rather than prospective longitudinal data . 
 the panel data that exist have been collected by economists and labor market researchers and do not always have the desirable demographic indicators . because income can not be measured well in a retrospective fashion , this has also meant that we know little about the income effects on marriage and divorce in europe . this is unfortunate because employment and income need to be examined simultaneously , especially if one wants to make a distinction between the neo - malthusian breadwinner hypothesis on the one hand , and oppenheimer s uncertainty hypothesis on the other . because in many european welfare states nonemployment does not , per se , 
 another drawback of the prevailing evidence is that most studies are based on single countries . 
 blossfeld s multi - nation project is clearly a major step forward in trying to summarize the evidence for europe as a whole , but the analyses are not pooled so the results can only be summarized verbally . 
 moreover , possible differences that exist between countries can be described but they can not be compared or tested in a more rigorous fashion . for these reasons , 
 in answering this question , i not only look at employment , but also i look at men s income , work experience , and type of labor contract . by looking at income and employment patterns simultaneously 
 , i obtain more direct evidence on the underlying mechanisms . the period for which i answer 
 unemployment rates increased substantially in the early 1990s before declining again in the mid to late 1990s ( oecd 2009 ) . 
 in italy , greece , and belgium , unemployment remained high in the late 1990s but began to decline later , in the early 2000s . 
 in other words , for most countries , the period that i examine covers a recovery stage of the economy , a stage which should have been positive for marriage and family formation . 
 second , are the effects of men s economic position similar or different for the chances of entering marriage and the chances of entering a cohabiting union ? with this part of the study , i replicate the last influential study of oppenheimer ( 2003 ) , in which she studied this issue for the united states 
 . we would expect effects to be weaker for cohabitation than for marriage : marriage would require a stronger economic underpinning than cohabitation ( kravdal 1999 ; oppenheimer 2003 ) . 
 in addition , i examine the chances that cohabiting unions turn into marriage . here too , oppenheimer expects men s economic position to have an effect , but because those who cohabit already have an independent household , the effects of income will probably be weaker . 
 third , to what extent are the effects of men s economic position on union formation different across societal contexts ? in this part , i focus on differences between traditional and egalitarian societies . 
 the expectation is that men s economic characteristics remain important in traditional societies but are less important in more modern , egalitarian ones . by looking at differences among societal contexts 
 , i try to generalize the cross - cohort comparison that sweeney ( 2002 ) made for the united states . answering this question is also of more general importance because if we find conditions under which the theory is ( not ) true , this could in principle lead to theoretical progress in the field . 
 i use panel data that are collected in the same format for a number of european countries , i.e. , the european community household panel ( echp ) . 
 the echp was an annual panel survey held between 1994 and 2001 ( behr et al . 
 samples are large and representative , and ( almost ) the same questionnaire was used each year . for the analyses in this article , i use data from 13 countries 
 : denmark , finland , germany , austria , the netherlands , belgium , france , the united kingdom , ireland , portugal , spain , italy , and greece . 
 my sample is limited to men who were never married in the first wave of the panel . 
 although i am able to exclude previously married men , i can not exclude men who ended a cohabiting union before the panel began . 
 never married men who were cohabiting in the first wave are also included because these men can make the transition from cohabitation to marriage . the first wave of data from the netherlands is excluded because no information is available on cohabitation status 
 there have been previous demographic analyses of the echp most notably by adsera ( 2011 ) who analyzes the effect of individual and aggregate labor market characteristics on fertility . 
 although adsera focused more on women than on men , her general conclusion is that labor market uncertainty is very influential in delaying fertility , in line with the perspective suggested above . in the current article , we go back one step by analyzing how labor market uncertainty affects union formation , a transition which probably remains the most important necessary condition for family formation . 
 i use discrete time event - history analysis by estimating logistic regression models on a person - year file . 
 the person - year file begins at the first wave and ends in the last wave or when a transition is made . as is the case with all event - history analyses of panel data , 
 such left truncation problems can be solved in principle , but not without losing our time - varying independent variables ( guo 1993 ) . 
 the first logit model is estimated for person - years in which men are single and never married . 
 the dependent variable is whether a man is living without a partner in wave t and living with a partner in wave t  +  1 ( union formation ) . in the second logit model , 
 i estimated which choice was made , cohabitation or marriage , using only the person - years in which the event occurred . 
 this sequential approach is slightly different from the approach taken by oppenheimer , who estimated multinomial logit ( i.e. , competing risk ) models ( 2003 ) . 
 the sequential approach does not need the assumption implicit in a competing risk model that the two choices 
 this assumption is problematic because it is plausible that unmeasured factors like personality , wealth , attractiveness , and the like , influence marriage and cohabitation to the same extent ( hill et al . 
 if a person is single in year t , missing in year t  +  1 , and married or cohabiting in year t  +  2 , i also regarded this as an event . 
 duration dependency is modeled with two age effects , according to the approach developed by blossfeld and huinink ( 1991 ) . 
 blossfeld used log ( age  a1 ) and log ( a2age ) to capture the nonlinear age - dependency of union formation , where a1 is the lowest and a2 the highest age in the sample . 
 there were 4,492 transitions to a first union of which 2,499 were to cohabitation and 1,993 to marriage . 
 the second logit model is estimated for person - years in which men were living with a partner without being married . 
 the dependent variable is defined as living with a partner unmarried in wave t and being married in wave t  +  1 ( marriage after cohabitation ) . respondents who were living alone in wave t  +  1 are truncated . 
 hence , separation is treated as a competing risk . if a person is cohabiting in year t , not in the panel in year t  +  1 , and married in year t  +  2 , i also regarded this as an event . 
 duration dependency could not be modeled directly because for those who are in a cohabitating union in the first wave of the panel , no data on the start of the union is available . as an alternative 
 age is obviously less ideal than duration since persons enter a cohabiting union at different ages . 
 it is noted that there was no question in the interview about whether the partner in one wave was the same partner as in the previous wave . 
 hence , i could not check if a man changed ( cohabiting ) partners between the subsequent waves , nor could i check if the married partner was the same person as the cohabiting partner in the previous wave . 
 all independent variables , except where noted , refer to time t. as the dependent variables refer to whether or not a transition was made between the time t and t  +  1 , the independent variables precede the dependent variable in time . 
 the first indicates a man is working on a paid job or is self - employed at the time of the interview for at least 15  h a week . 
 the second variable indicates other less common forms of employment , such as military service , apprenticeships , unpaid family work , and working less than 15  h. given that a man is employed , i also considered the type of contract he has . 
 temporary contracts include fixed - term or short - term contracts , casual work with no contract , and "" some other working arrangement "" that is not a permanent contract ( all included within the 15 + hours category ) . the coding is cumulative so that the effect of temporary work refers to the difference between the men with a temporary contract and men with a permanent contract . 
 i also include whether a person is enrolled in full - time schooling in the interview week . 
 data on work history are obtained from a monthly calendar that all respondents had to fill out . for the year 
 t    1 , i counted the number of months that a man was employed , self - employed , or in school ( hereafter called "" active "" ) . 
 i checked whether the effects of this variable were different when excluding the netherlands but this was not the case . 
 i consider personal income and not only income from work since social security income may also provide a stable source of income . 
 ideally , we would like to measure the income a man had in the interview month or in the 12  months before the interview . 
 unfortunately , incomes are measured for ( full ) calendar years only . to solve this 
 , i took a weighted average of the income in calendar year t    1 and the income in calendar year t , using information on the month of interview . 
 for example , if the interview was in september 1994 , income for that person - year is 9/12 of the income earned in 1994 plus 3/12 of the income earned in 1993 . 
 it is noted that some of the income in year t may be earned after a man marries . 
 marrying in the year of the interview is more likely when the interview took place early in the year , but then , this income receives a lower weight . 
 i corrected all incomes for changes in the consumer price index and converted them to pounds sterling . to estimate effects of income in a comparable way across countries 
 i first calculated income quintiles in each country using data from all men in the first wave of the echp . 
 next , i used these quintiles to categorize the men in the person - year file . 
 i also use a linear income variable which is coded 15 for the five quintiles . 
 three control variables are used which may affect union formation : general health ( time varying ) , the highest level of educational attainment , and the year of the interview ( using dummies ) . 
 health is controlled because having a poor health may be detrimental for finding a partner and is also related to job and income opportunities ( waldron et al . 
 descriptive findings for the independent variables for each country are presented in table  1.table  1means of independent variables in person - year file by countryyear ( 18)agegeneral health ( 15)secondary educationtertiary educationworking 15 + hoursother worktemporary contractenrolled in schoolmonths active ( 012)relative income ( 15)income in poundsuk ( n  =  7,585)4.0731.883.910.080.680.750.030.050.059.943.0410546ireland ( n  =  7,741)3.4032.314.370.440.230.620.050.080.129.522.707184germany ( 
 n  =  9,581)4.0630.123.790.640.260.680.090.070.1210.412.559536austria ( n  =  5,782)4.4829.124.400.790.080.700.080.060.1310.672.508995france ( n  =  11,385)3.9031.993.870.420.320.600.060.090.149.492.528537belgium ( n  =  3,842)3.8132.254.170.440.440.630.010.080.199.962.469182netherlands ( n  =  5,248)4.5232.664.110.540.220.730.030.100.149.002.449841italy ( n  =  18,316)4.0229.884.110.520.140.530.060.070.178.552.494696greece ( n  =  7,942)3.8929.644.690.470.280.610.110.120.119.572.574220spain ( n  =  12,764)3.8330.834.080.250.390.550.040.200.168.682.544553portugal ( n  =  10,097)4.0929.423.690.220.110.680.040.150.1210.062.883062finland ( n  =  5,360)5.0132.954.060.560.260.610.040.090.179.452.5511526denmark ( n  =  5,381)3.8833.384.420.450.400.710.060.080.1110.202.9912814total ( 
 n  =  111,024)4.0330.974.090.430.280.630.060.100.139.502.627227source : echp ( own calculations ) means of independent variables in person - year file by country source : echp ( own calculations ) to explore differences across societies , i constructed two measures at the macro level . 
 first , i considered a measure of the division of household labor in marriage in a society . 
 this measure was obtained from a article by knudsen and waerness ( 2008 ) and measures the extent to which husbands and wives share four household tasks ( i.e. , laundry , grocery shopping , meal preparation , caring for sick family members ) . 
 the second measure is the female labor force participation rate in the 1990s , which is defined as the percentage of women aged 2049 who are active in the labor force ( obtained from ilo , geneva ) . 
 . countries in which wives participate more often in the labor force are also countries in which the household tasks are divided more equally ( r  =  .74 ) . 
 the most traditional societies are southern european countries , the most egalitarian are northern european countries . 
 i construct a single macro - level indicator , which is the means of the two standardized items . 
 i use discrete time event - history analysis by estimating logistic regression models on a person - year file . 
 the person - year file begins at the first wave and ends in the last wave or when a transition is made . as is the case with all event - history analyses of panel data , 
 such left truncation problems can be solved in principle , but not without losing our time - varying independent variables ( guo 1993 ) . 
 the first logit model is estimated for person - years in which men are single and never married . 
 the dependent variable is whether a man is living without a partner in wave t and living with a partner in wave t  +  1 ( union formation ) . in the second logit model , 
 i estimated which choice was made , cohabitation or marriage , using only the person - years in which the event occurred . 
 this sequential approach is slightly different from the approach taken by oppenheimer , who estimated multinomial logit ( i.e. , competing risk ) models ( 2003 ) . 
 the sequential approach does not need the assumption implicit in a competing risk model that the two choices 
 this assumption is problematic because it is plausible that unmeasured factors like personality , wealth , attractiveness , and the like , influence marriage and cohabitation to the same extent ( hill et al . 
 if a person is single in year t , missing in year t  +  1 , and married or cohabiting in year t  +  2 , i also regarded this as an event . 
 duration dependency is modeled with two age effects , according to the approach developed by blossfeld and huinink ( 1991 ) . 
 blossfeld used log ( age  a1 ) and log ( a2age ) to capture the nonlinear age - dependency of union formation , where a1 is the lowest and a2 the highest age in the sample . 
 there were 4,492 transitions to a first union of which 2,499 were to cohabitation and 1,993 to marriage . 
 the second logit model is estimated for person - years in which men were living with a partner without being married . 
 the dependent variable is defined as living with a partner unmarried in wave t and being married in wave t  +  1 ( marriage after cohabitation ) . respondents who were living alone in wave t  +  1 are truncated . 
 hence , separation is treated as a competing risk . if a person is cohabiting in year t , not in the panel in year t  +  1 , and married in year t  +  2 , i also regarded this as an event . 
 duration dependency could not be modeled directly because for those who are in a cohabitating union in the first wave of the panel , no data on the start of the union is available . as an alternative , 
 age is obviously less ideal than duration since persons enter a cohabiting union at different ages . 
 it is noted that there was no question in the interview about whether the partner in one wave was the same partner as in the previous wave . 
 hence , i could not check if a man changed ( cohabiting ) partners between the subsequent waves , nor could i check if the married partner was the same person as the cohabiting partner in the previous wave . 
 all independent variables , except where noted , refer to time t. as the dependent variables refer to whether or not a transition was made between the time t and t  +  1 , the independent variables precede the dependent variable in time . 
 the first indicates a man is working on a paid job or is self - employed at the time of the interview for at least 15  h a week . 
 the second variable indicates other less common forms of employment , such as military service , apprenticeships , unpaid family work , and working less than 15  h. given that a man is employed , i also considered the type of contract he has . 
 temporary contracts include fixed - term or short - term contracts , casual work with no contract , and "" some other working arrangement "" that is not a permanent contract ( all included within the 15 + hours category ) . 
 the coding is cumulative so that the effect of temporary work refers to the difference between the men with a temporary contract and men with a permanent contract . 
 i also include whether a person is enrolled in full - time schooling in the interview week . 
 data on work history are obtained from a monthly calendar that all respondents had to fill out . 
 for the year t    1 , i counted the number of months that a man was employed , self - employed , or in school ( hereafter called "" active "" ) . 
 i checked whether the effects of this variable were different when excluding the netherlands but this was not the case . 
 i consider personal income and not only income from work since social security income may also provide a stable source of income . 
 ideally , we would like to measure the income a man had in the interview month or in the 12  months before the interview . 
 unfortunately , incomes are measured for ( full ) calendar years only . to solve this 
 , i took a weighted average of the income in calendar year t    1 and the income in calendar year t , using information on the month of interview . 
 for example , if the interview was in september 1994 , income for that person - year is 9/12 of the income earned in 1994 plus 3/12 of the income earned in 1993 . 
 it is noted that some of the income in year t may be earned after a man marries . 
 marrying in the year of the interview is more likely when the interview took place early in the year , but then , this income receives a lower weight . 
 i corrected all incomes for changes in the consumer price index and converted them to pounds sterling . to estimate effects of income in a comparable way across countries , 
 i first calculated income quintiles in each country using data from all men in the first wave of the echp . 
 next , i used these quintiles to categorize the men in the person - year file . 
 i also use a linear income variable which is coded 15 for the five quintiles . 
 three control variables are used which may affect union formation : general health ( time varying ) , the highest level of educational attainment , and the year of the interview ( using dummies ) . 
 health is controlled because having a poor health may be detrimental for finding a partner and is also related to job and income opportunities ( waldron et al . 
 descriptive findings for the independent variables for each country are presented in table  1.table  1means of independent variables in person - year file by countryyear ( 18)agegeneral health ( 15)secondary educationtertiary educationworking 15 + hoursother worktemporary contractenrolled in schoolmonths active ( 012)relative income ( 15)income in poundsuk ( n  =  7,585)4.0731.883.910.080.680.750.030.050.059.943.0410546ireland ( n  =  7,741)3.4032.314.370.440.230.620.050.080.129.522.707184germany ( n  =  9,581)4.0630.123.790.640.260.680.090.070.1210.412.559536austria ( n  =  5,782)4.4829.124.400.790.080.700.080.060.1310.672.508995france ( n  =  11,385)3.9031.993.870.420.320.600.060.090.149.492.528537belgium ( n  =  3,842)3.8132.254.170.440.440.630.010.080.199.962.469182netherlands ( n  =  5,248)4.5232.664.110.540.220.730.030.100.149.002.449841italy ( n  =  18,316)4.0229.884.110.520.140.530.060.070.178.552.494696greece ( n  =  7,942)3.8929.644.690.470.280.610.110.120.119.572.574220spain ( n  =  12,764)3.8330.834.080.250.390.550.040.200.168.682.544553portugal ( n  =  10,097)4.0929.423.690.220.110.680.040.150.1210.062.883062finland ( n  =  5,360)5.0132.954.060.560.260.610.040.090.179.452.5511526denmark ( 
 n  =  5,381)3.8833.384.420.450.400.710.060.080.1110.202.9912814total ( n  =  111,024)4.0330.974.090.430.280.630.060.100.139.502.627227source : echp ( own calculations ) means of independent variables in person - year file by country source : echp ( own calculations ) to explore differences across societies , i constructed two measures at the macro level . 
 first , i considered a measure of the division of household labor in marriage in a society . 
 this measure was obtained from a article by knudsen and waerness ( 2008 ) and measures the extent to which husbands and wives share four household tasks ( i.e. , laundry , grocery shopping , meal preparation , caring for sick family members ) . 
 the second measure is the female labor force participation rate in the 1990s , which is defined as the percentage of women aged 2049 who are active in the labor force ( obtained from ilo , geneva ) . 
 . countries in which wives participate more often in the labor force are also countries in which the household tasks are divided more equally ( r  =  .74 ) . 
 the most traditional societies are southern european countries , the most egalitarian are northern european countries . 
 i construct a single macro - level indicator , which is the means of the two standardized items 
 figure  1 shows that there are small differences within europe in the age at first union entry . in most countries , 
 male age at entry is low in portugal and austria , and high in the netherlands and italy . 
 for example , the age at union formation is quite high in italy and greece where the family is a strong institution . 
 europe , youth unemployment is high . when we look at the type of first union chosen by men , presented in the left side of fig .  1 
 even more in northern europe , direct marriage is a minority experience . in many countries , more than 80% of men cohabit first . in southern europe , the proportions are almost reversed : 6080% of the men in these countries marry directly . 
 the united kingdom resembles western europe but ireland resembles the south , with most men choosing marriage as their first way to enter a union . 
 1men s first union formation in 13 countries men s first union formation in 13 countries the effects of men s economic characteristics on union entry are presented in table  2 . 
 the odds of entering a union are 58% higher for employed men than for men who are not employed and not in school ( e ) . 
 the type of labor contract also matters for the choice between marriage and cohabitation . compared to men with a permanent job , men with a temporary job who enter a union are 23% less likely to choose marriage than cohabitation . 
 we also note that school enrollment has a negative effect on union entry while it does not affect the type of union.table  2discrete time event history models of union formation : logit regression coefficients and p - values in parentheses  union formationmarriage vs. cohabitationmarriage after cohabitationmodel amodel bmodel cmodel dmodel emodel fmodel gmodel hmodel iln ( age15)0.841 * * * ( 0.000)0.661 * * * ( 0.000)0.676 * * * ( 0.000)0.746 * * * ( 0.000)0.745 * * * ( 0.000)0.741 * * * ( 0.000)0.230 * ( 0.040)0.317 * * ( 0.006)0.316 * * ( 0.006)ln ( 65age)1.872 * * * ( 0.000)1.816 * * * ( 0.000)1.847 * * * ( 0.000)0.810 * * * ( 0.000)0.813 * * * ( 0.000)0.809 * * * ( 0.000)0.296 * ( 0.038)0.264 ( 0.061)0.268 ( 0.058)general health0.106 * * * ( 0.000)0.100 * * * ( 0.000)0.098 * * * ( 0.000)0.135 * ( 0.012)0.135 * ( 0.013)0.135 * ( 0.012)0.107 * * ( 0.008)0.104 * ( 0.010)0.103 * ( 0.011)education secondary versus primary0.029 ( 0.491)0.002 ( 0.955)0.007 ( 0.867)0.046 ( 0.655)0.035 ( 0.731)0.047 ( 0.648)0.005 ( 0.951)0.012 ( 0.890)0.014 ( 0.873)education tertiary versus primary0.225 * * * ( 0.000)0.141 * * ( 0.003)0.122 * * ( 0.010)0.113 ( 0.311)0.149 ( 0.194)0.111 ( 0.326)0.408 * * * ( 0.000)0.361 * * * ( 0.000)0.349 * * * ( 0.000)working 15 + hours versus no work / school0.457 * * * ( 0.000)0.193 * * ( 0.003)0.220 * * * ( 0.001)0.450 * * ( 0.003)0.419 * * ( 0.007)0.445 * * ( 0.004)0.178 ( 0.166)0.090 ( 0.495)0.102 ( 0.434)other employed versus no work / school0.144 ( 0.088)0.204 * ( 0.016)0.188 * ( 0.026)0.279 ( 0.176)0.350 ( 0.096)0.282 ( 0.173)0.237 ( 0.277)0.250 ( 0.254)0.245 ( 0.263)temporary versus fixed contract0.067 ( 0.182)0.026 ( 0.609)0.031 ( 0.544)0.253 * ( 0.033)0.262 * ( 0.028)0.251 * ( 0.035)0.120 ( 0.249)0.086 ( 0.410)0.090 ( 0.386)in school versus no versus no work / school0.623 * * * ( 0.000)0.431***(0.000)0.468 * * * ( 0.000)0.180 ( 0.388)0.089 ( 0.679)0.173 ( 0.412)0.515 * ( 0.015)0.410 ( 0.059)0.424 * ( 0.046)months worked last 2  years0.022 * * * ( 0.000)0.006 ( 0.243)0.007 ( 0.178)0.008 ( 0.518)0.011 ( 0.405)0.009 ( 0.504)0.031 * ( 0.010)0.021 ( 0.089)0.023 ( 0.066)first income quintile versus third quintile0.604 * * * ( 0.000)0.272 ( 0.054)0.256 * ( 0.044)second income quintile versus third quintile0.253 * * * ( 0.000)0.039 ( 0.735)0.182 * ( 0.037)fourth income quintile versus third quintile0.232 * * * ( 0.000)0.136 ( 0.210)0.094 ( 0.224)fifth income quintile versus third quintile0.366 * * * ( 0.000)0.127 ( 0.328)0.109 ( 0.233)linear income ( 15)0.239 * * * ( 0.000)0.007 ( 0.861)0.098 * * * ( 0.001)constant11.814 * * * ( 0.000)10.677 * * * ( 0.000)11.595 * * * ( 0.000)7.903 * * * ( 0.000)7.789 * * * ( 0.000)7.895 * * * ( 0.000)3.346 * * * ( 0.000)2.771 * * * ( 0.000)3.138 * * * ( 0.000)n776217762177621447244724472116781167811678224024602451162516301625335348346note : controlled for country and year dummies*p  <  .05 , * * p  <  .01 , * * * p  <  .001 discrete time event history models of union formation : logit regression coefficients and p - values in parentheses note : controlled for country and year dummies * p  <  .05 , * * p  <  .01 , * * * p  <  .001 next to the effects of current employment characteristics , we see effects of men s work history . 
 the more months men were employed or otherwise active in the past calendar year , the more likely they are to enter a union in the next two calendar years . 
 men who were active for an entire year were e  =  30% more likely to enter a union in the next 2  years than men who were inactive the entire year . 
 this effect comes on top of the effect of a man s current employment situation . 
 there is no effect of work history on the choice between marriage and cohabitation . in sum 
 , the findings from model a and model d confirm that men who are not yet settled in their career postpone union formation . 
 the findings also confirm that cohabitation is less sensitive to economic insecurity than marriage , although this applies more to employment per se and the type of employment than to a man s work history . in model 
 the poorest quintile of men are 45% less likely to enter a union than the middle quintile , while the richest quintile are 44% more likely to do so . 
 the income effects increase monotonically but the differences between the quintiles become smaller at higher income levels . a formal test shows 
 that the effect is not strictly linear : the nonlinear model for union formation ( model b ) has a better fit than the linear model ( model c ,   =  9.3 , p  =  .03 ) . 
 when we look at the income effect on choice between the marriage and cohabitation , we only see one marginally significant effect . 
 men in the lowest quintile who enter a union are 31% more likely than the middle quintile to choose cohabitation rather than marriage ( p  =  .054 ) . 
 this result seems to confirm the notion of cohabitation as the poor man s marriage , although the poor are still more likely not to enter a union in the first place . 
 when we compare the two models for union entry ( model a and b ) , the employment effects that we observed in model a are reduced by more than half once income is added to the model . in other words , the effects of employment and work history run in part via income . 
 nevertheless , the employment effect remains statistically significant even when income is included , which means that the influence of employment on union formation also has a non - financial element . 
 it is also interesting to observe that the effects of employment and temporary jobs on the choice between the marriage and cohabitation are not affected by whether or not income is added ( compare model d and e ) . 
 hence , the non - financial aspects of work are more important for the type of union than for the chance of entering a union in the first place . in this sense , 
 oppenheimer s uncertainty theory seems to work better for the type of union than for union formation per se . 
 this confirms theories about selection into marriage and suggests that screening or selection may be less strong for cohabitation . 
 education , finally , has a positive effect on union entry but these effects are reduced when income is controlled for . in the last columns of table  2 , we focus on marriage formation after cohabitation ( model g , h , and i ) . 
 we see that it is not affected by employment , but being enrolled in school does reduce the chance of marrying . 
 we also find that men who worked more months in the past are more likely to marry . 
 the higher the men s income during cohabitation , the more likely they are to change from cohabitation to marriage . 
 when comparing the linear income effect on marriage after cohabitation with the linear effect on initial union formation , it appears that income is less important for marriage after cohabitation ( b  =  .10 ) than it is for union formation after being single ( b  =  .21 ) . 
 this is in line with the neo - malthusian hypothesis , which suggests that it is primarily for initial union entry that the costs of setting up a household play a role ( although the wedding is a cost factor which applies specifically to marriage ) . 
 men in good health are more likely to experience a transition from cohabitation to marriage , suggesting that the selection effects also play a role in the decision to marry , not only in the decision to live together . 
 we also see that higher educated cohabiting men are more likely to marry , hence , for the higher educated , cohabitation is less often seen as a long - term option . 
 do the effects we found vary across societal contexts ? to assess this , i present interaction effects of key independent variables with the macro - level indicator of traditional versus egalitarian societies . 
 the p - values are based on standard errors that are corrected for the clustering of cases within countries . 
 this yields a more conservative test for macro - level effects and is a good alternative to multilevel models when the number of units at the macro level is limited . to check for outliers at the country level , 
 i calculated dfbetas for the interaction effects of modernization and employment , and of modernization and income , and re - estimated the models while leaving out countries for which dfbetas exceeded the critical value ( kalmijn 2010 ; ruiter and de graaf 2006 ) . dfbeta is calculated as the difference in an effect with and without the outlier divided by the standard error of the effect . 
 these outlier analyses are presented in the third and fourth columns of table  3.table  3discrete time event history models of union formation with interactions : logit regression coefficients and p - values in parenthesesmodel a all casesmodel b all casesmodel a w / o outliersmodel b w / o outliersln ( age15)0.953 * * * ( 0.000)0.781 * * ( 0.002)0.943 * * * ( 0.001)0.686 * ( 0.036)ln ( 65age)1.970 * * * ( 0.000)1.956 * * * ( 0.000)1.934 * * * ( 0.001)2.040 * ( 0.010)general health0.121 * * ( 0.001)0.107 * * ( 0.002)0.102 * * ( 0.006)0.119 * ( 0.016)education secondary versus primary0.071 ( 0.287)0.023 ( 0.732)0.031 ( 0.638)0.115 ( 0.079)education tertiary versus primary0.271 * * * ( 0.000)0.152 * ( 0.014)0.248 * * * ( 0.000)0.224 * * * ( 0.001)working 15 + hours versus no work / school0.501 * * * ( 0.000)0.194 * * * ( 0.000)0.549 * * * ( 0.000)0.175 * * * ( 0.000)in school versus no versus no work / school0.672 * * * ( 0.000)0.604 * * * ( 0.000)0.614 * * * ( 0.000)0.673 * * * ( 0.000)income linear0.242 * * * ( 0.000)0.243 * * * ( 0.000)index ( traditional  egalitarian)1.916 ( 0.177)2.075 ( 0.144)2.092 ( 0.262)2.039 ( 0.327)index  *  ln ( age15)0.278 ( 0.058)0.314 * ( 0.037)0.311 ( 0.110)0.217 ( 0.272)index  *  ln ( 65age)0.233 ( 0.442)0.262 ( 0.386)0.288 ( 0.471)0.309 ( 0.485)index  *  health0.003 ( 0.914)0.004 ( 0.872)0.007 ( 0.718)0.005 ( 0.897)index  *  secondary education0.079 ( 0.333)0.087 ( 0.285)0.086 ( 0.368)0.121 * ( 0.041)index  *  tertiary education0.119 ( 0.106)0.130 ( 0.079)0.139 ( 0.127)0.205 * * * ( 0.000)index  *  working0.158 * * ( 0.001)0.122 * * ( 0.001)0.152 * * * ( 0.000)0.098 * ( 0.043)index  *  enrolled0.236 * * ( 0.001)0.210 * * ( 0.002)0.215 * * ( 0.004)0.288 * * * ( 0.000)index  *  income0.002 ( 0.935)0.037 * * ( 0.008)constant13.107 * * * ( 0.000)12.953 * * * ( 0.000)12.853***(0.000)13.112 * * * ( 0.000)n77621776216883758354note : controlled for country and year dummies . 
 outliers for model a are denmark and greece , for model b these are italy and the united kingdom *  p  <  .05 , * *  p  <  .01 , * * *  p  <  .001 discrete time event history models of union formation with interactions : logit regression coefficients and p - values in parentheses note : controlled for country and year dummies . 
 outliers for model a are denmark and greece , for model b these are italy and the united kingdom *  p  <  .05 , * * 
 p  <  .01 , * * *  p  <  .001 in table  3 , we see a negative interaction of the societal index with men s employment ( b  =  .16 , p  <  .01 ) . 
 hence , the average effect of employment ( b  =  .50 ) is reduced by 32% for each standard deviation increase in the egalitarian context ( .16/.50 ) . 
 this shows that the effect of men s employment on the entry into a union is considerably weaker in more egalitarian countries than in more traditional countries . 
 i also present the effects for each country separately in a graph ( fig . 
 , the effect of selected independent variables is plotted against the macro - level index . 
 although the graph does not provide a test , like the interaction effect , it does give a good intuitive feel of the importance of the interaction effect . 
 in line with expectations , the graph shows that in more traditional societies like spain , italy , and ireland , the effect of men s employment is quite strong . in more egalitarian countries like the netherlands , denmark , and finland , 
 men s enrollment in school deters union formation but this effect is weaker in more egalitarian societies . 
 s economic characteristics on union entry effects of men s economic characteristics on union entry table  3 further shows no negative interaction of the societal index with men s income effect on union formation . after deleting two outliers ( italy and the united kingdom ) , 
 the interaction becomes negative and significant ( b  =  .037 , p  =  .01 ) . 
 hence , for these 11 countries , the effect of men s income on union formation is weaker in more egalitarian countries than in more traditional countries . 
 the magnitude of the effect is a 15% reduction in the income effect per standard deviation increase in the egalitarian context ( .037/.234 ) . 
 when we look at the graph , the pattern appears to be weaker than it was for employment . 
 spain and portugal reveal strong effects of men s income and denmark and the netherlands have weak effects . however , there are also outliers like the united kingdom ( stronger effect than expected ) and italy and belgium ( weaker effect than expected ) . 
 so far , these results apply to union formation regardless of the type of union . traditional and egalitarian societies also differ in the extent to which cohabitation occurs . the more egalitarian countries like denmark , finland , and 
 the netherlands also have the highest levels of cohabitation ( soons and kalmijn 2009 ) . 
 because the effects of men s economic position differ depending on whether marriage or cohabitation is the outcome , the results could in part be due to such compositional differences . for this reason 
 , i also look at the entry into marriage only ( either after being single or after cohabitation ) . 
 the interaction with employment is .066 ( p  =  .11 ) , the interaction with enrollment is .326 ( p  < 
 .01 ) , and the interaction with income is .073 ( p  <  .01 ) . hence , the employment interaction effect is weaker in this model , although still negative , while the other interaction effects are not affected . 
 we therefore conclude that part of the reason why the employment effects depend on the context lies in the fact that unions are more often unmarried unions in more egalitarian societies . even apart from that 
 , however , there is evidence that men s economic status matters more for marriage in more traditional societies . 
 this article has re - examined the importance of oppenheimer s theory on marriage timing in the european context . by and large 
 unemployment , little work experience , low income , and temporary employment on the part of men deter union formation . by analyzing income and employment effects simultaneously in a panel perspective , it was possible to obtain more direct evidence for the two contrasting hypotheses suggested by oppenheimer , i.e. , the neo - malthusian male breadwinner hypothesis and the career uncertainty hypothesis ( sometimes also called the career instability or immaturity hypothesis ) . 
 many previous european analyses have not been able to take income into account and have therefore not been able to separate these two mechanisms empirically . 
 my analyses first show that income effects are strong and significant , which supports the male breadwinner hypothesis . 
 second , the income effects explain about half of the effects of employment and work experience , suggesting that employment effects by themselves are not sufficient evidence for the uncertainty hypothesis . 
 this suggests that employment effects on union formation are more than just a matter of financial resources , in line with the uncertainty hypothesis . by analyzing the choice between cohabitation and marriage , 
 marriage in europe appears to be more sensitive to men s economic position than cohabitation . 
 men who are not employed or who have temporary jobs are more likely to choose cohabitation rather than marriage . 
 this finding provides additional evidence for the uncertainty hypothesis since cohabitation is more like a trial marriage and hence , more compatible with uncertainty in other life domains ( i.e. , employment ) . 
 we do not , however , see a clear income effect on the choice between marriage and cohabitation . 
 hence , the choice between marriage and cohabitation has more to do with employment uncertainty than with income . 
 there is a small effect of the lowest income group , however . among men who enter a union , 
 hence , there is some evidence that cohabitation is the "" poor man s marriage . "" 
 following oppenheimer s last study , i also examined the transition from cohabitation to marriage . 
 we would expect economic uncertainty to also reduce the chances of a transition from cohabitation to marriage , but in general , the effects we find are weaker for this transition 
 . the weaker income effect could be due to the fact that the cost of setting up a household plays no role for this transition . 
 while this is in line with the neo - malthusian argument , the finding that the employment effects on the transition from cohabitation to marriage are also weaker is unexpected . 
 other authors have found this too ( e.g. , bracher and santow 1998 ; liefbroer 2005 ) . 
 if couples buy a house or have a child , they may decide to marry . 
 although such transitions may be partly governed by economic uncertainty , they may also be exogenous and hence reduce the effects of other determinants like employment . 
 another speculation has to do with the lack of information on the ( female ) partner . 
 the partner may have become economically more secure during the cohabitation stage , thereby promoting the transition to marriage , but we do not observe such changes . 
 european countries differ considerably in terms of their economic , social , and cultural characteristics , and so , it is important to also examine country variation in the degree to which the theory applies . 
 we hypothesized that men s economic position would be less influential in more developed countries where gender roles are more symmetrical . 
 the effects of men s employment and school enrollment on union formation are stronger in more traditional societies than in more egalitarian ones . 
 income effects are also weaker in egalitarian societies but the evidence for this pattern is weaker . 
 our interpretation is that in egalitarian settings , the costs of setting up a household are more often shared between men and women . 
 hence , men can afford to experiment with their career if they have a partner who has a ( more ) stable career . 
 similarly , women may attach less weight to the career options of men than they did in more traditional circumstances and for instance , pay more attention to other traits , such as men s willingness to participate in child rearing . 
 my finding is in contrast to a previous cohort comparison for the united states which suggested that men s economic effects on marriage timing did not change over time ( sweeney 2002 ) . 
 perhaps this difference is due to the limited time period that was examined in sweeney s trend study , a design which may have reduced the variance in contextual gender roles . at the same time 
 , however , it needs to be investigated whether the patterns that i found still hold when a larger number of countries is considered . 
 the income interaction effects , for example , are sensitive to outliers and therefore less convincing . more units at the macro - level will be needed to confirm this finding . in closing , it is important to re - emphasize on the role of women . 
 although oppenheimer was justifiably "" bringing men back "" into the debate at a time when there was too little attention on the changing economic fate of young ( american ) men , the growing egalitarian model that is now supported by many couples in europe and the united states , suggests that men and women should be examined simultaneously for a better understanding of trends and differentials in marriage and cohabitation . 
 traditionally , studies of women were largely done from a beckerian perspective in which it was argued that women s status would lead to a decline of marriage ( and fertility ) . 
 currently , we can speculate that a strong economic position and career stability on the part of women might foster marriage . 
 it could be that certainity on just one side  either the man or the woman 
 alternatively , it could be that both the man s and the woman s career need to have been settled before couples enter a more committed union . in this respect 
 , it is somewhat unfortunate that analyses of marriage ( timing or formation ) have often been one - sided , focusing either on men or on women . 
 this design is unavoidable given that the partner of a respondent is typically observed too late , i.e. , when the respondent is married or living with the partner . 
 empirically , this problem does not exist when we observe cohabiting couples chances of marrying . 
 some authors in the past have regarded the transition from cohabitation to marriage as a two - sided problem and have analyzed economic characteristics of both partners in one model ( lichter et al . 
 these studies have not been able to examine the more important entry into a first union , however . for this transition , 
 the problem can , in principle , be solved by examining dating couples in a more systematic fashion . 
 for example , prospective surveys could collect data on the school and work careers of both members of a dating couple and subsequently analyze whether or not ( and when ) they begin to live together , while using the dissolution of the dating relationship as a competing risk . in this way 
 , the analysis of marriage and cohabitation can become truly two - sided and the economic characteristics of men and women can be included in one model . 
 there are some innovative sociological studies of transitions from dating to cohabitation and marriage , but so far , they have not focused on the partners economic characteristics ( joyner and kao 2005 ) .","this article discusses oppenheimer s theory on marriage timing , reviews the way this theory was received in european demography and family sociology , and develops a new test of the theory using annual panel data from 13 european countries for the period 19942001 . 
 several indicators of men s economic status are used , including school enrollment , employment , type of labor contract , work experience , income , and education . 
 effects of these indicators are estimated for the transition to marriage and cohabitation , as well as for the transition from cohabitation to marriage . 
 country differences in these effects are examined as well . 
 the evidence provides strong support for the male breadwinner hypothesis on the one hand , and for oppenheimer s career uncertainty hypothesis on the other . 
 however , the relevance of these hypotheses also depends on the national context , and especially on the way gender roles are divided in a society .",pubmed
"osteoporosis is a condition of impaired bone strength which leads to increased risk of fracture . 
 the enhanced bone fragility reflects the integration of the amount of bone ( bone mass ) and bone quality . 
 bone quality depends on its macro- and microarchitecture and on the intrinsic properties of the materials that comprise it ( e.g. , matrix mineralization , microdamage accumulation , or collagen quality ) . 
 bone is continually adapting to changes in its mechanical and hormonal environment via the process of bone remodeling . 
 bone remodeling maintains bone structure and its mechanical competence by removing damaged bone and replacing it with new bone and thus restores bone 's material composition , micro- , and macroarchitecture . 
 this process depends on the normal production , work and lifespan of osteoclasts , osteoblasts , and osteocytes . 
 thus , diseases and drugs that have an impact on bone cells and bone remodeling will influence bone 's structure and its resistance to fracture .   multiple sclerosis ( ms ) 
 is a chronic progressive disease affecting the myelin sheath covering of nerve fibers in the brain and spinal cord , leading to functional impairments such as visual impairment , abnormal walking mechanics , poor balance , muscle weakness , fatigue , and progressive immobilization . 
 the disease affects mainly young adults ( 20 to 40 years ) and its incidence is more frequent in women ( approximately 2  :  1 ) . 
 impaired mobility or lack of weight - bearing physical activity reduced mechanical stress on bone , which causes a marked imbalance in bone remodeling with a disruption of osteocytes network . 
 management of ms requires long - term disease - modifying therapy , such as glucocorticoids ( gcs ) with a further negative effect on bone remodeling and bone strength . 
 secondary osteoporosis may develop and low - trauma fractures occurring in patients with ms more frequently than in healthy controls [ 915 ] . 
 fractures and their sequelae can have important personal as well as ( economic ) implications for society . therefore , the attention on the issue of bone health among patients with ms is warranted . 
 this paper examines the underlying pathogenic mechanisms of osteoporosis in patients with ms as well as its management . 
 understanding the causes associated with decreased bone strength in patients with ms will help in the optimal therapeutic intervention . 
 the analysis of a registry of 9029 patients with ms in the usa found that 27.2% responders reported low bone mass , and more than 15% of responders reported a history of fracture . 
 most studies in patients with ms evaluated bmd in comparison with the control group of healthy subjects and showed significantly lower bmd in patients with ms than in controls [ 1116 ] . 
 several of these studies were shown that vertebral bmd is affected to a lesser degree than femoral bmd [ 11 , 12 , 16 ] . 
 interestingly , one study in men with ms reported low bmd ( osteoporosis ) in 37.5% ( 15 out of 40 ) and 21% ( 8 out of 38 patients ) had vertebral , rib , or extremities fractures 
 . patients with progressive forms of ms showed a more severe loss of bmd than those with relapsing - remitting ms . 
 cosman group found fracture rates of 22% in patients with ms compared with 2% in controls . 
 it remains unexplained whether all patients with ms are more susceptible to osteoporosis and fractures ; for example , there is evidence that patients with a low expanded disability status scale ( edss ) score did not show any significant difference in bmd with comparison with healthy control subjects [ 17 , 18 ] . 
 therefore , further elucidation is needed to qualify which risk factors are most responsible for a bone loss in patients with ms . 
 bone remodeling is under way throughout life and maintains bone strength by removing damaged bone and replacing it with new bone and thus restores bone 's micro- and macroarchitecture . 
 this process depends on the normal production , work , and lifespan of osteoclasts , osteoblasts , and osteocytes . 
 chronic diseases , such as ms , may significantly disturb the process of bone modeling and remodeling with resulting bone loss , deterioration of bone 's quality and increased frequency of fractures [ 20 , 21 ] . 
 secondary osteoporosis and low - trauma fractures occur in patients with ms more frequently than in healthy controls [ 9 , 11 ] 
 . the underlying pathogenic mechanisms of the osteoporosis in patients with ms are probably based on the progressive immobilization , long - term gcs treatment , vitamin d deficiency , skeletal muscle atrophy and possibly on the presence of various cytokines involved in the pathogenesis of ms . 
 in addition , chronic use of other drugs , such as antidepressants may contribute to the development of osteoporosis and fractures . 
 the functional impairments also leads to an increased risk of falling that , combined with bone loss and impaired quality of bone mass , can increase the frequency of bone fracture in individuals with ms . 
 increased bone loss in immobilized subjects is well - recognized complication in patients after spinal cord injury with tetraplegia [ 23 , 24 ] , in bedridden patients , or in astronauts , whereas localized bone loss is well documented in patients with regional disuse , for example , after fracture itself . 
 immobilization causes an overall progressive bone loss at a similar rate to osteoporosis caused by estrogen deficiency , but at the same amount of induced bone loss , disuse led to more deteriorated bone structure and mechanical properties than estrogen deficiency . 
 the available studies showed that cortical thinning and substantial decline of trabecular bone density account for increased bone fragility [ 2729 ] . 
 the duration and degree of motor disability appears to be a major contributor to the pathogenesis of secondary osteoporosis in patients with ms . 
 the degree of disability measured by the kurtzke edss score significantly correlated with bmd in patients with ms . 
 specifically , site - specific effects of motor disability were documented in ms patients , and edss correlated mainly with bmd in the hip but not in the lumbar spine . in wheelchair - bound patients , an atrophy of hip muscles affects proximal femur , while bmd of lumbar spine is not decreased because of its adequate mechanical stimulation by the trunk and back muscles in the upright position . 
 also , hemiplegic patients showed a significant loss of bmd in both trabecular and cortical bone at the forearm and at the neck and great trochanter on the paretic hip . 
 higher total body bone mineral content was documented in ambulatory patients ( edss score  6.5 ) compared with nonambulatory patients ( edss score  7.0 ) [ 12 , 13 ] . also , higher prevalence of osteoporosis was found in nonambulatory patients . in male patients 
 , a positive correlation has been observed between bmd and both edss score ( correlation with femoral and also vertebral bmd ) and bmi ( correlation with femoral bmd only ) . 
 there was shown that also edss score and bmi two years prior to the study could be used as future indicators of low bmd . 
 a reduced mechanical stress on bone causes a marked imbalance in bone remodeling with a transient increase in bone resorption ( which occurs initially ) and a decrease in bone formation ( which is sustained for a longer duration ) [ 25 , 33 , 34 ] ( table 1 ) . 
 the mechanism causing this decrease in bone formation probably lies in the reduction of mechanical stress during immobilization which results in a marked disruption of osteocytes network due to increase of osteocyte apoptosis . 
 osteocytes represent 95% of all bone cells and form a mechanosensory system which is based on a three - dimensional network of tightly interconnected osteocytes entombed in mineralized bone matrix . 
 disruption of this system affects probably several aspects of bone homeostatic system , such as mechanosensitivity , mechanotransduction , and basic multicellular units responsible for bone remodeling . 
 while molecular mechanisms of disuse osteoporosis are not well understood , recent evidence found that mechanical unloading caused upregulation of sost gene in osteocytes and increased levels of sclerostin ( product of sost gene ) . 
 sclerostin is responsible for the inhibition of wnt / beta - catenin signaling in vivo and for the suppressed viability of osteoblasts and osteocytes . 
 interestingly , sclerostin - deficient mice ( sost / ) were resistant to mechanical unloading - induced bone loss . 
 importantly , the administration of sclerostin neutralizing antibody in experimental model of immobilization resulted in a dramatic increase in bone formation and a decrease in bone resorption that led to increased trabecular and cortical bone mass . 
 osteocytes are also necessary for targeted bone remodeling to avoid microdamage accumulation , which could lead to whole - bone failure . 
 recently , waldorff et al . showed that osteocyte apoptosis may be insufficient for repair of microdamage without the stimulation provided through physiologic loading . 
 ms affects a wide range of neurological function and most of patients with ms have abnormal muscle strength , impaired balance , and gait control which leads to frequent falls [ 5 , 41 ] that combined with a bone loss increase the frequency of bone fractures . 
 it was demonstrated that changes in postural control in most patients with ms are probably the result of slowed afferent proprioceptive conduction in the spinal cord . 
 disuse , inflammatory changes , as well as gcs treatment or vitamin d deficiency , may also contribute to weakness and loss of muscle strength and thus to frequent falling . 
 gcs are frequently used to control ms relapses . oral gcs treatment in patients with ms may increase the risk of osteoporosis . 
 epidemiological studies showed that fracture risk is increased rapidly after starting oral gcs treatment and is related to the dose and duration of gcs exposure . 
 doses as low as 2.55  mg of prednisolone equivalents per day can be associated with a 2.5-fold increase in vertebral fractures , and the risk is greater with higher doses used for prolonged periods . 
 bone loss due to gcs treatment is steep during the first 12 months and more gradual but continuous in subsequent years . 
 however , the fracture risk returns towards baseline levels after discontinuation of oral gcs treatment . 
 the mechanism of osteoporosis in patients on gc treatment is complex   ( table 2 ) . 
 however , the contribution of other risk factors , such as vitamin d insufficiency and physical disability confounds the assessment of gcs effects on bone in patients with ms . 
 repeated pulses of high - dose methyprednisolone in ms patients did not result in a subsequent decrease in bmd ; however , the risk of osteoporotic fractures remains slightly increased in patients undergoing cyclic gcs treatment at high doses . 
 high - dose , short - term intravenous gc regimens cause an immediate and persistent decrease in bone formation and a rapid and transient increase of bone resorption . in fact , gcs may increase proresorptive il-6 signaling as well as increase the expression of receptor activator of nf-b ligand ( rankl ) and decrease the expression of its soluble decoy receptor , osteoprotegerin ( opg ) , in stromal and osteoblastic cells . 
 however , discontinuation of such regimens is followed by a high bone turnover phase . in physically active patients with ms treated with low - dose steroids , 
 the bone turnover markers were not different from controls . addressing the question of whether duration of low - dose gcs use in combination with other immunomodulators in patients with ms increases risk of osteoporosis requires further prospective study by taking into account other risk factors , particularly the level of disability . 
 although no harm effect of low - dose methotrexate was observed in patients with ms , several case reports have described associations between pathological nonvertebral fractures and low - dose methotrexate ( mtx ) in rheumatoid arthritis ( ra ) patients . 
 in addition , methotrexate osteopathy , characterized by pain , osteoporosis , and microfractures , has been very rarely observed in patients with low - dose mtx treatment . 
 other immune - modifying drugs , such as interferon - beta or azathioprine , which are used in conjunction with gcs have not been shown to promote bone loss experimentally or clinically . 
 on the contrary , interferon - beta may have favorable effect on bone metabolism in patients with ms , probably due to the inhibitory effect of interferon - beta on osteoclasts development . 
 experimentally , also treatment with the s1p(1 ) agonist fty720 , a new and promising drug for the treatment of ms , relieved ovariectomy - induced osteoporosis in mice by reducing the number of mature osteoclasts attached to the bone surface . 
 however , further investigation with regard to their effects on bone health is needed . 
 the role of vitamin d in bone homeostasis is well understood , and the use of vitamin d to prevent and treat osteoporosis was recently reviewed . 
 there is also evidence from both observational studies and clinical trials that hypovitaminosis d are predisposing conditions for various common chronic diseases . 
 in addition to skeletal disorders , vitamin d deficiency is associated with increase the risk of malignancies , particularly of colon , breast , and prostate gland cancer , of chronic inflammatory and autoimmune diseases ( e.g. , insulin - dependent diabetes mellitus , inflammatory bowel disease , or multiple sclerosis ) , as well as of metabolic disorders ( metabolic syndrome and hypertension ) . 
 vitamin d intake , decreasing latitude , increased sun exposure , and high serum vitamin d levels have all been shown to be associated with a decreased risk of ms . 
 patients with ms have more often vitamin d deficiency due to its low intake as well as limited sunlight exposure . 
 mean 25-hydroxyvitamin d3 ( 25ohd ) levels in patients with ms are more often lower ( below the level of 20  ng / ml ) than in age - matched controls [ 11 , 14 ] . 
 there was no significant correlation between 25(oh)d and bmd in patients with ms [ 11 , 14 ] . 
 thus , while patients with ms are susceptible to low 25ohd levels , the evidence implicating linking levels to reduced bmd and osteoporosis in patients with ms is unclear . 
 only a few studies have investigated this link [ 11 , 12 , 14 ] . 
 a low vitamin d state , from inadequate diet intake and decreased exposure to sunlight , contributes to malabsorption of calcium and vitamin d insufficiency in ms patients . 
 secondary hyperparathyroidism may develop , which can contribute to bone remodeling imbalance and bone loss in patients with ms . 
 moreover , patients with ms treated with gcs will be at greater risk for an imbalance between bone formation and bone resorption and , therefore , more susceptible to development of osteoporosis due to vitamin d insufficiency / deficiency . 
 gcs treatment is associated with reduced calcium absorption from the gastrointestinal tract by opposing vitamin d action . 
 in addition , gcs may affect pth secretory dynamic , with a decrease in the tonic release of pth and an increase in pulsatile burst of the hormone . 
 ms is an inflammatory disease of the central nervous system ( cns ) with a prominent role of immune cells and cytokines in degradation of the myelin sheaths . 
 recent evidence has indicated that a number of additional cell types , such as t cells , play a key role in bone loss . in inflammatory or autoimmune disease states , activated t - cells produce receptor activator of nuclear factor kappab ligand ( rankl ) and proinflammatory cytokines , such as tnf- , il-1 , or il-11 , all of which can induce rankl expression in osteoblasts and bone marrow stromal cells . 
 the systemic or local activation of t - cells may , therefore , trigger bone loss via the expression of rankl . 
 osteoprotegerin ( opg ) , a protein member of the tumor necrosis factor ( tnf ) receptor family and its ligand rankl were identified as a key cytokines that regulate osteoclastogenesis . significantly , 
 higher levels of rankl and opg were found in the patients with ms with low mean edss as compared to the age - matched controls . among other cytokines , 
 osteopontin ( opn ) has been studied in the shared pathogenesis of ms and osteoporosis . 
 opn is a member of the sibling ( small integrin - binding ligand n - binding glycoprotein ) family of noncollagenous matricellular proteins . 
 opn was identified as the most abundantly expressed cytokine in ms lesions , and opn levels were found to be increased in cerebrospinal fluid of ms patients [ 64 , 65 ] and in the plasma in patients with relapsing - remitting ms . however , other studies found that opn circulating levels are low in patients with ms . 
 it seems likely that further future studies experiments will uncover the role of opn and additional molecules mediating bone loss in inflammatory diseases , such as ms . 
 meta - analyses have revealed that barbiturate , antidepressant , antipsychotic , and benzodiazepine treatment increases patient 's risk of osteoporosis . 
 more recently , current use of antidepressant drugs with a high affinity for the 5-hydroxytryptamine reuptake transporter ( 5-htt ) was associated with a higher risk of osteoporotic fractures compared to use of antidepressants with a medium or low affinity . 
 bmd was lower among those reporting current selective serotonin reuptake inhibitors ( ssri ) use but not among users of other antidepressants [ 72 , 73 ] . 
 in vivo studies have found that 5-ht could alter bone architecture and could reduce bone mass and density . 
 the 5-htt has been located in osteoclasts , osteoblasts , and osteocytes , and the the inhibition of 5-htt using a ssri ( fluoxetine hydrochloride ) had antianabolic skeletal effects in rats . 
 further research is needed to confirm this finding in light of widespread ssri use and potentially important clinical implications . 
 despite the fact that patients with ms can develop osteoporosis and fractures more often than their age - matched healthy controls , many patients with ms are not evaluated for their bone status , and there are no clinical guidelines for prevention and treatment of osteoporosis in patients with ms . 
 patients with ms are also at a higher risk of falls that can increase the frequency of bone fracture combined with bone loss and impaired bone 's quality . 
 clinical evaluation in all patients with ms should include the assessment of the clinical risk factors for osteoporosis and fractures , such as the hereditary disposition of osteoporosis , previous low trauma fractures , and smoking or alcohol habits . 
 the specific risk factors of the osteoporosis in patients with ms are the level of disability ( specifically motor disability ) and possibly a long - term gcs treatment , vitamin d deficiency , skeletal muscle atrophy , and increased risk of falling . 
 the examination of the motor function using the edss score could provide a useful indicator for further evaluation . 
 cutoff edss 6 represents reasonable end of motor performance of the patient ; 6.5 means only several meters with bilateral support , and 7 is only the ability of transfer to wheelchair from the bed . 
 the edss scores of 6 or greater has been found to correlate well with decreased bmd [ 12 , 15 ] , and bmd should be routinely measured in these patients . 
 on the other hand , patients with a good physical activity and low edss score ( < 5 ) may have normal bmd   as well as markers of bone turnover . 
 bmd measurement should be also performed in all patients who are receiving 5  mg of prednisone equivalents daily for more than 3 months . 
 bmd testing using dual - energy x - ray absorptiometry ( dxa ) should be conducted at the lumbar spine and hip . 
 this measure provides an assessment of fracture risk prior to the occurrence of a fragility fracture as well as monitors the course of the disease and response to therapy . 
 no consensus exists as to how frequently patients at risk osteoporosis should have followup scans . 
 however , bmd should be remeasured after 1 or 2 years to ascertain that it is stable or to identify the patient with ongoing bone loss , especially in patients treated with long - term gcs treatment . in the presence of clinical risk factors 
 therefore , combination of bmd with clinical risk factors is recommended to identify a risk patient and to target pharmacologic therapy . in postmenopausal women and men ( between 40 and 90 ) , the assessment of individualized 10-year absolute fracture risk ( frax , fracture prediction algorithm ) 
 the identification of previous low - trauma fractures , especially vertebral fractures is important for the decision - making process as a previous vertebral fracture is a particularly strong risk factor . 
 importantly , vertebral fractures may occur in 3050% of patients receiving chronic gcs therapy   and up to 50% of vertebral fractures are asymptomatic and , therefore , do not come to the attention of physicians . 
 spinal x - rays should be performed in those with localized back pain or a loss of more than 3  cm in height in order to detect prevalent vertebral fractures . 
 alternatively , the vertebral fracture assessment tool of the bone densitometer , which is associated with low radiation , may be useful screening test for vertebral fractures assessment . 
 laboratory tests are indicated to exclude other secondary causes of osteoporosis , such as vitamin d deficiency , renal insufficiency , malabsorption , and hypogonadism . 
 useful biochemical tests include routine standard tests to exclude renal or hepatic impairment , blood count , serum calcium , 24-hour urinary calcium , 25-hydroxyvitamin d3 ( to exclude vitamin d deficiency ) , and gonadal hormones ( to exclude hypogonadism ) . 
 nonpharmacological therapies should be considered for prevention of skeletal fragility , including adequate weight - bearing exercise , nutrition ( protein , calcium , vitamin d ) , and lifestyle modifications . as reviewed above 
 , disability is the most often cause of bone loss in patients with ms , and mechanical loading and exercise interventions can prevent osteocyte apoptosis and bone loss [ 77 , 78 ] . 
 exercises have beneficial effects on strength , physical endurance , mobility - related activities ( transfer , balance , and walking ) , and on mood , without any evidence of detrimental effects ; however , there was no evidence that any particular exercise programs were more effective in improving or maintaining function . 
 whole - body vibration is a new approach to improve neuromuscular functions and bone strength , but there is limited evidence that whole body vibration provides any additional improvements . 
 further experimental studies are necessary to identify optimal physical activities for the prevention of osteocyte apoptosis and bone loss . 
 recurrent falls may be an important risk factor for fracture in disabled patients with ms . in patients with ms , falls are related to the level of disability , and possibly other factors may contribute to muscle weakness and imbalance , such as vitamin d deficiency or gcs treatment . 
 calcium and vitamin d supplementation has been routinely provided in most clinical trials of bone protective therapy for both primary and secondary osteoporosis , for example , in glucocorticoid - induced osteoporosis ( gio ) . 
 the effect of calcium and vitamin d supplementation is maximized in patients whom baseline intake is low . as patients with ms are at a higher risk of calcium and vitamin d deficiency should have their calcium and vitamin d status checked and intake must be individualized . those with a personal or family history of nephrolithiasis 
 an increase in serum calcium is provoked by bed rest alone and additional calcium intake would not be helpful and might be harmful and provoke an increased risk of kidney stone formation . 
 however , calcium and vitamin d should be used as an adjunct treatment , because a low calcium intake may exacerbate calcium loss during low mechanical loading . in general , the amount of vitamin d supplementation should aim at achieving serum 25ohd levels above 50  nmol / 
 a daily dose of 8001000  iu of vitamin d3 should be able to obtain this minimal 25ohd target . 
 due to vitamin d resistance in patients receiving gcs , those patients may require amounts of 10002000  iu of vitamin d3 daily . 
 measurement of serum 25oh vitamin d is recommended , especially in gcs - treated patients . 
 although some evidence suggests that daily supplemental intake of 20004000  iu colecalciferol is required to obtain at least 75  nmol / l 25ohd , which may be optimum for many health outcomes , prospective trials showing that higher 25ohd levels ( > 7580 
 although a number of drugs have been evaluated for the prevention and treatment of postmenopausal osteoporosis and gio , the evidence of their efficacy in patients with ms , especially in premenopausal women and younger men is less strong . as osteoporosis in ms patients 
 have multiple pathogenesis , medical interventions used in women with postmenopausal osteoporosis may not be similarly efficient . 
 patients requiring long - term gcs treatment and those being immobilized may require pharmacological therapy to prevent excessive bone loss and fractures . 
 options for treatment include antiresorptive drugs , such as estrogen , or aminobisphosphonates , or anabolic agents such as teriparatide . 
 although the use of bps may be appropriate , the etiology of osteoporosis in patients with ms is fundamentally different from the osteoporosis commonly found in the postmenopausal women for whom these drugs were originally developed . 
 as immobilization in patients with ms can cause substantial bone loss and increase in the risk of fractures , bps may be option for treatment for those patients . 
 although bps have not been systematically evaluated in the therapy of these conditions , some studies support the potential benefit of bps in the management of bone loss associated with immobilization [ 24 , 85 , 86 ] . in immobilized patients , bps is known to reduce immobilization - induced hypercalcaemia by inhibiting bone resorption of calcium . 
 an immobilization - related elevated serum calcium level may inhibit parathyroid hormone ( pth ) secretion , and hence renal 1 , 25(oh)2d3 production , in disabled long - standing ms patients . 
 if oral therapy of bps can not be tolerated or excluded due to gastroesophageal disease , intravenous route of administration of ibandronate or zolendronate may be applied . however 
 , acute phase reaction with fever , particularly after the first application of bp , may occur . 
 bps ( alendronate , risendronate , or zolendronate ) were also approved for the treatment of gio . 
 these drugs were shown to improve bmd , whereas the data on fractures were scanty in gio , particularly in premenopausal or younger men . 
 the mechanism by which bps reduce the adverse skeletal effects of gcs have not been elucidated . 
 the disadvantage of long - term bps treatment is that it may lead to a reduction in bone turnover to a level inadequate to support normal bone remodeling . 
 although experimental data showed that bps also prevents osteocyte apoptosis , there is also experimental evidence of increased accumulation of microdamage with long - term bps therapy . 
 also , as bps accumulate in the skeleton ( with a long - term residual time ) , they cross the placenta , accumulate in fetal skeleton , and cause toxic effects in pregnant rats . 
 therefore , bps should be used with caution in women who may become pregnant . 
 drugs , such as bps , that suppress bone resorption have been proposed as interventions for prevention of gio as well as disuse osteoporosis . 
 the disadvantage of this approach is that it may lead to a reduction in bone turnover to a level inadequate to support normal bone remodeling . 
 an alternative approach is to maintain a normal level of bone formation using a bone anabolic agent such as pth . 
 the human recombinant n - terminal parathyroid hormone ( pth 134 or teriparatide ) is a potent osteoanabolic agent , which decreases osteoblast and osteocyte apoptosis and increases bone formation and bone strength . because of gcs - induced decrease in the number of osteoblasts and rate of bone remodeling , anabolic , and antiapoptotic treatment with teriparatide may directly counteracts the key pathogenetic mechanisms of gcs excess on bone , thus , it may be a more effective treatment than bps . 
 the same rationale applies to immobilization - induced osteoporosis , as progressive immobilization as well as long - term gcs exposure results in osteocyte apoptosis and reduced bone formation . 
 as sclerostin augments osteocyte apoptosis , the antibody - mediated blockade of sclerostin represents a promising new therapeutic approach for the anabolic treatment of immobilization - induced osteoporosis and probably also for gcs - induced osteoporosis . 
 indeed , more recently , experimental data showed that administration of sclerostin neutralizing antibody in rat model of right hindlimb immobilization resulted in a dramatic increase in bone formation and a decrease in bone resorption that led to increased trabecular and cortical bone mass . 
 we have described a spectrum of pathogenetic factors which may contribute to the development of osteoporosis and low - trauma fractures in patients with ms . whilst there is evidence to support an important role for many of the risk factors 
 , the most significant etiology of bone loss in patients with ms seems to be the level of motor disability and reduced bone load within individual patients . 
 other risk factors , such as long - term gcs treatment , hypovitaminosis d , or inflammation , may also play an important part in subset of patients with ms ; however , further examinations in prospective studies are required . with regard to diagnostic as well as therapeutic interventions 
 , there are currently no specific recommendations in patients with ms ; however , identification and treatment of underlying cause should be the goal of therapeutic management . 
 optimally , the patients in a higher risk of osteoporosis should be early identified and preventively promptly treated to avoid the bone loss and fractures . because the long - term disability and long - term gcs are probably two most significant etiologic risk factors for osteoporosis development in the majority of the patients with ms , the interventions which can counteract the osteocyte apoptosis as well as loss of muscle mass and muscle weakness will be promising .","multiple sclerosis ( ms ) is a gait disorder characterized by acute episodes of neurological defects leading to progressive disability . 
 patients with ms have multiple risk factors for osteoporotic fractures , such as progressive immobilization , long - term glucocorticoids ( gcs ) treatment or vitamin d deficiency . the duration of motor disability appears to be a major contributor to the reduction of bone strength . 
 the long term immobilization causes a marked imbalance between bone formation and resorption with depressed bone formation and a marked disruption of mechanosensory network of tightly connected osteocytes due to increase of osteocyte apoptosis . 
 patients with higher level of disability have also higher risk of falls that combined with a bone loss increases the frequency of bone fractures . 
 there are currently no recommendations how to best prevent and treat osteoporosis in patients with ms 
 . however , devastating effect of immobilization on the skeleton in patients with ms underscores the importance of adequate mechanical stimuli for maintaining the bone structure and its mechanical competence . the physical as well as pharmacological interventions which can counteract the bone remodeling imbalance , particularly osteocyte apoptosis , 
 will be promising for prevention and treatment of osteoporosis in patients with ms .",pubmed
"malaria is one of the main scourges of the world , affecting half a million people a year . 
 it is caused by parasites belonging to the phylum apicomplexa , family plasmodiidae , and genus plasmodium . 
 there are five known species that parasitize man , the most prevalent being plasmodium falciparum , p. vivax , and p. malariae in tropical areas such as latin america , the indian subcontinent , southeast asia , and parts of africa . 
 p. ovale has been reported in africa , middle east , and the indian subcontinent , while p. knowlesi has been recently described as responsible for large foci of infections in southeast asia . 
 the natural transmission occurs through the bite of infected female anopheles mosquito , anopheles darlingi being the most prevalent species in the country . 
 the ministry of health directs therapy and provides free antimalarial medicines used throughout the national territory , in units of the unified health system ( sus ) , through a national policy on malaria treatment . 
 malaria caused by p. vivax ( or p. ovale ) is treated with chloroquine in brazil at a dose of 30  mg / kg divided into three days and primaquine in the 30  mg dose / day for 7 days or 15  mg / day for 14 days . 
 also , low doses of chloroquine can be used for the prevention of vivax malaria . 
 pregnant women and children under 6 months of age with vivax malaria receive only chloroquine in different dosage schedules . 
 primaquine is the only antimalarial drug with effective activity against all the species gametocytes of plasmodium which cause malaria in humans , as well as against hypnozoites , latent forms of p. vivax and p. ovale responsible for relapses . 
 however , it has hemolytic effects , especially in people with glucose-6-phosphate dehydrogenase deficiency ( g6pd ) , in which primaquine as a stressor to the erythrocyte induces hemolysis . 
 hemolysis induced by primaquine can result in discoloration of urine and faeces and it is dose related [ 8 , 9 ] . 
 gastrointestinal disorders , such as nausea , dizziness , and vomiting , are common but usually not present as severe complications , especially when primaquine is administered with food . 
 with respect to chloroquine , there are relatively few adverse effects with use in usual doses , of which the most serious are retinopathy , cardiomyopathy , myopathy , and neuromyopathy . prolonged treatment or use of high level doses 
 may cause retinal toxicity , long subtle symptoms of decreased visual acuity , diplopia , and bilateral loss of vision . 
 adverse reactions of the gastrointestinal tract are the most common side effects , usually controlled by reducing drug dose . 
 there are also reports of ototoxicity , tingling , itching , and change in skin color , in addition to seizures , insomnia , and paresthesia [ 12 , 13 ] . 
 the occurrence of symptoms specifically related to the intake of antimalarials and the overlap of symptoms of malaria with the side effects of primaquine and chloroquine may decrease adherence to treatment . 
 therefore , it becomes important to assess the main side effects of antimalarial drugs in patients from endemic areas , where treatment is aimed not only at improving the clinical status but also at reducing the transmission of plasmodium . 
 this study evaluated the frequency of the symptoms that commonly occur in vivax malaria before and after treatment and the frequency of side effects reported during the term of antimalarial treatment with chloroquine and primaquine in standardized doses according to the protocol of the brazilian ministry of health . 
 the study was conducted in the urban area of the municipality of mncio lima , located in the western part of the brazilian amazon , in the state of acre . 
 this municipality , with 5000  km , is bordered by the cities of cruzeiro do sul and rodrigues alves and the republic of peru . 
 mncio lima has 14,884 inhabitants living in urban areas ( 57.3% ) , rural or riparian areas ( 37.9% ) , and indian villages ( 4.8% ) . 
 the county is located 38  km from cruzeiro do sul and 650  km from rio branco . 
 patients with microscopic diagnosis of vivax malaria were identified in microscopy stations of the city of mncio lima before the medication and invited to participate in the study . 
 after signing the informed consent or having it signed by parents or legal guardians , patients answered a questionnaire on malaria symptoms that occurred up to the time of diagnosis ( before antimalarial treatment ) , to verify the occurrence of the following symptoms : nausea , vomiting , diarrhea , pale stools , loss of appetite , mesogastric and/or hypogastric pain , pain in hypochondrium , headache , myalgia , arthralgia , back pain , retroocular pain , fever , chills , sweating , malaise or weakness , itchy throat , jaundice , choluria , dyspnea , and bitter taste sensation , covering up thereby symptoms commonly occurring in malaria and overlapping with possible side effects of chloroquine and primaquine . between 3 and 5 days after the start of medication , a second interview 
 was conducted asking about the occurrence of the same symptoms plus some symptoms related to the use of chloroquine that are infrequent in malaria : blurred vision , itching , stinging sensation of the skin , convulsions , numbness , and insomnia . 
 the distributions of relative and absolute frequencies , median , mean , and standard deviations of the variables of interest were calculated . as primaquine and chloroquine 
 may lead to the onset of symptoms and also intensify or reduce some of the symptoms of malaria , the occurrence of symptoms before and after drug treatment was analyzed with the mcnemar 's test for paired samples . 
 some of the symptoms considered to be side effects of chloroquine and primaquine and that are not usual in malaria were assessed only after drug treatment was started . 
 the research protocol was submitted to the ethics committee for experimentation with human beings of universidade federal do acre and approved , cae number 22876013.9.0000.5010 ( 2013 ) . 
 informed consent was obtained from each adult participant or legal guardian in case of minors , before the start of the study . 
 the study included 50 people with vivax malaria , 28 men and 22 women , aged between 7 and 68 years ( average age 28.38 years ) . 
 the average length of stay in an endemic area was 24.79 years , and about one - third of the study population ( 30% ) lived in an endemic area for malaria between 11 and 20 years . 
 of the 50 patients evaluated , only one had a primary infection , and all the others had had between 1 and 41 episodes of previous malaria , and 36% report having had more than 10 episodes of malaria . 
 about 52% had an episode in the 12 months leading up to the current episode , ranging from one to 10 , the number of malaria events in 2012 . 
 most of the patients had low parasitemia ( less than 200 parasites / mm in 66% of cases ) , 12% of them had between 301 and 500 parasites / mm , and 12% were found to have 50110000 parasites / mm ( table 1 ) . 
 the main symptoms observed in untreated malaria were fever ( 72% ) , chills ( 54% ) , sweating ( 54% ) , and weakness ( 42% ) , as well as pain symptoms including headache ( 76% ) , arthralgia ( 48% ) , and lower back pain ( 52% ) . 
 less commonly reported symptoms were retroocular pain ( 36% ) and myalgia ( 30% ) . 
 gastrointestinal symptoms occurred in low frequency , such as bitter taste ( 30% ) , decreased appetite ( 32% ) , nausea ( 22% ) , vomiting ( 16% ) , and pain in hypogastric or mesogastric region ( 20% ) . before treatment , only 20% of patients reported dark urine , and no patient reported having had pale stools . 
 after initiation of treatment , a significant reduction in the prevalence of fever ( 38% ) , arthralgia ( 26% ) , back pain ( 32% ) , and retroocular pain ( 18% ) ( p < 0.05 , table 2 ) was noted . 
 however , there was significant increase in the frequency of nausea ( 46% ) , diarrhea ( 26% ) , pale stools ( 12% ) , abdominal pain ( 38% ) , bitter taste in the mouth ( 60% ) , weakness ( 36% ) , and hypochondrial pain ( 32% ) ( p < 0.05 , table 2 ) . the frequency of choluria increased to 56% after initiation of antimalarial treatment ( p = 0.01 , table 2 ) . 
 other symptoms such as vomiting , loss of appetite , headache , myalgia , chills , and sweating , as well as respiratory symptoms and upper respiratory tract symptoms , suffered no major change in frequency ( table 2 ) . among the adverse effects of chloroquine described in the literature and which are not often reported in malaria patients without treatment , there were reported changes in visual acuity ( 54% ) , insomnia ( 46% ) , pruritus ( 22% ) , the feeling of  stings  into the skin ( 22% ) , and paresthesias ( 6% ) . 
 most gastrointestinal symptoms appeared after the patient started the antimalarial treatment ( table 3 ) . 
 the symptoms most commonly seen in patients with malaria reported in the literature are fever , chills , sweating , headache , myalgia , and arthralgia . 
 da silva - nunes and ferreira   evaluated 326 cases of untreated malaria , reporting high frequency of headache ( 84.8% ) , fever ( 80.9% ) , and myalgia ( 68.7% ) , while gastrointestinal symptoms were infrequent ( < 20% ) . in pregnant women with vivax or falciparum malaria , the most frequent symptoms were also fever ( 97.9% ) , chills ( 71.7% ) , and headache ( 62.4% ) . 
 the treatment of vivax malaria with chloroquine , a blood schizonticide , and primaquine ( active against latent tissue forms of p. vivax or so - called hypnozoites ) is very effective in reducing parasitemia and some of the symptoms caused by the plasmodium infection , such as fever and algic symptoms . 
 fever tends to disappear between 24 and 48 hours after the beginning of treatment ; parasitemia decreases significantly at about 48 to 72 hours , and pain symptoms decrease rapidly . 
 reported the disappearance of fever in 91.2% of patients , chills in 86% of patients , and headaches in 65.6% of patients at the end of the third day of treatment with chloroquine and primaquine and significant reduction of peripheral parasitemia between the second and third days of treatment . 
 however , chloroquine can cause side effects or intensify symptoms already present , such as abdominal discomfort , nausea , vomiting , and diarrhea . 
 these side effects can also occur during the use of primaquine   being suitable to ingest the medication with food to avoid such side effects . 
 chloroquine can rarely cause neurological symptoms , such as mental confusion , seizures , and coma , and cardiovascular symptoms such as hypotension , vasodilation , suppression of myocardial function , and cardiac arrhythmias , usually associated with rapid infusion of the drug parenterally . in usual doses for treatment of malaria , headache , blurred vision , and lichenoid rash can occur . 
 chloroquine may block the entrance of potassium into the cells and primaquine also has an effect on blocking sodium channels . both may also have an effect on the chloride channels present in cardiac myocytes , which may explain the occurrence of cardiac and gastrointestinal side effects . in studies conducted in belm ( par ) , silva et al 
 .   found , as major side effects of chloroquine , the following symptoms : itching ( 5.55% ) , epigastric pain ( 1.28% ) , diarrhea ( 0.85% ) , and choluria ( 3.42% ) . 
 the itching and stinging sensation are explained by formation of haptens during the metabolism of chloroquine , which bind to degraded products erythrocytes or phospholipids involved in allergic reactions , stimulating the production of ige - like antibodies and degranulation of mast cells and basophils . 
 also , the binding of chloroquine at the dermoepidermal junction may possibly result in stimulation of nerve fibers , causing these symptoms . 
 regarding primaquine use , the most prevalent side effect is hemolytic anemia , resulting in jaundice and choluria . 
 data on primaquine use worldwide indicates that , among 200 million people , there were 14 deaths in six decades related to its use , from which 12 were caused by severe hemolysis . 
 hemolytic anemia can also occur in people without g6pd deficiency when using primaquine , although it is a very scarce event . 
 the gene encoding the g6pd is located on the x chromosome ( locus xq28 ) , with a recessive pattern of inheritance linked to sex . 
 in heterozygous females normal red blood cells can coexist with red blood cells deficient in g6pd , in variable proportion , and this can cause a milder hemolytic anemia when exposed to primaquine . at the same time 
 , there are 180 different genetic variants of g6pd , resulting in different levels of hemolysis . 
 the mediterranean variant , which predominates in europe , western and central asia , and north of india , is more severe , while the african variant ( common in sub - saharan africa , amazon , and afro - americans ) is a milder variant . in these milder variants 
 , hemolysis will be clinically detected only after one or two days of exposure to primaquine . 
 the prevalence of g6pd deficiency is between 3 and 30% in endemic countries for malaria , corresponding to 350 million people . in the americas 
 , it has a lower prevalence , varying between 5 and 10% in the amazon region and higher levels in tropical africa , middle east , and the mediterranean basin , such as sudan and congo , where it is present in more than 20% of the population . in brazil 
 , the frequency of g6pd deficiency varies between 1.7% and 6.0% , prevailing the african variant . in manaus , 
 a city with high levels of transmission of malaria in the brazilian amazon , the prevalence of g6pd was 2.5% . 
 this difference in prevalence may explain why , in brazil , primaquine is administered to all nonpregnant patients older than 6 months with an unknown g6pd deficiency status , while in other countries with higher prevalence of this enzymatic deficiency ( or in which more severe variants are common ) primaquine use is very much debated and controversial . 
 these events are dependent on the extent of hemolysis , which is related to the severity of g6pd deficiency and the dose of primaquine . recommended primaquine doses used to block falciparum malaria transmission ( 0.25  mg of base per kilogram ) are usually considered to have low toxicity , because it is a single - dose treatment . 
 however , primaquine use in vivax malaria , aimed at preventing relapsing events , is a longer course ( between 7 and 14 days ) , with varying dosage among malaria control policies , and therefore hemolytic events are more prone to happen . in a systematic revision , monteiro et al .   detected 47 cases of hemolytic anemia in patients using primaquine in latin america and caribbean , and 23 occurred in brazil . 
 however , brazil has the largest population at risk for malaria ; thus it is expected to have more cases reported . 
 a few cases of primaquine - induced hemolysis have also been reported in other countries , such as cuba , el salvador , puerto rico , and trinidad and tobago , between 1963 and 2013 . in our study , the frequency of choluria (  dark urine  ) increased significantly from 6% to 56% in malaria patients after primaquine was started . since it is an event referred by the patient , we can not be sure how many of the patients really presented with choluria , or how severe was the choluria , but it is known that g6pd deficiency occurs among amazonian residents . 
 it is possible that the hemolytic anemia is the cause of weakness and malaise , reported by these patients . 
 however , none of the patients studied suffered from severe hemolysis or severe anemia ( data not shown ) or required hospitalization during or after malaria treatment . 
 although severe events related to the use of primaquine were infrequent in our and other studies in the amazon , it is important to bear in mind the possible adverse side effects of primaquine and also mild anemia occurring in patients with the less severe forms of g6pd deficiency , which can be associated or not with weakness . 
 reported that subjects with no g6pd deficiency showed reduction of 1 to 2  g / dl of hemoglobin after primaquine use , while in g6pd deficient patients hemoglobin levels decreased between 3 and 5  g / dl , with fast normalization after treatment was over . 
 reported abdominal pain as a side effect for primaquine in falciparum malaria patients from myanmar , occurring in 16% of patients receiving a single dose . 
 there is no clear evidence why primaquine can cause abdominal pain ; it can be related to the direct effect of the drug on an empty stomach   or related to the release of pharmacological substances during hemolysis . despite its cause , 
 it is noteworthy that , in our study , abdominal pain increased significantly from 6% to 32% after primaquine was started . 
 although the present study has a limitation in the number of subjects studied , it was possible to detect significant association of symptoms and malaria treatment . thus , while the treatment of vivax malaria with a combination of chloroquine and primaquine leads to decrease in fever and pain symptoms , it can lead to the onset of gastrointestinal symptoms and hemolysis or enhances these symptoms when already present in the patient with malaria , contributing to reducing treatment adherence , especially during the use of primaquine , which has a longer course of treatment . 
 moreover , the sensation of itching and stinging , although they do not occur in all patients , can be quite intense , hindering treatment with chloroquine . 
 it is important to warn the patient of these possible side effects and seek measures to minimize them , either with concomitant use of antihistamines or antiemetics , according to the individual needs of each patient . 
 the occurrence of severe hemolysis after primaquine use , although not common in brazil , can still occur , and since brazilian control programs currently do not have a policy for g6pd deficiency screening before malaria treatment is offered , health care personnel must be aware of it to interrupt primaquine use and manage its side effects properly .","side effects of antimalarial drug can overlap with malaria symptoms . 
 we evaluated 50 patients with vivax malaria in mncio lima , acre , treated with chloroquine and primaquine . 
 patients were evaluated for the presence of 21 symptoms before and after treatment and for reported side effects of these drugs after treatment was started . 
 the most frequent symptoms before medication were headache , fever , chills , sweating , arthralgia , back pain , and weakness , which were present in between 40% and 76% of respondents . 
 the treatment reduced the occurrence of these symptoms and reduced the lack of appetite , but gastrointestinal symptoms and choluria increased in frequency . 
 there were no reports of pale stools before medication , but 12% reported the occurrence of this symptom after treatment started . 
 other symptoms such as blurred vision ( 54% ) , pruritus ( 22% ) , paresthesia ( 6% ) , insomnia ( 46% ) , and  stings  into the skin ( 22% ) were reported after chloroquine was taken . 
 the antimalarial drugs used to treat p. vivax malaria reduce much of the systemic and algic symptoms but cause mainly gastrointestinal side effects that may lead to lack of adherence to drug treatment . 
 it is important to guide the patient for the appearance and the transience of such side effects in order to avoid abandoning treatment .",pubmed
"from a worldwide perspective , the life span of humans has been prolonged , and the proportion of elderly people in the total population has increased . 
 various changes occur in the human body with age . in elderly men , lower urinary tract symptoms ( luts ) , erectile dysfunction ( ed ) , and decreased libido often appear concurrently . 
 several community - based studies have shown a strong correlation among sexual dysfunction , increasing age , and the severity of luts . 
 this coexistence of sexual problems with luts and benign prostatic hyperplasia ( bph ) further affects quality of life ( qol ) . 
 luts and ed share increased prevalence as age increases , and in many cases , the two are synchronously present . 
 thus , continuous efforts have been made to disclose the common pathophysiology between these two conditions . 
 no definite explanations have been given to clarify the pathophysiological mechanism of the correlation between luts and ed , but several hypotheses have been proposed [ 2 - 5 ] . 
 one possible cause is overactivation of the autonomic nervous system and increased tension of the sympathetic nervous system . 
 according to this hypothesis , luts increases sympathetic nervous system activity , which induces the occurrence of urinary storage symptoms due to the contraction of smooth muscles in the prostate gland and urinary bladder . 
 ed occurs as the result of smooth muscle contractions in the corpus spongiosum , and the myosin light chain is phosphorylated because of increased rho - kinase activity , which can cause smooth muscle layer contraction . 
 it has been proposed that endothelial dysfunction in the prostate gland and corpus spongiosum and hormonal imbalance are physiological causes for the correlation between luts and erectile dysfunction . 
 changes in lifestyle due to nocturia and rapid eye movement sleep disorder may affect the occurrence of erectile dysfunction . 
 also , the result of our preliminary study showed that age , international prostate symptom score ( ipss ) , nocturia , and the uroflow rate correlated significantly with the 5-item version of the international index of erectile function ( iief-5 ) . even in cases in 
 which age was controlled for , qol was found to have a significant correlation with sexual function . 
 prostate gland volume was not significantly correlated with sexual function , implying that erectile function is not affected by subjective luts even with a relatively greater prostate size . 
 irwin et al reported that overactive bladder ( oab ) was significantly associated with increased prevalence of ed and that sexual activity , sexual enjoyment , and sexual satisfaction were reduced as the result of urinary symptoms . in recent years , many studies have reported that administration of alpha - blockers improves luts due to bph and sexual dysfunction . 
 these findings suggest that rudimentary treatments of luts , a risk factor for erectile dysfunction , would be mandatory to effectively improve sexual function in patients with bph who concurrently have luts and ed . 
 the primary treatment regimen for bph is to administer alpha - blockers , for which the main mode of action is based on smooth muscle relaxation in the prostate and the resulting improvement in the urinary flow rate and voiding symptoms . 
 however , a synchronous improvement of irritative and voiding symptoms may be the most effective treatment regimen for luts / bph . 
 it is expected that the concomitant administration of alpha - blockers and anticholinergics would improve luts more effectively in patients with luts / bph . 
 these effects are also expected to have a positive effect in secondarily improving sexual function . 
 thus , the aim of this study was to examine the effects on erectile function of a combination of the alpha - blocker tamsulosin to improve luts and the antimuscarinic agent solifenacin to improve irritative symptoms of the bladder . 
 male patients aged 40 years or older who had concurrent luts / bph and ed were enrolled in this study . 
 inclusion criteria were an ipss total score > 12 , an ipss - qol score > 3 , and an iief-5 score < 20 . 
 exclusion criteria were as follows : 1 ) antiandrogens administered in the 4 weeks previously for the management of bph or luts ; 2 ) sex hormone agents or pde-5 inhibitors administered in the 4 weeks previously for the management of male sexual dysfunction ; 3 ) surgical treatment of the prostate gland or urethra ; 4 ) diagnosis of urethral stricture , urinary tract infection , prostatitis , prostate cancer , or bladder cancer ; 5 ) psa > 4 mg / dl ; 6 ) severe renal dysfunction or hepatic dysfunction ; 7 ) residual urine > 100 ml ; and 8) ineligibility for the current study as judged by the investigators . 
 sixty male patients between the ages of 41 and 76 years with luts / bph and ed were divided into two groups by using a table of random sampling numbers : group 1 ( the alpha - blocker treatment group ; n=30 ) and group 2 ( the alpha - blocker+solifenacin treatment group ; n=30 ) . in group 1 , an alpha - blocker ( tamsulosin 0.2 mg q.d . ) 
 was solely administered for 3 months . in group 2 , an alpha - blocker and an antimuscarinic drug ( tamsulosin 0.2 mg and 
 solifenacin 5 mg q.d ) were concomitantly administered during the same period . in patients who were suspected of having a significant bladder outlet obstruction ( postvoiding residual urine > 100 ml or maximum flow rate < 5 ml / s ) , solifenacin was not administered . to examine the correlation between luts and the degree of erectile function , ipss and iief-5 
 then , prostate - specific antigen ( psa ) , transrectal ultrasonography ( trus ) , urine flowmetry ( ufm ) , and residual urine ( ru ) were measured . 
 ipss was categorized into total scores , storage symptoms ( st ) , voiding symptoms ( vd ) , and quality of life ( qol ) . in group 1 , in which tamsulosin was solely administered ( n=30 ) for 3 months , and group 2 , in which tamsulosin and solifenacin were concomitantly administered ( n=30 ) for 3 months , 
 comparative analyses were also done to examine the linear relationship between ipss scores and iief-5 scores before and after treatment in the two groups . the t - test ( t ) and mann - whitney u - test were used to compare parameters indicating the degree of luts and sexual function between the two groups , such as trus , psa , ipss ( total , vd , and st ) , ipss - qol , iief-5 total , amount of urination , amount of ru , maximal urinary flow rate ( qmax ) , and mean urinary flow rate ( qave ) . by use of the paired t - test ( t ) and wilcoxon signed rank test ( z ) 
 , a comparative analysis was performed on each group for ipss ( vd , st , total ) , ipss - qol , iief-5 total , the amount of urination , the amount of ru , qmax , and qave before and after treatment . 
 regression analysis was used to inspect the linear relationship between ipss scores and iief-5 scores before and after treatment in the two groups . 
 the t - test ( t ) and mann - whitney u - test were used to compare parameters indicating the degree of luts and sexual function between the two groups , such as trus , psa , ipss ( total , vd , and st ) , ipss - qol , iief-5 total , amount of urination , amount of ru , maximal urinary flow rate ( qmax ) , and mean urinary flow rate ( qave ) . by use of the paired t - test ( t ) and 
 wilcoxon signed rank test ( z ) , a comparative analysis was performed on each group for ipss ( vd , st , total ) , ipss - qol , iief-5 total , the amount of urination , the amount of ru , qmax , and qave before and after treatment . 
 regression analysis was used to inspect the linear relationship between ipss scores and iief-5 scores before and after treatment in the two groups . 
 of a total of 60 study patients , 1 patient in group 1 and 3 patients in group 2 dropped out of the 3-month follow - up study . 
 therefore , analyses were performed on 29 patients in group 1 and 27 patients in group 2 . 
 the incidences of hypertension and diabetes were 11 and 3 in group 1 and 9 and 5 in group 2 , respectively . before treatment , no significant differences ( p<0.05 ) were found between groups 1 and 2 , respectively , in age ( 59.176.73 yr vs. 56.410.80 yr ) , prostate gland size ( 27.18.0 g vs. 28.438.18 g ) , psa levels ( 1.090.97 mg / dl vs. 1.180.80 mg / dl ) , amount of urination ( 245.86160.97 ml vs. 223.36125.76 ml ) , amount of ru ( 2434.50 ml vs. 32.239.79 ml ) , qmax ( 14.417.16 ml / s vs. 14.806.1 ml / s ) , or qave ( 7.333.62 ml / s vs. 8.063.53 ml / s ) . 
 no significant differences were found in iief-5 ( 12.456.85 vs. 12.047.65 ) or ipss - qol ( 4.280.88 vs. 3.820.88 ) scores . 
 however , the ipss total score was significantly higher ( p<0.05 ) in group 1 than in group 2 ( 20.287.39 vs. 15.225.55 ) . 
 this was due to the exclusion of patients in group 2 who were suspected of having significant bladder outlet obstruction ( postvoiding residual urine > 100 ml or maximum flow rate < 5 ml / s ) . in both groups 1 and 2 , the ipss total , 
 ipss - vd , ipss - st , and ipss - qol scores were significantly improved after the 3-month treatment ( p<0.05 ) . however , no significant differences in the iief-5 total score , amount of urination , amount of ru , qmax , or qave were found before or after treatment ( table 1 ) . a comparison of the change (  ) in luts and sexual function after treatment between group 1 and group 2 revealed no significant differences in ipss total ( 5.726.90 vs. 4.155.72 ) , ipss - vd ( 3.694.77 vs. 2.044.71 ) , ipss - st ( 2.243.50 vs. 2.112.33 ) , ipss - qol ( 0.831.47 vs. 0.821.04 ) , or iief-5 
 a comparison of the degree of improved sexual function ( iief-5 ) associated with improved luts in each patient ( ipss total , ipss - vd , ipss - st , and ipss - qol ) showed significant improvement in the iief-5 score associated with the degree of improvement in the ipss - st domain ( ipss - st ) in group 1 , but no significant associations were found in group 2 ( table 3 , fig . 
 1 ) . in cases in which tamsulosin was administered , the iief-5 score significantly improved as the ipss - st domain score improved . in the group in which tamsulosin and solifenacin were concomitantly administered , 
 improvement of the ipss - st domain score had no significant effect on the iief-5 score . 
 luts / bph is the most common disease of lower urinary tract obstruction that occurs in men aged 60 years or older . in most cases , it develops slowly , but in some patients , it progresses rapidly . 
 the incidence of both bph and ed is increased in elderly people , and the synchronous occurrence of luts and ed is considered to happen as the result of aging . even in cases 
 in which age and underlying disease such as cardiovascular disease , diabetes mellitus , and hypertension were statistically controlled , epidemiologic studies have shown a definite correlation between bph and ed . 
 these two disease entities are not due to aging , but they share a common pathophysiology . 
 thus , a synergistic effect whereby the treatment of one disease would lead to improvement in the other disease is expected . 
 conversely , khler and mcvary reported that the improved sexual function resulting from treatment with pde-5 inhibitors led to improved luts . 
 a questionnaire study of 12,815 men aged 50 years or older found a significant correlation between bph and ed . 
 furthermore , age and luts were found to be greater risk factors for ed than were organic factors such as diabetes mellitus , hypertension , and hyperlipidemia . in our series , age had the highest degree of correlation with sexual function . voiding symptoms of the ipss , storage symptoms , qol , 
 nocturia symptoms , residual urine , maximal urinary flow rate , and mean urinary flow rate were significantly correlated with the degree of sexual satisfaction as well as with erectile function . 
 luts accompanied by bph can be divided mainly into voiding ( obstructive ) symptoms and storage ( irritative ) symptoms . 
 however , little is known about which of the various symptoms are more closely associated with ed . it remains unclear which luts might effectively improve the concurrent presence of ed , and a substantial number of studies in recent years have aimed to resolve this issue . 
 this study was also performed to investigate which symptoms of bph , obstructive or irritative symptoms , are closely related to erectile function . 
 morant et al reported that both irritative and voiding symptoms are significantly correlated with sexual function . 
 tsai et al reported that irritative symptoms are more highly correlated with ed than are obstructive symptoms . 
 a preliminary study that we conducted in patients with bph found a significant correlation between sexual function and various irritative and voiding symptoms , qol , nocturia , residual urine , and urine flow rates . 
 following a 3-month treatment of luts with alpha - blockers , the improvement in voiding symptoms , qol , and average flow rate was significantly correlated with improved sexual function . to date , however , few studies have reported similar results , and controversy remains . 
 storage symptoms due to oab may cause a higher degree of pain than do voiding symptoms . in cases of bph accompanied by oab , 
 for this reason , the use of anticholinergic agents ( or antimuscarinic agents ) has been attempted in patients with oab accompanied by bph . 
 several previous studies have confirmed the efficacy and safety of concomitant administration of alpha - blockers and anticholinergic agents . 
 even so , in some cases , residual urine is increased or voiding symptoms are aggravated following the use of anticholinergic agents , and in patients with luts / bph , anticholinergic agents are used selectively . in our series , we excluded patients who likely had a significant bladder outlet obstruction ( pvr > 100 ml or qmax < 5 ml / s ) and who had luts / bph ; we therefore found significantly improved luts with no complications such as acute urinary retention . 
 common side effects following the use of anticholinergic ( or antimuscarinic ) agents are dry mouth , constipation , blurred vision , headache , and dry eye , but almost no reports have described the effects on sexual function . 
 some concern exists that the use of anticholinergic ( or antimuscarinic ) agents in patients with bph may lead to aggravation of voiding difficulty or the development of urinary retention . to date , urologists have not actively used anticholinergic ( or antimuscarinic ) agents , but many types of antimuscarinic agents have been used in recent years to treat oab in a clinical setting . 
 as related experiences have accumulated , these drugs have been selectively used for patients with luts / bph . 
 almost no reports have addressed the effects of antimuscarinic agents on sexual function in luts / bph patients . in a study of 39 men with bph and luts 
 in whom previous alpha - blocker therapy had failed , kaplan et al found normal sexual function in 27 ( 63% ) and 29 ( 67% ) patients before and after administration , respectively . 
 no reports have indicated that the administration of anticholinergic ( or antimuscarinic ) agents significantly impairs sexual function . in our series , a comparison of iief-5 scores before and after concomitant drug treatment with solifenacin and tamsulosin revealed no statistically significant changes . 
 thus , it can be inferred that administration of anticholinergic ( or antimuscarinic ) agents had no significant effect on sexual function . in patients with luts 
 / bph , concomitant treatment with alpha - blockers and antimuscarinics may lead to improvement in luts , but this treatment can not be expected to improve sexual function . 
 in patients with luts / bph and ed , concomitant use of tamsulosin and solifenacin was effective for improving luts . 
 however , sexual function was not significantly improved by the concomitant administration of alpha - blockers and antimuscarinics .","purposeto examine the effects on erectile function of concomitant treatment with an alpha - blocker ( tamsulosin ) and an antimuscarinic agent ( solifenacin ) in patients with lower urinary tract symptoms ( luts)/benign prostatic hyperplasia ( bph).materials and methodsfifty - seven male patients with luts / bph were assessed for the degree of luts and erectile function . in group 1 ( tamsulosin ) and group 2 ( tamsulosin and solifenacin ) , changes in the international prostate symptom score [ ipss : total scores , storage symptoms ( st ) , voiding symptoms ( vd ) , and quality of life ( qol ) ] , prostate - specific antigen , trans - rectal ultrasonography , urine flowmetry , residual urine , and a 5-item version of the international index of erectile function ( iief-5 ) were assessed after a 3-month treatment period . in both groups , it was determined whether treatment was associated with changes in luts and erectile function and whether improvement in the ipss was correlated with the iief-5 . 
 comparative analysis was also done to examine the linear relationship between improved ipss scores and iief-5 scores.resultsa comparison of the degree of improvement in all the parameters indicated that both groups showed significant improvement in total ipss , ipss - st , ipss - vd , and ipss - qol ( p<0.05 ) . 
 a comparison of the degree of improved sexual function associated with improved luts in each patient showed significant improvement in the iief-5 score associated with the degree of improvement in the ipss - st domain in group 1 , but no significant associations were found in group 2 . in cases 
 in which tamsulosin was administered , the iief-5 score significantly improved as the ipss - st domain score improved . in the group in which tamsulosin and solifenacin were concomitantly administered , 
 improvement of the ipss - st domain score had no significant effect on the iief-5 
 score.conclusionsin patients with luts / bph , tamsulosin and solifenacin combination therapy was effective for luts , but erectile function was not significantly improved . 
 therefore , although effective for improving luts , combination therapy with an alpha - blocker and an antimuscarinic agent was not effective for improving erectile function .",pubmed
"specific mirnas have been found to be expressed in cell type - specific manner , at specific developmental stages , and in disease states including cancer [ 1 , 2 ] . during the initiation and progression of cancer , mirnas 
 have been observed to act as oncogenes or tumor suppressors [ 3 , 4 ] . 
 while some mirnas are overexpressed in cancers , the majority appear to be lost and often localize to fragile sites . 
 differences in the mirna expression profiles of normal compared to cancerous tissue of the endometrium , breast and ovary have been documented [ 611 ] . 
 mirnas can affect the expression of a large number of proteins , including those involved in pathways relevant to cancer , such as apoptosis , migration and metastatis . 
 thus , mirnas hold promise as biomarkers for several types of cancer [ 12 , 13 ] . 
 epithelial to mesenchymal transition ( emt ) is a normal process that occurs during development in which individual cells or groups of cells become motile . 
 the same process is thought to be used by cancer cells during tumor progression to enable them to become more motile and thus more metastatic . 
 emt involves reprogramming of the cells by transcription factors such as zeb1 , sip1 ( zeb2 ) , twist , snail , and slug . 
 a hallmark of emt is loss of e - cadherin expression , loss of polarity , acquisition of mesenchymal markers , and increased motility [ 16 , 17 ] . 
 both zeb1 and the closely related zeb2 bind e - box like sequences in the e - cadherin promoter , recruit the corepressor ctbp and thereby repress e - cadherin . 
 zeb1 expression is confined to cells of mesenchymal origin , while normal epithelial cells and low grade carcinomas do not express zeb1 . 
 however , we and others have shown that in high grade , aggressive carcinomas that have undergone emt , zeb1 can be expressed , leading to loss of e - cadherin [ 1922 ] . 
 several mirnas have been implicated in the process of emt , among them are the members mir-200 family [ 2325 ] . 
 this family contains five members ( mir-200a , -200b , -200c , -141 and , -429 ) which are highly homologous . 
 originally , mir-200c was reported to directly bind zeb1 and cause degradation of the mrna , resulting in an upregulation of e - cadherin . 
 subsequently , other reports have shown that all members of the mir-200 family , since they share a high degree of homology especially in their seed sequence , are capable of repressing both zeb1 and zeb2 [ 2729 ] . 
 we have demonstrated that mir-200c represses not only zeb1/2 , but a program of transcripts normally expressed only in cells of mesenchymal origin . since members of the mir-200 family are responsible for repressing zeb1 and zeb2 as well as other mesenchymal genes , these mirnas are considered 
 mir-200 family members are therefore thought to be expressed in an epithelial cell - specific manner in normal tissues . 
 recently , the ability of zeb1 to transcriptionally repress expression of mir-200 family members has been documented [ 31 , 32 ] . 
 this double negative feedback loop between mir-200 family members and zeb1 allows for plasticity between the epithelial and mesenchymal states . in this paper , we focus on the role of mir-200c in breast , ovarian , and endometrial cancers . 
 the mutual repression between zeb1 and mir-200c is functional in some , but not all cells that we have tested . 
 we demonstrate that the decrease in migration and invasion observed when mir-200c is reintroduced to cancer cells that lack it is independent of restoration of e - cadherin . 
 lastly , we have previously demonstrated that class iii beta tubulin ( tubb3 ) is directly controlled by mir-200c . 
 expression of tubb3 is known to be a common mechanism of resistance to microtubule - targeting agents in many types of cancer . here , 
 we present conclusive data that repression of tubb3 is the mechanism whereby mir-200c restores sensitivity to paclitaxel . taken together , these data demonstrate that loss of mir-200c is a marker for chemoresistance and aggressiveness in breast , ovarian , and endometrial cancers . 
 hec50 cells , representing the more aggressive type ii endometrial cancers , were cultured in dmem with 10% fbs , and 2  mm l - glutamine . 
 mda - mb-231 are a triple negative breast cancer cell line and were grown in media containing 5% fbs , hepes , nonessential amino acids , l - glutamine , penicillin , streptomycin , and insulin . 
 the identity of all the cell lines was confirmed by dna profiling using the identifiler kit ( applied biosystems ) . 
 lipofectamine 2000 ( invitrogen ) was combined with pre-200c ( mirna mimic ) or scrambled negative control ( ambion ) at a concentration of 60  nm and incubated in serum free rpmi for 20 minutes prior to addition to hey cells . 
 cells were incubated at 37c for 4 hours before replacement of fbs to 10% . protein and rna were harvested 48 hours posttransfection . 
 tubb3 ( from fernando cabral , university of texas - houston medical school ) was cloned into pci - neo . 
 transient transfection of 3.3  g of tubb3 plasmid or empty vector ( pci - neo , promega ) per well in a 6-well plate was performed using lipofectamine 2000 . 
 transduction of cells was performed using smartvectortm shrna lentivral particles ( thermo scientific dharmacon ) . 
 each cell line was transduced with 3 separate lentiviral constructs targeting zeb1 as well as two controls : smartvector empty vector particles and smartvector firefly luciferase control particles . 
 the former is a negative control and does not correlate with gene silencing and the latter is a positive control targeting firefly luciferase plasmids pgl2 and pgl3 . 
 all vectors are packaged and contain a turbogfp and an scmv promoter , as well as a puromycin - resistance selectable marker . mda231 and 
 hec50 cells were plated at 3000  cells / well and 1500  cells / well , respectively , in triplicate using 96 well plates . 
 the following day , media was replaced with 80  l of fresh media containing 10  g / ml polybrene ( sigma ) .   the amount of viral particles / well was determined using the following calculation : ( moi      cn)/vt , where moi ( multiplicity of infection )   =   10  tu / cell , cn   =   number of cells / well , and vt   =   stock viral titer of 10  tu/l . 
 viral particles were added in a total volume of 20  l to each well .   the following day , 
 transduction media was removed and wells were rinsed with pbs and replaced with regular media . 
 once confluent , cells were trypsinized and replated in 48 well plates .   at this point 
 , antibiotic selection was initiated and cells were ultimately expanded and maintained using 1  g / ml of puromycin ( sigma ) . 
 whole cell protein extracts were denatured and 50  g separated on 8% sds page gels and transferred to pvdf membranes . 
 the membranes were blocked in 5% milk in tbs - t , and then probed overnight at 4c . 
 doug darling , university of kentucky , 1 : 1500 dilution ) , e - cadherin ( clone nch-38 from dako , 1  g / ml ) , tubb3 ( rabbit polyclonal prb-435p from covance , 1 : 5000 dilution ) , and -tubulin ( clone b-5 - 1 - 2 from sigma , 1 : 20000 dilution ) . 
 after incubation with appropriate hrp - conjugated secondary antibody , bands were detected using western lightning chemiluminescence reagent plus ( perkin elmer ) . 
 rna was harvested from cells using trizol ( invitrogen ) as per the manufacturer 's instructions . prior to generating cdna 
 , mrna was treated with dnase1 ( invitrogen ) for 15 minutes at room temperature . 
 rna was reverse transcribed into cdna in a reaction containing reaction buffer , dntps , rnase inhibitor ( applied biosystems ) , random hexamers , and 200  u of mulv - rt ( applied biosystems ) . 
 the reaction proceeded at 22c for 10 minutes , then at 37c for one hour . for normalization 
 , real time rt - pcr was performed on the cdna using eukaryotic 18s rrna endogenous control primers and fam - mgb probe ( applied biosystems ) . 
 taqman microrna reverse transcription kit was used to generate cdna for real time rt - pcr reaction in conjunction with a mir-200c specific primer and probe ( abi , assay i d 002300 ) . the reverse transcription primer for mir-200c is a hairpin primer which is specific for the mature mirna and will not bind to the precursor molecules . reported values are the means and standard errors of 3 biological replicates . 
 the relative mrna or mirna levels were calculated using the comparative ct method ( ct ) . 
 briefly , the cycle threshold ( ct ) values for the rrna were subtracted from ct values of the target gene to achieve the ct value . 
 the 2 was calculated for each sample and then each of the values was then divided by a control sample to achieve the relative mirna levels ( ct ) . 
 the assays were performed on mda - mb-231 stable empty vector or shzeb1 # 2 , or hey cells transiently transfected with the mir-200c mimic for 48 hours . 
 bd biocoat control insert chambers 24-well plate with 8 micron pore size and bd biocoat matrigel invasion chambers were used for migration and invasion assays , respectively . 
 after starvation , cells were removed from the plate and 50000 hey cells or 250000 mda - mb-231 cells were plated in 0.5  ml media with 0.5% fbs in the upper chamber . in the lower chamber 0.8  ml of 50% conditioned media plus 50% complete media containing an additional 10% fbs 
 hey cells were incubated for 24 hours and mda - mb-231 cells for 48 hours at 37c . migrating or invading cells on the lower surface of the membranes were stained with diff - quik stain ( fisher ) and counted manually using imagepro plus software ( mediacybernetics inc . ) . 
 adhesion assays were performed using innocyte ecm cell adhesion assays ( calbiochem ) for collagen iv , fibronectin , basement membrane complex and laminin . to each well 50000 cells 
 the plates were incubated for 1 hour at 37c , and fluorescence was read with an excitation wavelength of 485  nm and an emission wavelength of 528  nm . 
 the relative fluorescent units were plotted , and the error bars represent standard error of the mean over four replicates . 
 hey cells were plated into 6-well plates at a density of 2000 cells per well . 
 twenty - four hours after plating , the cells were treated with 0 , 1 , 2 , 3 , 4 , or 5  nm paclitaxel ( sigma ) in triplicate . 
 the cells were incubated at 37c for 8 days before fixing and staining with crystal violet . 
 photos were taken of the plates and the images analyzed using imagej software ( nih ) . the average number of colonies and the average total area was plotted , with error bars representing the standard error of the mean over the three replicates . 
 hec50 cells were transfected with the mir-200c mimic as described previously for the hey cells . 
 twenty - four hours after transfection , cells were treated with 0 , 15 , 20 or 25  nm paclitaxel ( sigma ) . 
 twenty - four hours after treatment , the cell death elisa ( roche ) which recognizes mono- and oligonucleosomes in the cytoplasm of dying cells was performed as per manufacturer 's instructions . 
 cells were plated into 6-well plates ( 4000 hey cells / well and 6000 mda - mb-231 or hec50 cells / well ) . at time 
 points indicated , cells were trypsinized and counted using the vi - cell cell viability counter ( beckman coulter ) . 
 just as members of the mir-200 family can repress zeb1 by degradation of its transcript , zeb1 can repress expression of the mir-200 family members by binding to e - boxes within their promoter regions [ 31 , 32 ] ; see figure 1(a ) . directly increasing mir-200c levels in hey cells ( aggressive serous ovarian cell line ) by transfection of a mir-200c mimic ( pre-200c ) results in repression of zeb1 expression ( figure 1(b ) ) . 
 although zeb1 is a repressor of e - cadherin , we did not observe e - cadherin expression induced by the repression of zeb1 in these cells ( data not shown ) . 
 however there are several other mechanisms through which e - cadherin can be lost including methylation of the promoter [ 34 , 35 ] and chromosomal deletion [ 36 , 37 ] . 
 in contrast , we have previously shown that transient transfection of the mir-200c mimic into mda - mb-231 ( an aggressive triple negative breast cancer cell line ) and hec50 ( an aggressive type 1 endometrial cancer cell line ) causes a marked repression of zeb1 and a restoration of e - cadherin expression . presently , we stably transduced lentiviral shrnas targeting zeb1 into these two cell lines ( hec50 and mda - mb-231 ) . 
 while two of the shrnas did not decrease zeb1 protein , shrna # 2 caused an almost complete repression of zeb1 expression resulting in re - expression of e - cadherin in both cell lines ( figures 2(c ) and 2(d ) ) . 
 intriguingly , while knock down of zeb1 in mda - mb-231 cells causes the expected increase in mir-200c levels ( indicative of the reciprocal regulation ) , no such increase is observed in hec50 cells . 
 this suggests that while reciprocal repression of mir-200c and zeb1 occurs in some cell lines , it does not occur in all . 
 it has been previously shown that the mir-200 family members cause a decrease in cell migration and invasion [ 27 , 29 , 30 ] . 
 we observe a decrease in migration and invasion in the mda - mb-231 cells in which zeb1 has been knocked down , resulting in an increase in mir-200c levels . in the mda - mb-231 cells there is 52% decrease in migration and a 50% decrease in invasion in the shzeb1 # 2 containing cells in which zeb1 is completely knocked down versus luciferase control ( figures 2(a ) and 2(b ) ) . 
 we show here that the same holds true in the aggressive ovarian cancer hey cell line . 
 this cell line is highly migratory and invasive , and reintroduction of mir-200c to these cells results in an 83% decrease in migration and a 7986% decrease in invasion compared to negative controls ( figures 2(c ) and 2(d ) ) . 
 however , it is interesting to note that the effect on migration and invasion caused by mir-200c is independent of the e - cadherin status of the cells , since unlike the mda - mb-231 cells , hey cells do not regain e - cadherin expression in response to decreased zeb1 levels . 
 while e - cadherin protein affects epithelial cell - cell contact , we also wished to determine if mir-200c affects adhesion to substrates as measured by fluorescent adhesion assays . 
 hey cells transiently transfected with the mir-200c mimic showed a small but statistically significant decrease in adhesion to basement membrane complex ( bmc ) and laminin ( figures 3(a ) and 3(b ) ) . 
 there is also a trend towards decreased adhesion to collagen iv ( figure 3(c ) ) ; however , this did not reach statistical significance . 
 no difference in adhesion to fibronectin was observed ( figure 3(d ) ) . since there was an affect on adhesion to bmc and laminin in ovarian cancer cells with high mir-200c levels , we performed the adhesion assay with the hec50 and mda - mb-231 cells in which zeb1 had been stably knocked down . 
 we again see a decrease in adhesion to bmc and laminin in the mda - mb-231 cells ; however , only the decrease in bmc binding is statistically significant ( figures 3(e ) and 3(f ) ) . 
 in contrast to the hey and mda - mb-231 cells , there was no decrease in adhesion to either substrate in the hec50 cells in which zeb1 is knocked down ( data not shown ) , but there is not a concomitant increase in mir-200c , as shown in figure 1(d ) . 
 we have previously demonstrated that mir-200c expression causes increased chemosensitivity to microtubule targeting agents such as paclitaxel . while the elisa cell death assay that we have used previously to demonstrate this property of mir-200c is a short - term assay , we confirm here , in a relatively long - term clonogenic assay , that there is increased sensitivity of hey cells to paclitaxel when transfected with pre-200c ( figure 4(a ) ) . 
 we observe a 4955% decrease in total area and a 6770% decrease in the number of colonies in the pre-200c treated cells versus the negative control with 5  nm paclitaxel treatment ( figures 4(b ) and 4(c ) ) . as the assay 
 is conducted over a relatively long period of time , the maximum dose of paclitaxel used is relatively small compared to what is used in the assays that look at acute toxicity ( i.e. , 24 hours ) . at doses of paclitaxel of 10  nm and higher , 
 we have previously implicated the ability of mir-200c to directly target tubb3   ( class iii beta tubulin ) as being the mechanism responsible for the increased chemosensitivity to microtubule targeting agents . 
 tubb3 is normally only expressed in neuronal cells ; however aberrant expression of tubb3 in several different types of cancers has been shown to cause resistance to paclitaxel [ 3843 ] . 
 to definitively test whether tubb3 is responsible for the mir-200c - mediated increase in chemosensitivity to paclitaxel , we transfected cells with a tubb3 construct lacking its 3 utr ( containing the mir-200c binding site ) which is therefore not able to be targeted by mir-200c . 
 transfection of this exogenous tubb3 construct does not affect the transfection of the mir-200c mimic , nor its ability to downregulate zeb1 and upregulate e - cadherin ( figures 5(a ) and 5(b ) ) in hec50 cells . 
 when the hec50 cells are transfected with an empty vector ( no exogenous tubb3 ) in addition to mir-200c mimic , there is a statistically significant increase in sensitivity to paclitaxel as measured in a cell death elisa ; see figure 5(c ) . 
 however , when the cells are transfected with the tubb3 expression vector lacking its 3 utr , the enhanced sensitivity to paclitaxel is lost ; see figure 5(d ) . 
 therefore , expression of exogenous tubb3 lacking the mir-200c target site reverses the chemosensitivity to paclitaxel caused by increased mir-200c expression . 
 it can be argued that cells with increased proliferation would be more sensitive to microtubule poisons and that could be an alternative explanation for the observed chemosensitivity upon restoration of mir-200c . 
 we therefore performed proliferation assays in the three cell types and found decreased proliferation in all three ( figures 6(a ) , 6(b ) , and 6(c ) ) . since the decrease in proliferation 
 is observed in all three cell types , including the hec50s where there was no increase in mir-200c levels , it is likely that the effects on proliferation occur via zeb1 and not by mir-200c . 
 the fact that the increase in chemosensitivity is found in cells that are proliferating more slowly than the negative controls demonstrates that increased proliferation is not the mechanism behind the increase in chemosensitivity . 
 in this paper we build on our previous work to further characterize the role that loss of mir-200c plays in generating an aggressive cancer phenotype . 
 zeb1 is normally only expressed in cells of mesenchymal origin ; however , its aberrant expression is observed in cancers that have undergone emt . 
 zeb1 ( and the closely related zeb2 ) transcripts are targeted by mir-200c and the other mir-200 family members . 
 interaction of any of the mir-200 family members with the zeb transcripts results in degradation and inhibition of translation . 
 therefore the maintenance of mir-200c expression in normal epithelial cells serves to prevent zeb1 and zeb2 from being expressed . 
 since both zeb1 and zeb2 repress genes involved in polarity , repression of these proteins serves to maintain polarity , an important epithelial cell characteristic . 
 we have recently shown that in addition to repressing zeb1 and 2 , mir-200c represses a program of transcripts normally only expressed in cells of mesenchymal and neuronal origin , such as fibronectin ( fn1 ) , neurotrophic tyrosine kinase ( ntrk2 ) , quaking 1 ( qki ) , and tubb3 . 
 thus , mir-200c maintains epithelial cell characteristics not only by maintaining polarity via repression of zeb1 and zeb2 , but also by repressing additional non - epithelial genes . 
 it has been recently demonstrated that mir-200c and zeb1 regulate each other in a double - negative feedback loop [ 31 , 32 ] . 
 the mir-200 family of mirnas is expressed in two clusters , one on choromosome 1p36.33 and the other on chromosome 12p12.31 . 
 therefore , in cells that have undergone emt , zeb1 and 2 not only serve to repress genes involved in polarity , but also repress the mir-200 family and thereby release the repression of many genes characteristic of the mesenchymal phenotype . 
 central to the double feedback loop between mir-200c and zeb1 is tgf-. during tgf-   -induced emt , there is an increase in ets1 which binds to and activates the promoter of zeb1 . 
 therefore , in a tumor microenvironment , increased tgf- levels are thought to result in an increase of zeb1 transcription to a point where it can overcome the repression caused by mir-200c . 
 as zeb1 protein begins to be made , it can then repress the mir-200 family members , resulting in progression through emt . 
 we have previously shown that restoration of mir-200c in hec50 endometrial cells and mda - mb-231 breast cancer cells causes repression of zeb1 and re - expression of e - cadherin protein . here 
 we show that transfection of mir-200c mimic into hey cells , an aggressive serous ovarian cell line , also causes a dramatic repression of zeb1 ; however no expression of e - cadherin was observed . to test whether the double - negative feedback loop is intact in the hec50 and mda - mb-231 cells , 
 these cells were infected with lentivirus expressing an shrna against zeb1 . in both cell lines , efficient knock down of zeb1 was achieved , as was re - expression of e - cadherin . in mda - mb-231 cells , zeb1 knock down resulted in an increase in mir-200c levels , as would be expected from the negative feedback loop . however , this was not the case in hec50 cells , where there was no increase in mir-200c . 
 whether the break in the negative feedback loop is an anomaly of this particular cell line remains to be tested . 
 the mechanism behind the phenomenon is also unknown ; however , it does offer an opportunity to dissect the contribution of zeb1 versus that of mir-200c to the phenotype of the cells . 
 for example , significantly decreased proliferation was observed in the hey cells transiently transfected with the mir-200c mimic as well as in the mda - mb-231 and hec50 cells that have zeb1 stably knocked down , although mir-200c levels did not rise in the hec50s . therefore it is likely that the decrease in proliferation is due to the lack of zeb1 , not an increase in mir-200c . 
 conversely , the decrease in adhesion to the basement membrane complex and laminin was only observed in the hey and mda - mb-231 cells , and not the hec50s , suggesting that this phenotype is a function of mir-200c expression rather than zeb1 . 
 we and others have previously shown that restoration of mir-200c to cancer cells that do not express it causes a decrease in invasion and migration [ 27 , 30 ] . here 
 we show that knock down of zeb1 in mda - mb-231 cells , which causes an increase in mir-200c , negatively affects migration and invasion . 
 furthermore , we show that restoration of mir-200c in hey ovarian cancer cells results in a dramatic decrease in migration and invasion even though e - cadherin is not restored in these cells , despite complete repression of zeb1 . 
 loss of e - cadherin expression can result from mechanisms other than zeb1 transcription repression , including chromosomal deletion and promoter hypermethylation [ 3437 ] . 
 possibilities for its continued absence in these cells include promoter methylation such that even when repression by zeb1 is relieved , e - cadherin will not be expressed , or perhaps levels of another transcriptional repressor such as snail or twist remain high and repress e - cadherin . 
 regardless of the mechanism , the effects of mir-200c on invasion and migration appear to be independent of e - cadherin status . 
 while e - cadherin is involved in epithelial cell - cell adhesion and its expression has been shown to negatively affect migration and invasion [ 45 , 46 ] , increased mir-200c is able to decrease migration and invasion on its own . 
 we have previously observed that restoration of mir-200c affects genes involved in cell motility and invasion such as arhgdib , ntrk2 , ephb1 , and fn1 . 
 we demonstrate that mir-200c causes a decrease in adhesion to basement membrane complex , laminin , and perhaps collagen type iv . 
 this observation is particularly relevant to ovarian cancer because the cancerous cells adhere to sites within the peritoneal cavity . during the progression of cancer 
 there is switching of the expression patterns of the cell surface adhesion molecules , such as the cadherins and integrins [ 4749 ] . 
 although the change in the number of adherent cells appears modest , this might play a significant role in developing a potential treatment for ovarian cancer . 
 the ability of ovarian cancer cells to spread and adhere to the peritoneal cavity is one of the major phenotypes of this disease . 
 a small change in ability of the cells to adhere might reflect a great decrease in the tumor burden and/or increase the ability to debulk the tumor . 
 these results are independent of e - cadherin expression since the decrease in adhesion is observed in both the mda - mb-231 cells ( where e - cadherin expression is regained with increased mir-200c ) and in hey cells , where it is not . 
 clonogenic assays reveal that there is an increase in chemosensitivity to paclitaxel with increased mir-200c levels . 
 indeed , acquired resistance to paclitaxel in ovarian cancer cells has been shown to be associated with emt , resulting in an aggressive phenotype . clinically , 
 aberrant expression of tubb3 ( not normally expressed in epithelial cells ) has been found to be associated with resistance to taxanes [ 3841 ] . 
 we have previously shown that tubb3 is a direct target of mir-200c and suggested that its repression by mir-200c is the mechanism behind the ability of mir-200c to increase chemosensitivity to microtubule targeting agents . here , we perform the definitive experiment to prove that mir-200c - mediated tubb3 downregulation is indeed the cause of the enhanced chemosensitivity . 
 we utilized exogenous tubb3 lacking its 3utr such that it can not be targeted by mir-200c and show that resistance to paclitaxel is maintained even in the presence of mir-200c . 
 in contrast , endogenous tubb3 is reduced when mir-200c is added , resulting in enhanced chemosensitivity to paclitaxel . microtubule targeting agents such as paclitaxel work more efficiently in cells that are rapidly dividing . 
 consequently , it could be argued that the increase in chemosensitivity caused by mir-200c is due to increased proliferation . 
 however , we show that increase of mir-200c or direct knockdown of zeb1 results in decreased proliferation in three different types of cancer cells . 
 it is therefore the downregulation of tubb3 , not an increase in proliferation that is responsible for the enhanced chemosensitivity to taxanes observed with restoration of mir-200c to resistant cancer cells . 
 mir-200c expression serves to maintain the epithelial phenotype in well - differentiated , low - grade , breast , ovarian , and endometrial cancer cells . 
 furthermore we find that not all of mir-200c 's actions can be attributed to the restoration of e - cadherin via targeting of zeb1 . 
 we further prove that mir-200c - mediated repression of tubb3 is the cause of enhanced chemosensitivity to microtubule targeting agents . 
 lastly we demonstrate that not all cells exhibit the double negative feedback loop between mir-200c and zeb1 and that this can be exploited to identify the distinct roles of mir-200c as compared to zeb1 .","we focus on unique roles of mir-200c in breast , ovarian , and endometrial cancers 
 . members of the mir-200 family target zeb1 , a transcription factor which represses e - cadherin and other genes involved in polarity . 
 we demonstrate that the double negative feedback loop between mir-200c and zeb1 is functional in some , but not all cell lines . 
 restoration of mir-200c to aggressive cancer cells causes a decrease in migration and invasion . 
 these effects are independent of e - cadherin status . 
 additionally , we observe that restoration of mir-200c to ovarian cancer cells causes a decrease in adhesion to laminin . 
 we have previously reported that reintroduction of mir-200c to aggressive cells that lack mir-200c expression restores sensitivity to paclitaxel . 
 we now prove that this ability is a result of direct targeting of class iii beta - tubulin ( tubb3 ) . 
 introduction of a tubb3 expression construct lacking the mir-200c target site into cells transfected with mir-200c mimic results in no change in sensitivity to paclitaxel . 
 lastly , we observe a decrease in proliferation in cells transfected with mir-200c mimic , and cells where zeb1 is knocked down stably , demonstrating that the ability of mir-200c to enhance sensitivity to paclitaxel is not due to an increased proliferation rate .",pubmed
"the transobturator approach to treat female stress urinary incontinence by means of a midurethral sling ( mus ) placement was first described by delorme to potentially prevent complications associated with the retropubic mus placement originally described by ulmsten ( delorme , 2001 ; ulmsten et al . , 1996 ) . 
 a similar transobturator passage utilizing an inside - out approach , as opposed to delorme s outside - in approach , was later described by de leval , aiming to further reduce the potential injury to the urethra and bladder ( de leval , 2003 ) . 
 a recent meta - analysis found similar cure rates between the 3 most commonly used surgical approaches for treating stress urinary incontinence , namely the retropubic , outside - in transobturator , and inside - out transobturator mus procedures ( latthe et al . , 2010 ) . 
 multiple studies have discussed the anatomical trajectories of both obturator approaches and confirmed their anatomical safety from a neurovascular perspective ( bonnet et al . , 2005 ; achtari et al . , 2006 ; hinoul et al . 
 , 2007 ; zahn et al . , 2007 ; reisenauer et al . , 2006 ) . 
 the large body of clinical evidence supports these findings . to date , anatomical studies have focused on the mus s trajectory in relation to the obturator foramen , the muscles and especially the neuro - vascular structures . 
 traditional anatomical dissections do not allow the study of the mus s configuration in relation to the urethra , as a progressive dissection of the entire tape s trajectory would be required , inherently leading to a distortion of the relevant anatomy . 
 the aim of this cadaveric study was to compare the specific relationship between the tape and the urethra in both the inside - out and outside - in approach . to allow such a comparison , 
 ct - scan image analysis was performed after tantalum wire marked mesh slings were implanted in a series of cadavers . 
 all 10 cadavers were operated upon , by independent surgeons , one experienced with the  inside - out ( in - out ) and one with 
 cadavers were positioned in the dorsal supine lithotomy position with the legs in abduction and at a 100 flexion position ( at the level of the hips ) on the in - outside and at 90 flexion on the out - in side . the surgical technique for the transobturator mus procedures were performed according to the instructions for use as provided by the respective manufacturers ( out - in : monarc , american medical systems , minnetonka , mn and in - out : tvt - obturator system , ethicon inc . , 
 all cadavers were catheterized using a foley catheter with its lumen filled with a radio - opaque solution ( hypaquemeglumine 60% , amersham health , princeton , nj ) to allow subsequent identification of the urethral position . 
 both the in - out and the out - in transobturator procedures were performed on each cadaver , alternating the type of procedure and tape between the right and left sides in the consecutively operated cadavers . to be able to do this with a single tape in each cadaver , 
 a device was conceived for this study in which the tape on one side consisted of an in - out mus device , while an out - in mus device was attached to the other side at the midpoint . 
 to allow radiographic visualization of these tapes , a double tantalum wire was threaded through each side of both midurethral tapes as shown in figure 1 . 
 this design allowed for an intra - cadaveric comparison between 10 independent in - out transobturator mus trajectories and 10 out - in transobturator mus trajectories . 
 care was taken to have a flat positioning of both tape ends at the end of the procedures . 
 the vaginal incision sites were closed and the mus tapes were cut 2  cm from the level of the skin . to exclude the possible influence of the surgeon s right handedness , an equal number of both procedures was performed on the right and left sides ; 
 bmi of the cadavers ranged between 15 and 33  kg / m2 ( mean : 25.3 ; median : 26 ) . 
 post capture image analysis was performed using the vitrea core 5.1.1618.1808 image analysis software ( vital , 5850 opus parkway , suite 300 , minnetonka , mn 55343 - 4414 ) . 
 plane that included the urethra at the point where it crossed the urethra and the two arms of the sling extending from the urethra to the point where it crossed the obturator membrane . 
 this was done to prevent parallax effects that might alter the measured angle ( hoyte and ratiu , 2001 ) . 
 on the mips view of the images , the relative angulations between a line through the urethral axis and the slope drawn through each mus tape s mean upward pathway ( forming the hammock part of its trajectory ) were measured ( table i ) . 
 the angle inside the upward trajectory of the tape constituted the angle under consideration for this study and is referred to as the tape s hemi inner angle , as only half a procedure was performed on each cadaver ( fig . 
 for the study s purposes the assumption was made that a symmetric , bilateral placement of a full length tape would yield the full inner angle of the mus hammock corresponding to double the hemi - inner angle measurement . 
 2a . ventro - dorsal view ( frontal view ; l = left , r = right ) . 
 note : dotted lines schematically demonstrates the hemi - inner angle but do not correspond to the mips view used to measure the actual angles . 
 statistical analyses were performed using the wilcoxon signed - rank test for matched paired data for non - parametric testing of 2 dependent samples . 
 statistical analyses were performed using the wilcoxon signed - rank test for matched paired data for non - parametric testing of 2 dependent samples . 
 three cadavers were noted to have stage 2 prolapse and 1 had a notably high vaginal sulcus . 
 the 3-dimensional ct images revealed that the mus tapes lie as a band posterior / dorsal to the urethra rather than inferior . 
 the out - in muss were more closely wrapped around the inferior ischio - pubic ramus than the in - out muss . 
 qualitative assessment of the images demonstrated a safe distance from the tapes to the obturator canal for both techniques . 
 distances of the tapes trajectories to the individual obturator nerve branches could not be determined . 
 the mean full inner angle in a strict antero - posterior view formed by the in - out mus measured 122 ( 95%ci : 107-136 ) ; versus 144 ( 95%ci : 131-151 ) for the out - in mus ( p  =  0.02 ) . 
 the ( paired ) differences between the tapes inner angles were significantly different ; with a mean difference of 19.8 ( median 19.0 ) , ( wilcoxon signed rank test : p  =   0.008 ) . 
 correlation analysis only showed a trend between the out - in and the in - out mus angle ( correlation coefficient : 0.6 , p  =  0.08 ) . 
 a similar trend was observed between the patient s bmi and the in - out mus angle ( correlation coefficient : 0.6 , p  =  0.08 ) , but not for bmi and the out - in mus angle ( correlation coefficient : 0.08 , p  =  0.8 ) . 
 there was no correlation between the infra - pubic angle measurement and the out - in mus angle nor the in - out mus angle . 
 the use of tantalum wires threaded through the polypropylene-/ prolene - tape in combination with three - dimensional ct - scanning yielded a unique method to visualize midurethral tapes throughout the entirety of their trajectory . 
 this technique can be applied to different types of non radio - opaque implants , allowing for a better spatial understanding of the anatomical position within the pelvis . 
 this technique circumvents the technical problems encountered by ultrasound where visualization is partially obstructed by the bony pelvis and the fact that traditional anatomical techniques rely on a progressive dissection intrinsically only allowing visualization at a single level each time . 
 this is the first controlled anatomical study to compare the spatial relationships between the in - out and out - in mus tapes and the female urethra . 
 the difference could be objectified by measuring the angle formed between the two legs of the mus tape . 
 the in - out approach angulated around the urethra in a more acute fashion than the out - in approach , which seemed to follow a more horizontal , flatter , trajectory ( 122 versus 142 ) . 
 these findings were counter - intuitive as the exit - points of both devices at the level of the skin would suggest the opposite . 
 the three dimensional radiographic visualization of the tape s trajectory also allowed for an appreciation of the mus tapes spatial configuration in relation to the bony pelvis and the urethra . 
 contrary to the belief that the urethra is suspended in a cranially directed u - shaped band , both obturator mus approaches were demonstrated to be u - shaped bands that head backwards or dorsally . 
 the tape is not positioned caudally but lies as a band behind the urethra , suggestive of its passive ( non obstructive ) role in achieving continence . as observed in the different measurements of the tapes angles , there seemed to be a higher variability in the in - out s than the out - in trajectory ( 58 versus 40 variation respectively ) , as previously described by hinoul et al . in a traditional anatomic study , underscoring the need for strict adherence to the prescribed operative technique ( hinoul et al . , 2007 ) . 
 the importance of this becomes clear when comparing it to two ultrasound studies investigating the angles formed by the two arms of mus tapes . 
 lin et al described an angle at rest for the in - out tape to be 115 ( sd13 ) while chene et al , measured the same angle at rest also using ultrasound to be 138(sd7 ) ( lin et al . 
 the differences in outcomes obtained in these studies demonstrate that ( inter- and intra - individual ) comparisons may prove to be difficult to interpret . 
 the cadaveric study design was also the study s limitation as no clinical findings could be correlated to the static anatomic findings . a direct meta - analysis comparing both routes of transobturator tapes by madhuvrata et al . 
 ( 2012 ) reported no significant differences in the objective and subjective efficacy but showed that the  inside - out 
 route was associated with significantly fewer vaginal angle injuries but with a non - significanttrend towards higher risk of postoperative groin pain . in a small retrospective study , out - in mus tapes 
 were more frequently associated with a finding of superficial placement at the level of the lateral vaginal sulcus , described as paraurethral banding ( cholhan et al . , 2010 ) . 
 coincidentally , the only cadaver with a notably high vaginal sulcus was found to have a very palpable out - in mus tape post placement . increased rates of dyspareunia and tape exposures after the out - in compared to the in - out approach 
 ( 2012 ) , but these finding were not confirmed in an rct by abdel - fattah et al . 
 the more horizontal positioning of the out - in tape s trajectory , taking the mesh closer to the vaginal wall , as described in this study could explain these clinical observations . 
 the radiographic images explain the increased risk of  button - holing during an out - in procedure because the more acute in - out mus trajectory buries the tape deeper inside the internal adductor muscles . on the other hand 
 , this study also confirmed that the out - in mus trajectory stayed closer to the ischiopubic ramus . 
 the slightly more lateral in - out mus trajectory causes the tape to be seated in more muscular tissue . 
 the corresponding inflammatory reaction might explain the trend to a slightly higher risk of transient thigh pain . 
 the clinical implications from this cadaveric imaging study are that surgeons must pay more attention to the 2nd half of the helical pass , whichever way they chose to go . for the in - out passage 
 , one must rotate hard and carefully around the ischiopubic ramus and not get lazy with the rotation ; while for the out - in pass , care must be taken that dissection of the peri - urethral tunnel provides for a full thickness vaginal epithelial layer . 
 the advent of three - dimensional ultrasonography will allow for similar measurements of the different tapes trajectories in vivo in a dynamic setting , possibly allowing correlation of these findings to nuances in clinical outcomes . 
 ultrasound will not be able to provide the same holistic visualization within the bony pelvis , but mesh - tape beyond the obturator membrane probably does not contribute to the mus functional effect or possible adverse events at the level of the vagina . 
 recently , a new approach , incorporating iron particles into polymer - based implants , allowed in vivo visualization of mesh by magnetic resonance imaging ( mri ) following inguinal hernia surgery ( hansen et al . , 2013 ) . 
 approval of these types of products for human use in the future will bring new insights into the action mechanism of mesh in the pelvic floor as well as a better understanding of the mesh- tissue interaction .","the three - dimensional configuration of mid - urethral sling tapes is difficult to demonstrate in traditional anatomical dissections or imaging studies . the aim of this study was to test the utility of a novel technique using mesh tapes to assess spatial differences between the in - out and out - in transobturator mid - urethral slings . 
 two independent surgeons performed their usual transobturator mid - urethral sling placement on 10 fresh thawed cadavers , alternating sides in the consecutive cadavers . 
 tantalum wires threaded through the polypropylene - tapes rendered them radio - opaque . following placement , ct scans 
 were obtained to generate 3-d and mips images for analysis . 
 results showed that the mean angle formed by the in - out sling measured 122 ( 95%ci : 107-136 ) ; versus 144 ( 95%ci : 131-151 ) for the out - in sling ( p  =  0.02 ) . 
 the paired differences between the tapes inner angles were significantly different ; with a mean difference of 20 ( median 19.0 ) , ( p  =  0.008 ) . there was no significant correlation between either approach and bmi or angle of the pubic arch . 
 the images revealed that the tapes lie as a band posterior / dorsal to the urethra rather than inferior . in conclusion : marking mesh with tantalum wire , in combination with 3-d and mips ct - scan reconstruction images , provided a unique method to visualize the entire sling trajectory . 
 the clinical implications of the more horizontal positioning after the out - in approach remain to be determined .",pubmed